Nucleic acid molecules for use in the production of recombinant AAV virions

Abstract

To provide a method for increasing production efficiency of recombinant AAV particles.SOLUTION: A nucleic acid molecule including at least the following base sequences: (a) a base sequence coding Rep protein of AAV or functional equivalents thereof; (b) a base sequence coding Cap protein of AAV or functional equivalents thereof; (c) a base sequence including inverted terminal repeat (ITR) of first AAV or functional equivalents thereof; (d) a base sequence including inverted terminal repeat (ITR) of second AAV or functional equivalents thereof; (e) a base sequence for inserting a base sequence coding foreign protein or / and a base sequence coding foreign protein which is positioned between the first ITR and the second ITR; (f) a base sequence including an E2A region of an adenovirus or functional equivalents thereof; (g) a base sequence including an E4 region of an adenovirus or functional equivalents thereof; and (h) a base sequence including a VA1 RNA region of an adenovirus or functional equivalents thereof.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] In one embodiment, the present invention relates to the field of recombinant adeno-associated virus (rAAV) virions that can be used in gene therapy, and more particularly to nucleic acid molecules that can be used to produce rAAV virions and methods for producing rAAV virions using the nucleic acid molecules. [Background technology]

[0002] Adeno-associated virus (AAV) belongs to the Parvoviridae family, which is a linear single-stranded DNA virus that is one of the smallest viruses found in nature, and its viral genome is approximately 4.7 kb. AAV does not have an envelope and forms regular icosahedral particles with a diameter of approximately 22 nm. AAV is also classified as a defective virus that cannot grow by infection alone, and requires co-infection with a helper virus that supports AAV replication in order to replicate efficiently in animal cells. Examples of helper viruses that support AAV replication include adenoviruses and herpes viruses. In the absence of a helper virus, AAV replication is not observed, and the AAV genome is integrated into the host genome.

[0003] When wild-type AAV infects a human host cell alone, the viral genome is site-specifically integrated into chromosome 19 via the inverted terminal repeats (ITRs) present at both ends of the viral genome. The genes of the viral genome integrated into the genome of the host cell are hardly expressed, but when the cell is infected with a helper virus, AAV is excised from the host genome and replication of the infectious virus begins. When the helper virus is an adenovirus, the genes responsible for the helper function are E1A, E1B, E2A, VA1, and E4. Here, when the host cell is a HEK293 cell, which is a cell derived from human fetal kidney tissue transformed with adenovirus E1A and E1B, the E1A and E1B genes are originally expressed in the host cell.

[0004] The AAV genome also contains two genes, rep and cap. The rep proteins (rep78, rep68, rep52, and rep40) produced by the rep gene are essential for capsid formation and mediate the integration of the viral genome into the chromosome. The cap gene is responsible for the production of three capsid proteins (VP1, VP2, and VP3).

[0005] In the wild-type AAV genome, there are ITRs at both ends, and the rep and cap genes are present between the ITRs. A recombinant AAV virion (rAAV virion) is a virion in which the region containing the rep and cap genes has been replaced with a gene encoding a foreign protein, and the virion is encapsulated in a capsid.

[0006] rAAV virions are used as vectors to deliver exogenous genes into cells in gene therapy, and have the potential to provide therapeutic options for previously incurable diseases.

[0007] Three types of plasmids are generally used to produce rAAV virions. These are a plasmid containing a gene encoding a foreign protein flanked by ITRs (Patent Document 1), a plasmid containing a gene encoding a Rep protein and a gene encoding a Cap protein (Patent Document 2), and a plasmid containing genes encoding adenovirus-derived E2A, E4, and VA1 RNA (Patent Document 3). In order to produce rAAV virions containing a target gene in a gene-transfected cell, it is necessary to introduce these three types of plasmids into a host cell such as a HEK293 cell. rAAV virions are synthesized in a host cell into which the three types of plasmids have been introduced. At this time, in addition to rAAV virions, empty particles that do not contain DNA are generated in the host cell. It has also been reported that in a transient expression system of plasmid DNA, nearly 80% are empty particles that do not contain DNA. (Non-Patent Document 1) [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO 95 / 34670 [Patent Document 2] International Publication No. 97 / 06272 [Patent Document 3] International Publication No. 97 / 17458 [Non-patent literature]

[0009] [Non-Patent Document 1] Maria S. et al., HUMAN GENE THERAPY METHODS, 28, 101-108 (2017) Summary of the Invention [Problem to be solved by the invention]

[0010] In one embodiment, the present invention relates to a nucleic acid molecule used for efficiently producing rAAV virions in which a nucleic acid sequence including a base sequence encoding a foreign protein that can be used for gene therapy, etc. is packaged in an AAV capsid, and a method for producing such rAAV virions. [Means for solving the problem]

[0011] In the research for the above-mentioned object, the present inventors have conducted intensive studies and found that rAAV virions can be efficiently produced by introducing into a host cell a nucleic acid molecule comprising a base sequence encoding the Rep protein of an adeno-associated virus, a base sequence encoding the Cap protein of an adeno-associated virus, a base sequence including an inverted terminal repeat (ITR) of a first adeno-associated virus, a base sequence including an inverted terminal repeat (ITR) of a second adeno-associated virus, a base sequence including an E2A region of an adenovirus, a base sequence including an E4 region of an adenovirus, and a base sequence including a VA1 RNA region of an adenovirus, and a base sequence encoding a foreign protein between the first and second ITRs of the nucleic acid molecule, thereby completing the present invention. That is, the present invention includes the following. 1. A nucleic acid molecule comprising at least the following base sequence: (a) a nucleotide sequence encoding the Rep protein of the adeno-associated virus or a functional equivalent thereof, (b) a nucleotide sequence encoding the Cap protein of an adeno-associated virus or a functional equivalent thereof, (c) a nucleotide sequence containing the inverted terminal repeat (ITR) of a first adeno-associated virus or a functional equivalent thereof, (d) a nucleotide sequence containing the inverted terminal repeat (ITR) of a second adeno-associated virus or a functional equivalent thereof, (e) a base sequence for inserting a base sequence encoding a foreign protein located between the first and second ITRs and / or a base sequence encoding a foreign protein; (f) a nucleotide sequence containing the E2A region of an adenovirus or a functional equivalent thereof, (g) a nucleotide sequence containing the E4 region of an adenovirus or a functional equivalent thereof, and (h) A nucleotide sequence containing the VA1 RNA region of an adenovirus or a functional equivalent thereof. 2. A base sequence including a first gene expression control site that controls the expression of the Rep protein, A base sequence including a second gene expression control site that controls the expression of the Cap protein, and The nucleic acid molecule of the above 1, further comprising a base sequence including a third gene expression control site that controls the expression of the foreign protein. 3. A nucleic acid molecule according to 1 or 2 above, which further comprises a base sequence containing a drug resistance gene. 4. A nucleic acid molecule according to 2 or 3 above, which comprises, downstream of the base sequence including the first gene expression control site, a base sequence encoding the Rep protein, further downstream a base sequence including the second gene expression control site, and further downstream a base sequence encoding the Cap protein. 5. A nucleic acid molecule of any of 2 to 4 above, which comprises, downstream of the base sequence including the first inverted terminal repeat (ITR), a base sequence including the third gene expression control site, and further downstream, a base sequence for inserting a base sequence encoding the foreign protein and / or a base sequence encoding the foreign protein, and further downstream, a base sequence including the second inverted terminal repeat (ITR). 6. A nucleic acid molecule according to any one of 2 to 5 above, which comprises a base sequence of the E4 region of the adenovirus or a functional equivalent thereof downstream of the base sequence of the E2A region of the adenovirus or a functional equivalent thereof, and further downstream of the base sequence of the VA1 RNA region of the adenovirus or a functional equivalent thereof. 7. A nucleic acid molecule as set forth in 4 above, which comprises a base sequence including the first inverted terminal repeat (ITR) downstream of the base sequence encoding the Cap protein, a base sequence further downstream including the third gene expression control site, and a base sequence for inserting a base sequence encoding the foreign protein further downstream and / or a base sequence encoding the foreign protein and a base sequence further downstream including the second inverted terminal repeat (ITR). 8. A nucleic acid molecule as set forth in 7 above, which comprises, downstream of the base sequence including the second inverted terminal repeat (ITR), a base sequence of the E2A region of the adenovirus or a functional equivalent thereof, further downstream, a base sequence of the E4 region of the adenovirus or a functional equivalent thereof, and further downstream, a base sequence of the adenovirus VA1 RNA region or a functional equivalent thereof. 9. A nucleic acid molecule according to any one of 1 to 8 above, further comprising a base sequence containing a drug resistance gene. 10. The nucleic acid molecule according to 9 above, which contains a base sequence including a drug resistance gene downstream of the base sequence encoding the Cap protein and upstream of the base sequence including the first ITR. 11. A nucleic acid molecule according to any one of 1 to 10 above, which comprises a base sequence downstream of the base sequence encoding the Cap protein, the base sequence having an enhancer function for increasing the expression levels of the Rep protein and the Cap protein. 12. A nucleic acid molecule according to any one of 1 to 11 above, wherein the Rep protein is selected from the group consisting of adeno-associated viruses of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. 13. The nucleic acid molecule according to claim 12, wherein the Rep protein comprises Rep68 or / and Rep78. 14. A nucleic acid molecule according to any one of 1 to 13 above, wherein the Cap protein is selected from the group consisting of adeno-associated viruses of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. 15. The nucleic acid molecule of claim 14, wherein the Cap protein comprises VP1. 16. Any of the nucleic acid molecules according to 2 to 15 above, wherein the first gene expression control site is controlled by the E2A region of the adenovirus or a functional equivalent thereof. 17. The nucleic acid molecule of claim 16, wherein the first gene expression control site is the p5 promoter of the adeno-associated virus. 18. Any of the nucleic acid molecules according to 2 to 17 above, wherein the second gene expression control site is controlled by adenovirus VA1 RNA or a functional equivalent thereof. 19. The nucleic acid molecule of claim 18, wherein the second gene expression control site is the p40 promoter of an adeno-associated virus. 20. Any of the nucleic acid molecules 1 to 19 above, wherein the adeno-associated virus serotypes of the first and second ITRs are selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. 21. A nucleic acid molecule according to any one of 1 to 19 above, wherein the adeno-associated virus serotype of the ITR is 2. 22. A nucleic acid molecule according to any one of 1 to 21 above, wherein the base sequence for encoding the foreign protein contains a multicloning site. 23. A nucleic acid molecule according to any one of 1 to 22 above, wherein the adenovirus serotype is selected from the group consisting of 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24 to 30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3 type, canine Ad2 type, ovine Ad, and porcine Ad3 type. 24. Any of the nucleic acid molecules 1 to 23 above, wherein the functional equivalent of the E2A region of the adenovirus is derived from any one selected from the group consisting of herpes simplex virus, vaccinia virus, cytomegalovirus, and pseudorabies virus. 25. Any of the nucleic acid molecules 1 to 24 above, wherein the functional equivalent of the E4 region of the adenovirus is derived from any one selected from the group consisting of herpes simplex virus, vaccinia virus, cytomegalovirus, and pseudorabies virus. 26. Any of the nucleic acid molecules 1 to 25 above, wherein the functional equivalent of the VA1 RNA region of the adenovirus is derived from any one selected from the group consisting of herpes simplex virus, vaccinia virus, cytomegalovirus, and pseudorabies virus. 27. A nucleic acid molecule according to any one of 2 to 26 above, wherein the E2A region of the adenovirus is controlled by E1B of the adenovirus or a functional equivalent thereof. 28. A nucleic acid molecule according to any one of 2 to 27 above, wherein the E4 region of the adenovirus is controlled by E1B of the adenovirus or a functional equivalent thereof. 29. A nucleic acid molecule according to any one of 9 to 28 above, wherein the drug resistance gene confers resistance to a drug corresponding to the drug resistance gene to a prokaryotic cell. 30. Any of the nucleic acid molecules 2 to 29 above, wherein the third gene expression control site is selected from the group consisting of a promoter derived from a cytomegalovirus, an SV40 early promoter, a human elongation factor-1 alpha (EF-1α) promoter, a human ubiquitin C promoter, and a β-actin promoter, a promoter comprising the base sequence shown in SEQ ID NO: 47, and a promoter comprising the base sequence shown in SEQ ID NO: 39 downstream of a promoter comprising the base sequence shown in SEQ ID NO: 47. 31. A nucleic acid molecule according to any one of 11 to 30 above, wherein the base sequence having an enhancer function is the p5 promoter of an adeno-associated virus or a functional equivalent thereof. 32. A nucleic acid molecule according to any one of 1 to 31 above, wherein the foreign protein is human. 33. A nucleic acid molecule according to any one of 1 to 31 above, wherein the foreign protein is selected from the group consisting of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody. 34. The foreign protein is growth hormone, lysosomal enzyme, somatomedin, insulin, glucagon, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of antibody and other protein, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle stimulating hormone (FSH), gonadotropin releasing hormone (GnRH), gonadotropin, DNasel, thyroid stimulating hormone (TSH), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell any of the nucleic acid molecules 1 to 32 above, which is selected from the group consisting of growth factor deficiency (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-α receptor), an enzyme having an activity of degrading beta-amyloid, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and GLP-1 receptor agonist. 35. The foreign protein is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. A nucleic acid molecule according to any one of 1 to 32 above, which is selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1 and CLN2. 36. A nucleic acid molecule according to any one of 1 to 33 above, wherein the foreign protein is selected from the group consisting of anti-IL-6 antibodies, anti-beta amyloid antibodies, anti-BACE antibodies, anti-EGFR antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-HER2 antibodies, anti-PCSK9 antibodies, anti-TNF-α antibodies, and antibodies having affinity for proteins present on the surface of vascular endothelial cells. 37. The nucleic acid molecule of 36 above, wherein the protein present on the surface of the vascular endothelial cell is selected from the group consisting of transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor. 38. The foreign protein is a fusion protein of an antibody and another protein, the antibody being selected from the group consisting of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody, and the other protein is a growth hormone, a lysosomal enzyme, a cytokine, a lymphokine, a blood coagulation factor, an antibody, a fusion protein of an antibody and another protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), erythropoietin, darbepoietin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone, DNasel, thyroid-stimulating hormone (T SH), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), and an enzyme having the activity of degrading beta-amyloid, any of the nucleic acid molecules 1 to 31 above. 39. The foreign protein is a fusion protein of an antibody and another protein, the antibody is selected from the group consisting of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody, and the other protein is a lysosomal enzyme, the lysosomal enzyme being α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, α-galactosidase A nucleic acid molecule according to any one of 1 to 31 above, which is selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1 and CLN2. 40. The nucleic acid molecule of 38 or 39 above, wherein the other protein is human. 41. A nucleic acid molecule according to any one of 38 to 40 above, wherein the antibody has affinity for a protein present on the surface of a vascular endothelial cell. 42. The nucleic acid molecule of 41 above, wherein the protein present on the surface of the vascular endothelial cell is selected from the group consisting of transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor. 43. A nucleic acid molecule according to any one of 1 to 42 above, which is a circular plasmid. 44. A method for producing a recombinant AAV virion, comprising the step of introducing any one of the nucleic acid molecules according to 1 to 43 above into a host cell. 45. The production method according to claim 44, wherein the genome of the host cell contains a base sequence including the E1A and E1B regions of adenovirus. 46. ​​The production method according to claim 45, wherein the host cell is a HEK293 cell. Effect of the Invention

[0012] According to one embodiment of the present invention, rAAV virions packaged with nucleic acid sequences including base sequences encoding foreign proteins that can be used for gene therapy, etc. can be efficiently produced, making it possible to supply rAAV virions at low cost and reducing the medical costs required for gene therapy. [Brief description of the drawings]

[0013] [Figure 1] Schematic diagram showing the structure of the pHelper(mod) vector. [Diagram 2] Schematic diagram showing the structure of the pAAV-CMV-GFP(mod) vector. [Diagram 3] Schematic diagram showing the structure of the pR2(mod)C6 vector. [Figure 4] Schematic diagram showing the structure of the pR2(mod)C9 vector. [Diagram 5] Schematic diagram showing the structure of the pHelper-ITR / CMV / GFP vector. [Figure 6] Schematic diagram showing the structure of the pHelper-R2(mod)C6 vector. [Figure 7] Schematic diagram showing the structure of the pHelper-R2(mod)C9 vector. [Figure 8] Schematic diagram showing the structure of the pR2(mod)C6-ITR / CMV / GFP vector. [Figure 9] Schematic diagram showing the structure of the pR2(mod)C9-ITR / CMV / GFP vector. [Figure 10]Schematic diagram showing the structure of the pHelper-ITR / CMV / GFP-R2(mod)C6 vector. [Figure 11] Schematic diagram showing the structure of the pHelper-ITR / CMV / GFP-R2(mod)C9 vector. [Figure 12] 12 is a diagram showing the results of measuring the amount of rAAV genome and the amount of total capsid contained in the culture supernatant obtained in Example 16. The vertical axis indicates the amount of rAAV genome (x1011) or the amount of total capsid (x1011). The black bars indicate the amount of rAAV genome, and the white bars indicate the amount of total capsid. From the left, (1) shows the results when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, were introduced, (2) shows the results when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, were introduced, (3) shows the results when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, (4) shows the results when pHelper-ITR / CMV / GFP-R2(mod)C9 vector, and (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced. Measurement results are shown only for rAAV9. [Figure 13]13 is a diagram showing the results of measuring the rAAV genome amount and total capsid amount contained in the purified cell lysate solution obtained in Example 18. The vertical axis indicates the rAAV genome amount (x1011) or the total capsid amount (x1011). The black bars indicate the rAAV genome amount, and the white bars indicate the total capsid amount. From the left, (1) shows the measurement results when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, were introduced, (2) shows the measurement results when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, were introduced, (3) shows the measurement results when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, (4) shows the measurement results when pHelper-ITR / CMV / GFP-R2(mod)C9 vector, and (5) shows the measurement results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced. Measurement results are shown only for rAAV9. [Figure 14] FIG. 14 is a diagram showing the results of calculating the abundance ratio in the total capsid of rAAV virions obtained in Example 19. The vertical axis shows the abundance ratio (%). From the left, the values ​​are shown for each of the following cases: (1) when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, are introduced; (2) when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, are introduced; (3) when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, are introduced; (4) when pHelper-ITR / CMV / GFP-R2(mod)C9 vector, is introduced; and (5) when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, are introduced. Measurement results are shown only for rAAV9. [Figure 15]15 is a diagram showing the results of measuring the infectivity of rAAV6 virions purified from the cell lysate solution obtained in Example 20. The vertical axis shows the percentage of GFP-positive cells, i.e., the infection efficiency (%) of rAAV6 virions. From the left, (1) two types of plasmids, pHelper-ITR / CMV / GFP vector and pRC6 vector, were introduced; (2) two types of plasmids, pHelper-R2(mod)C6 vector and pAAV-CMV-GFP vector, were introduced; (3) two types of plasmids, pR2(mod)C6-ITR / CMV / GFP vector and pHelper vector, were introduced; (4) pHelper-ITR / CMV / GFP-R2(mod)C6 vector was introduced; and (5) three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pRC6 vector, were introduced. For each, black bars indicate the amount of rAAV genome infected per well was 2 x 108 vg (i.e., the amount of rAAV virions was 2 x 108), and white bars indicate the amount of rAAV genome infected per well was 2 x 109 vg (i.e., the amount of rAAV virions was 2 x 109). [Figure 16]16 is a diagram showing the results of measuring the infectivity of rAAV9 virions purified from the cell lysate solution obtained in Example 21. The vertical axis shows the percentage of GFP-positive cells, i.e., the infection efficiency (%) of rAAV9 virions. From the left, (1) when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, were introduced; (2) when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, were introduced; (3) when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, were introduced; (4) when an integrated rAAV plasmid (pHelper-ITR / CMV / GFP-R2(mod)C9 vector) was introduced; and (5) when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced. For each case, the black bars represent the case where the amount of rAAV genome infected per well was 2x109 vg (i.e., 2x109 rAAV virions), and the white bars represent the case where the amount of rAAV genome infected per well was 1x1010 vg (i.e., rAAV virion amount: 2 x 1010). [Figure 17] 17 is a diagram showing the results of calculation of the Transducing Unit of the rAAV6 virion obtained in Example 22. The vertical axis shows the Transducing Unit value (x105). From the left, the Transducing Unit values ​​are shown for (1) when two types of plasmids, pHelper-ITR / CMV / GFP vector and pRC6 vector, were introduced, (2) when two types of plasmids, pHelper-R2(mod)C6 vector and pAAV-CMV-GFP vector, were introduced, (3) when two types of plasmids, pR2(mod)C6-ITR / CMV / GFP vector and pHelper vector, were introduced, (4) when an integrated rAAV plasmid (pHelper-ITR / CMV / GFP-R2(mod)C6 vector) was introduced, and (5) when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pRC6 vector, were introduced. [Figure 18] 18 is a diagram showing the results of calculation of the Transducing Unit of the rAAV9 virion obtained in Example 22. The vertical axis shows the Transducing Unit value (x105). From the left, the Transducing Unit values ​​are shown for (1) when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, were introduced, (2) when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, were introduced, (3) when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, (4) when integrated rAAV plasmid (pHelper-ITR / CMV / GFP-R2(mod)C9 vector) was introduced, and (5) when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced. [Figure 19] Schematic diagram showing the structure of the pAAV-CBA(BAM)-I2S vector. [Figure 20] Schematic diagram showing the structure of the pHelper-ITR / CBA(BAM) / I2S-R2(mod)C9 vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Three types of plasmids are typically used to produce recombinant adeno-associated virus (rAAV) virions used to introduce foreign genes into cells, tissues, or living organisms: (1) a plasmid (plasmid 1) having a structure containing a base sequence including a first inverted terminal repeat (ITR) and a base sequence including a second inverted terminal repeat (ITR) derived from a virus such as AAV, and a gene encoding a desired protein located between these two ITRs; (2) a plasmid (plasmid 2) containing an AAV Rep gene having the function required to integrate the base sequence of the region (including the ITR sequence) sandwiched between the ITR sequences into the genome of a host cell, and a gene encoding the capsid protein of AAV; and (3) a plasmid (plasmid 3) containing the E2A region, E4 region, and VA1 RNA region of adenovirus.

[0015] In general, to produce recombinant adeno-associated virus (rAAV) virions, these three types of plasmids are first introduced into host cells such as HEK293 cells in which the adenovirus E1a and E1b genes have been incorporated into the genome. Then, a region containing a base sequence containing the first inverted terminal repeat (ITR), a base sequence containing the second inverted terminal repeat (ITR), and a gene encoding a desired protein arranged between these two ITRs is incorporated into the genome of the host cell. Single-stranded DNA is replicated from this region and packaged into the capsid protein of AAV to form recombinant adeno-associated virus (rAAV) virions. These recombinant adeno-associated virus (rAAV) virions have infectivity and can be used to introduce foreign genes into cells, tissues, or living organisms.

[0016] In one embodiment, the present invention relates to a nucleic acid molecule comprising components of the above-mentioned three types of plasmids, plasmids 1 to 3, necessary for producing recombinant adeno-associated virus (rAAV) virions, namely, (a) a base sequence encoding an adeno-associated virus Rep protein or a functional equivalent thereof, (b) a base sequence encoding an adeno-associated virus Cap protein or a functional equivalent thereof, (c) a base sequence including a first inverted terminal repeat (ITR), (d) a base sequence including a second inverted terminal repeat (ITR), (e) a base sequence for inserting a base sequence encoding a foreign protein located between the first ITR and the second ITR and / or a base sequence encoding a foreign protein, (f) a base sequence encoding an adenovirus E2A protein or a functional equivalent thereof, (g) a base sequence encoding an adenovirus E4 protein or a functional equivalent thereof, and (h) a base sequence encoding an adenovirus VA1 RNA or a functional equivalent thereof.

[0017] In one embodiment of the present invention, the term "integrated rAAV plasmid" refers to a plasmid containing the above components (a) to (h). The integrated rAAV plasmid may be linear or circular.

[0018] Furthermore, in one embodiment, the present invention relates to a nucleic acid molecule further comprising a base sequence including a base sequence comprising a first gene expression control site that controls the expression of the Rep protein, a base sequence including a second gene expression control site that controls the expression of the Cap protein, and a base sequence including a third gene expression control site that controls the expression of the foreign protein.

[0019] The Rep protein of adeno-associated virus (AAV) is encoded by the rep gene of AAV. The Rep protein has a function required for integrating, for example, the AAV genome into the genome of a host cell via the ITR present in the genome. There are multiple subtypes of Rep proteins, but two types, Rep68 and Rep78, are required for the integration of the AAV genome into the genome of a host cell. Rep68 and Rep78 are the translation products of two types of mRNA transcribed by alternative splicing from the same gene. In the present invention, the Rep protein of AAV includes at least the two types of proteins, Rep68 and Rep78.

[0020] In one embodiment of the present invention, the base sequence encoding the Rep protein of an adeno-associated virus refers to a base sequence encoding at least Rep68 and Rep78, or a base sequence with a mutation added thereto. The Rep protein is preferably that of an AAV of serotype 2, but is not limited thereto, and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11. The base sequence encoding the Rep protein of a wild-type AAV of serotype 2 has the base sequence shown in SEQ ID NO: 1. Rep68 and Rep78 of a wild-type AAV of serotype 2 have the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0021] In addition, as long as Rep68 exerts its function, it may be modified by substitution, deletion, addition, or the like in the amino acid sequence of wild-type Rep68 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In the present invention, Rep68 with these mutations is also included in Rep68.

[0022] When amino acids in the amino acid sequence of wild-type Rep68 are replaced with other amino acids, the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of wild-type Rep68 are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Rep68 with mutations that combine these amino acid substitutions and deletions is also included in Rep68. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added in the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep68. Rep68 with mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep68. The amino acid sequence of the mutated Rep68 preferably exhibits greater than 85% homology to the amino acid sequence of wild-type Rep68, more preferably greater than 90% homology, even more preferably greater than 95% homology, and even more preferably greater than 98% homology.

[0023] In addition, as long as Rep78 exerts its function, it may be modified by substitution, deletion, addition, or the like in the amino acid sequence of wild-type Rep78 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In the present invention, Rep78 with these mutations is also included in Rep78.

[0024] When amino acids in the amino acid sequence of wild-type Rep78 are replaced with other amino acids, the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of wild-type Rep78 are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Rep78 with mutations that combine these amino acid substitutions and deletions is also included in Rep78. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added in the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep78. Rep78 with mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep78. The amino acid sequence of the mutated Rep78 preferably exhibits greater than 85% homology to the amino acid sequence of wild-type Rep78, more preferably greater than 90% homology, even more preferably greater than 95% homology, and even more preferably greater than 98% homology.

[0025] A functional equivalent of the AAV Rep protein means, in the case of Rep68, something that can be used functionally in place of Rep68, and in the case of Rep78, something that can be used functionally in place of Rep78.

[0026] The base sequence shown in SEQ ID NO: 1 can be modified by substitution, deletion, addition, etc., so long as it encodes functional Rep68 and Rep78. When a base in the base sequence shown in SEQ ID NO: 1 is substituted with another base, the number of bases to be substituted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When a base in the base sequence shown in SEQ ID NO: 1 is deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a base sequence obtained by adding a mutation that combines the substitution and deletion of these bases can also be used as a base sequence encoding a Rep protein. When adding a base, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 1 or to the 5'-end or 3'-end. A base sequence obtained by adding a mutation that combines the addition, substitution, and deletion of these bases can also be used as a nucleic acid molecule encoding a Rep protein. The mutated base sequence preferably exhibits 85% or more homology, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology to the base sequence shown in SEQ ID NO: 1. However, when these mutations are added, it is preferable that the start codons of the genes encoding the Rep proteins Rep68 and Rep78 are ACG.

[0027] In a preferred embodiment of the present invention, an example of a nucleic acid molecule encoding a Rep protein includes the base sequence shown in SEQ ID NO: 4. This nucleic acid molecule encodes Rep68, a Rep protein having the amino acid sequence shown in SEQ ID NO: 2, and Rep78, a Rep protein having the amino acid sequence shown in SEQ ID NO: 3. In this nucleic acid molecule, the initiation codon of the genes encoding the Rep proteins Rep68 and Rep78 is ACG. A nucleic acid molecule encoding a Rep protein is also one in which this base sequence has been modified by substitution, deletion, addition, or the like, so long as it encodes a Rep protein.

[0028] In one embodiment of the present invention, the base sequence encoding the Cap protein of an adeno-associated virus refers to a base sequence encoding at least VP1, which is one of the proteins constituting the capsid of AAV, or a base sequence including a mutated base sequence thereof. VP1 is preferably that of AAV serotype 9, but is not limited thereto, and may be any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 10, or 11. The base sequence encoding VP1 of wild-type AAV serotype 6 has the base sequence shown in SEQ ID NO:5. VP1 of wild-type AAV serotype 6 has the amino acid sequence shown in SEQ ID NO:6. The base sequence encoding VP1 of wild-type AAV serotype 9 has the base sequence shown in SEQ ID NO:7. VP1 of wild-type AAV serotype 9 has the amino acid sequence shown in SEQ ID NO:8.

[0029] In addition, as long as VP1 exerts its function, it may be modified by substitution, deletion, addition, or the like in the amino acid sequence of wild-type VP1 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In one embodiment of the present invention, VP1 with these mutations is also included in VP1.

[0030] In the case where an amino acid in the amino acid sequence of wild-type VP1, for example, the amino acid sequence of wild-type serotype 6 AAV VP1 shown in SEQ ID NO: 6 or the amino acid sequence of wild-type serotype 9 AAV VP1 shown in SEQ ID NO: 9, is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. In the case where an amino acid in the amino acid sequence of wild-type VP1 is deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. In addition, VP1 with a mutation that combines the substitution and deletion of these amino acids is also VP1. In the case where an amino acid is added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added in the amino acid sequence or at the N-terminus or C-terminus of wild-type VP1. VP1 with a mutation that combines the addition, substitution, and deletion of these amino acids is also included in VP1. The amino acid sequence of the mutated VP1 preferably exhibits greater than 85% homology to the amino acid sequence of wild-type VP1, more preferably greater than 90% homology, even more preferably greater than 95% homology, and even more preferably greater than 98% homology.

[0031] A functional equivalent of AAV VP1 refers to a substance that can be used functionally in place of VP1.

[0032] In a preferred embodiment of the present invention, a nucleic acid molecule encoding a Cap protein includes one containing the base sequence shown in SEQ ID NO: 5 or 7. These nucleic acid molecules are, respectively, a base sequence encoding VP1 of wild-type AAV serotype 6 and a base sequence encoding VP1 of wild-type AAV serotype 9. Nucleic acid molecules in which these base sequences have been modified by substitution, deletion, addition, or the like are also nucleic acid molecules encoding Cap proteins, so long as they encode functional VP1.

[0033] When bases in the base sequence shown in SEQ ID NO: 5 or 7 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 5 or 7 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a mutation that combines the substitution and deletion of these bases can also be used as a base sequence encoding a Cap protein. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 5 or 7 or to the 5'-end or 3'-end. A mutation that combines the addition, substitution, and deletion of these bases can also be used as a nucleic acid molecule encoding a Cap protein. The mutated base sequence preferably exhibits 85% or more homology to the base sequence shown in SEQ ID NO: 5 or 7, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0034] In one embodiment of the present invention, the term "inverted terminal repeat (ITR)" of the adeno-associated virus refers to a base sequence essential for the incorporation of an adeno-associated virus gene into the genome sequence of a host cell by non-homologous recombination. In one embodiment of the present invention, there are two adeno-associated virus inverted terminal repeats (ITR) in a nucleic acid molecule, which are called the first adeno-associated virus inverted terminal repeat (ITR) and the second adeno-associated virus inverted terminal repeat (ITR). Here, when a gene encoding a foreign protein is placed between the two ITRs, the ITR located on the 5' side is called the first adeno-associated virus inverted terminal repeat (ITR), and the ITR located on the 3' side is called the second adeno-associated virus inverted terminal repeat (ITR).

[0035] The inverted terminal repeats (ITRs) are preferably those of AAV of serotype 2, but are not limited thereto and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10 or 11. The inverted terminal repeats (ITRs) of AAV of serotype 2 include a first ITR containing the base sequence shown in SEQ ID NO: 9 and a second ITR containing the base sequence shown in SEQ ID NO: 10.

[0036] In addition, as long as the ITR functions, it may be modified by substitution, deletion, addition, or other such changes to the base sequence of the wild-type ITR of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. ITRs with such mutations are also included in the ITR.

[0037] When bases in the base sequence of the wild-type ITR are replaced with other bases, the number of bases to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When bases in the base sequence of the ITR are deleted, the number of bases to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. In addition, an ITR with a mutation that combines the substitution and deletion of these bases is also an ITR. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added in the base sequence of the wild-type ITR or to the 5'-end or 3'-end. An ITR with a mutation that combines the addition, substitution, and deletion of these bases is also included in the ITR. The base sequence of the mutated ITR preferably exhibits 85% or more homology with the base sequence of the wild-type ITR, more preferably exhibits 90% or more homology, even more preferably exhibits 95% or more homology, and even more preferably exhibits 98% or more homology.

[0038] A functional equivalent of an AAV ITR is one that can be used functionally in place of an AAV ITR. In addition, an artificially constructed ITR based on an AAV ITR is also a functional equivalent of an AAV ITR as long as it can replace an AAV ITR.

[0039] An example of such an artificially constructed first inverted terminal repeat (first ITR) is one having the base sequence shown in SEQ ID NO: 11. This base sequence shown in SEQ ID NO: 11 with substitution, deletion, or mutation is also included in the functional equivalent of the ITR of AAV, so long as it can be used functionally in place of the ITR of AAV. When a base in the base sequence shown in SEQ ID NO: 11 is replaced with another base, the number of bases to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When a base in the base sequence shown in SEQ ID NO: 11 is deleted, the number of bases to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. An ITR with a mutation that combines the substitution and deletion of these bases is also the first ITR. When a base is added to the base sequence shown in SEQ ID NO: 11, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added in the base sequence or to the 5'-end or 3'-end. The first ITR also includes an ITR having a mutation resulting from a combination of addition, substitution, and deletion of these bases. The base sequence of the mutated ITR preferably exhibits 85% or more homology with the base sequence shown in SEQ ID NO: 11, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0040] Moreover, such an artificially constructed second inverted terminal repeat (second ITR) may have the base sequence shown in SEQ ID NO: 12. The base sequence shown in SEQ ID NO: 12 to which substitution, deletion or mutation has been added is also included in the functional equivalent of the ITR of AAV, so long as it can be used functionally in place of the ITR of AAV. When a base in the base sequence shown in SEQ ID NO: 12 is replaced with another base, the number of bases to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When a base in the base sequence shown in SEQ ID NO: 12 is deleted, the number of bases to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. An ITR to which a mutation has been added by combining the substitution and deletion of these bases is also the second ITR. When a base is added to the base sequence shown in SEQ ID NO: 12, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added in the base sequence or to the 5'-end or 3'-end. The second ITR also includes an ITR having a mutation resulting from a combination of addition, substitution, and deletion of these bases. The base sequence of the mutated ITR preferably exhibits 85% or more homology with the base sequence shown in SEQ ID NO: 12, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0041] In one embodiment of the present invention, between the inverted terminal repeat (ITR) of a first adeno-associated virus and the inverted terminal repeat (ITR) of a second adeno-associated virus, there is a base sequence for inserting a base sequence encoding a foreign protein and / or a base sequence encoding a foreign protein.

[0042] In one embodiment of the present invention, a base sequence for inserting a base sequence encoding a foreign protein refers to a base sequence that includes a base sequence that can be specifically cleaved with a restriction enzyme. This also includes a so-called multicloning site. This base sequence can be cleaved with a restriction enzyme, and a nucleic acid molecule encoding a desired foreign protein can be inserted into the cleavage site.

[0043] In one embodiment of the present invention, there is no particular limitation on the foreign protein that can be encoded in the nucleic acid molecule. When the foreign protein is derived from a specific biological species, there is no particular limitation on the biological species, and it may be any of the proteins encoded in the genome of a prokaryotic cell or a eukaryotic cell. Here, eukaryotic cells are, for example, fungi, yeast, insects, protozoa, amphibians, reptiles, birds, mammals, and plants. When the biological species is a mammal, it is, for example, a human, a non-human primate, livestock such as cows, horses, pigs, and sheep, and pets such as cats and dogs. Furthermore, the foreign protein may be a protein obtained by adding a mutation such as a substitution, deletion, or addition to the amino acid sequence of a wild-type protein derived from a specific biological species. Furthermore, the foreign protein may be an artificial protein that includes an amino acid sequence that does not exist in nature.

[0044] When a foreign protein replaces an amino acid in the amino acid sequence of a wild-type protein with another amino acid, the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. When an amino acid in the amino acid sequence of a wild-type protein is deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Mutations combining the substitution and deletion of these amino acids can also be added. When amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added in the amino acid sequence or at the N-terminus or C-terminus of the wild-type protein. Mutations combining the addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated protein exhibits a homology of preferably 85% or more, more preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more with the amino acid sequence of the corresponding wild-type protein.

[0045] In one embodiment of the present invention, the foreign protein is not particularly limited, and examples thereof include proteins that are partially or completely functionally deficient in genetic diseases. Examples of such genetic diseases include lysosomal diseases, cystic fibrosis, and hemophilia. Other foreign proteins include growth hormone, somatomedin, insulin, glucagon, lysosomal enzymes, cytokines, lymphokines, blood coagulation factors, antibodies, fusion proteins of antibodies and other proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, DNasel, thyroid-stimulating hormone (TSH), nerve growth factor (NGF), ciliary neurotrophic factor (C-CSF), and the like. Examples include neurotrophin 3 (NTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), enzymes with beta-amyloid degrading activity, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and GLP-1 receptor agonists.

[0046] Examples of foreign proteins include mouse antibodies, humanized antibodies, human-mouse chimeric antibodies, and human antibodies. Examples of foreign proteins include anti-IL-6 antibodies, anti-beta amyloid antibodies, anti-BACE antibodies, anti-EGFR antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-HER2 antibodies, anti-PCSK9 antibodies, and anti-TNF-α antibodies.

[0047] In the case where the foreign protein is a lysosomal enzyme, the gene for the foreign protein may be selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. Examples include A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

[0048] The foreign protein may be a fusion protein of an antibody and another protein. In such a fusion protein, the antibody is any one of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody, and the other protein is a growth hormone, a lysosomal enzyme, a cytokine, a lymphokine, a blood coagulation factor, an antibody, a fusion protein of an antibody and another protein, a granulocyte macrophage colony stimulating factor (GM-CSF), a granulocyte colony stimulating factor (G-CSF), a macrophage colony stimulating factor (M-CSF), an erythropoietin, a darbepoietin, a tissue plasminogen activator (t-PA), a thrombomodulin, a follicle stimulating hormone, a DNase, a thyroid stimulating hormone (TSH), a neurotrophin, a thyroid hormone ... Examples include growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), and enzymes that have the activity of degrading beta-amyloid.

[0049] The foreign protein may be a fusion protein of an antibody and a lysosomal enzyme. In such a fusion protein, the antibody is any one of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody, and the lysosomal enzyme is any one of α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. Examples of the enzyme include A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2.

[0050] When the foreign protein is a fusion protein of an antibody and another protein or an antibody and a lysosomal enzyme, the antibody is, for example, an antibody having a specific affinity for a protein present on the surface of a vascular endothelial cell. Examples of such proteins include transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor. Furthermore, examples of organic anion transporters include OATP-F, and examples of monocarboxylate transporters include MCT-8.

[0051] A region containing a base sequence including the inverted terminal repeat (ITR) of a first adeno-associated virus or a functional equivalent thereof, a base sequence including the inverted terminal repeat (ITR) of a second adeno-associated virus or a functional equivalent thereof, and a base sequence for inserting a base sequence encoding a foreign protein and / or a base sequence encoding a foreign protein, located between the first ITR and the second ITR, is called a transfer region.

[0052] Adenovirus provides the functions necessary for the replication and packaging of the AAV genome into capsids to form viral virions in host cells, which are exerted by the E1, E2A, E4, and VA1 RNA regions of the adenovirus genome.

[0053] In one embodiment of the present invention, a recombinant AAV virion (rAAV virion) refers to a nucleic acid molecule in which a wild-type AAV genome has been modified, such as a rAAV genome, packaged in an AAV capsid protein (including its functional equivalent). Here, a rAAV genome (recombinant AAV genome) refers to a nucleic acid molecule in which a wild-type AAV genome has been modified. The rAAV genome packaged in the rAAV virion is single-stranded DNA. Such a nucleic acid molecule includes single-stranded DNA that includes, from the 5' end, a base sequence including an inverted terminal repeat (ITR) of a first adeno-associated virus or a functional equivalent thereof, a region including a base sequence encoding a foreign protein, and an inverted terminal repeat (ITR) of a second adeno-associated virus. However, the nucleic acid molecule that is not packaged in the rAAV virion can also be referred to as a rAAV genome. Therefore, the rAAV genome may be single-stranded DNA or double-stranded DNA.

[0054] In one embodiment of the present invention, the amount of rAAV genome means the number of rAAV genomes, and its unit is vg.

[0055] In one embodiment of the present invention, an empty capsid refers to a particle composed of capsid protein in which the rAAV genome is not packaged. In addition, total capsid is a general term for empty capsids and rAAV virions, and the total capsid amount refers to the total number of empty capsids and rAAV virions.

[0056] In addition, a recombinant AAV virion whose capsid protein is derived from AAV of serotype 6 can be called a recombinant AAV6 virion (rAAV6 virion), and whose capsid protein is derived from AAV of serotype 9 can be called a recombinant AAV9 virion (rAAV9 virion). The same applies to AAV of other serotypes.

[0057] Further, such nucleic acid molecules include a base sequence including, from the 5' end, the inverted terminal repeat (ITR) of a first adeno-associated virus or a functional equivalent thereof, a base sequence including a gene expression control site that controls the expression of the foreign protein, a region including a base sequence encoding the foreign protein, and a single-stranded DNA including the inverted terminal repeat (ITR) of a first adeno-associated virus.

[0058] Further, such a nucleic acid molecule includes a single-stranded DNA including, from the 5' end, a base sequence including the inverted terminal repeat (ITR) of the first adeno-associated virus or a functional equivalent thereof, a base sequence including a gene expression control site that controls the expression of the foreign protein, an intron sequence, a region including a base sequence that codes for the foreign protein, a polyA sequence, and the inverted terminal repeat (ITR) of the first adeno-associated virus. Here, there is no particular limitation on the base sequence that can be used as the intron sequence, but a synthetic human β-globin intron obtained by introducing a mutation into the human β-globin gene, for example, an intron including the base sequence shown in SEQ ID NO: 25, can be preferably used. In addition, there is no particular limitation on the base sequence that can be used as the polyadenylation signal sequence (polyA sequence), but there is a base sequence of a synthetic polyadenylation signal sequence (synthetic polyA sequence) including the base sequence shown in SEQ ID NO: 26.

[0059] The function of the E1 region of adenovirus is required for the formation of recombinant AAV virions in host cells. Generally, host cells having all or part of the E1 region are used to produce recombinant AAV virions. HEK293 cells are known as such cells. The genome of HEK293 cells contains at least the coding regions of E1A and E1B.

[0060] When using a host cell having all or a part of the E1 region, the regions of the adenovirus genome necessary for the AAV genome to be replicated and packaged into a capsid to form a viral virion are the E2A region, the E4 region, and the VA1 RNA region. These regions code for proteins and RNAs required for AAV replication. Regarding the functions provided by the E4 region, the E4 34 kD protein encoded by the open reading frame 6 (ORF6) of the E4 region is required for AAV replication.

[0061] In one embodiment of the present invention, the E2A region may be from any adenovirus of serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3, so long as it exhibits the original functions required for AAV replication. One of the preferred regions in the present invention is the E2A region of adenovirus of serotype 2. The E2A region of adenovirus of serotype 2 contains the base sequence shown in SEQ ID NO:13.

[0062] Furthermore, as long as the E2A region exerts its inherent function, it is also possible to use the E2A region of a wild-type adenovirus that has been modified by substitution, deletion, addition, etc. In one embodiment of the present invention, the E2A region with these mutations is also included in the E2A region.

[0063] When bases in the base sequence of the wild-type E2A region are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the E2A region are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, an E2A region in which a mutation has been added that combines the substitution and deletion of these bases is also included in the E2A region. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the base sequence or the 5'-end or 3'-end of the wild-type E2A region. An E2A region in which a mutation has been added that combines the addition, substitution, and deletion of these bases is also included in the E2A region. The base sequence of the mutated E2A region preferably exhibits 85% or more homology to the wild-type E2A base sequence, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0064] In one embodiment of the present invention, the E4 region may be that of any of the adenoviruses whose serotypes are 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3, as long as it exerts the original function required for AAV replication. The E4 region of an adenovirus of serotype 2 is one of the preferred ones in the present invention. The E4 region of serotype 2 adenovirus contains the nucleotide sequence shown in SEQ ID NO:14.

[0065] Furthermore, as long as the E4 region exerts its inherent function, it is also possible to use the E4 region of a wild-type adenovirus that has been modified by substitution, deletion, addition, etc. In one embodiment of the present invention, the E4 region includes the E4 region to which such mutations have been added.

[0066] When bases in the base sequence of the wild-type E4 region are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the E4 region are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. In addition, an E4 region to which a mutation has been added by combining the substitution and deletion of these bases is also included in the E4 region. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added in the base sequence of the wild-type E4 or to the 5'-end or 3'-end. E4 to which a mutation has been added by combining the addition, substitution, and deletion of these bases is also included in E4. The base sequence of the mutated E4 preferably shows 85% or more homology with the base sequence of the wild-type E4, more preferably shows 90% or more homology, even more preferably shows 95% or more homology, and even more preferably shows 98% or more homology.

[0067] A suitable example of the E4 region is one that contains the base sequence shown in SEQ ID NO: 15. Modifications such as substitutions, deletions, and additions to the base sequence shown in SEQ ID NO: 15 can also be used as the E4 region, so long as the region exerts its inherent function.

[0068] When bases in the base sequence shown in SEQ ID NO: 15 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the E4 region are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations combining the substitution and deletion of these bases can also be added. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 15 or to the 5'-end or 3'-end. Mutations combining the addition, substitution, and deletion of these bases can also be added. The base sequence with the mutation preferably exhibits 85% or more homology with the base sequence shown in SEQ ID NO: 15, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology. The one with these mutations is also the E4 region.

[0069] In one embodiment of the present invention, the VA1 RNA region may be that of any adenovirus of serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad3, canine Ad2, ovine Ad, or porcine Ad3, so long as it exhibits the original functions required for AAV replication. One of the preferred regions in the present invention is the VA1 RNA region of adenovirus of serotype 2. The VA1 RNA region of adenovirus of serotype 2 contains the base sequence shown in SEQ ID NO:16.

[0070] Furthermore, as long as the VA1 RNA region exerts its inherent function, it is also possible to use the VA1 RNA region of a wild-type adenovirus that has been modified by substitution, deletion, addition, etc. In one embodiment of the present invention, the VA1 RNA region to which these mutations have been added is also included in the VA1 RNA region.

[0071] When bases in the base sequence of the wild-type VA1 RNA region are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the VA1 RNA region are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The VA1 RNA region includes a VA1 RNA region that has been mutated by combining the substitution and deletion of these bases. When bases are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the base sequence of the wild-type VA1 RNA region or to the 5'-end or 3'-end. The VA1 RNA region includes a VA1 RNA region that has been mutated by combining the addition, substitution, and deletion of these bases. The base sequence of the mutated VA1 RNA region preferably exhibits greater than 85% homology to the base sequence of the wild-type VA1 RNA region, more preferably greater than 90% homology, even more preferably greater than 95% homology, and even more preferably greater than 98% homology.

[0072] In a preferred embodiment of the present invention, the E2A region, the E4 region, and the VA1 RNA region may be located in any order. A base sequence including the E2A region, the E4 region, and the VA1 RNA region is called a helper region.

[0073] A preferred embodiment of the helper region includes a region including the base sequence shown in SEQ ID NO: 17, in which the E4 region is located downstream of the E2A region and the VA1 RNA region is located further downstream. A region in which this base sequence has been modified by substitution, deletion, addition, or the like can also be used as a helper region, so long as each of the E2A region, E4 region, and VA1 RNA region exhibits its original function.

[0074] When bases in the base sequence of the helper region containing the base sequence shown in SEQ ID NO: 17 are replaced with other bases, the number of bases to be replaced is preferably 1 to 60, more preferably 1 to 30, and even more preferably 1 to 10. When bases in the base sequence of the helper region containing the base sequence shown in SEQ ID NO: 17 are deleted, the number of bases to be deleted is preferably 1 to 60, more preferably 1 to 30, and even more preferably 1 to 10. In addition, a region in which a mutation is added by combining the substitution and deletion of these bases can also be used as the helper region. When bases are added, preferably 1 to 60, more preferably 1 to 30, and even more preferably 1 to 10 bases are added to the base sequence of the helper region containing the base sequence shown in SEQ ID NO: 17 or to the 5'-end or 3'-end. A region in which a mutation is added by combining the addition, substitution, and deletion of these bases can also be used as the helper region. The base sequence of the mutated helper region preferably exhibits 85% or more homology to the base sequence of the helper region containing the base sequence shown in SEQ ID NO:17, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0075] The above rules apply to modifications in the E2A region, E4 region, and VA1 RNA region contained in the helper region.

[0076] In one embodiment of the present invention, the base sequence that can be used as the base sequence containing the first gene expression control site that controls the expression of the Rep protein is not particularly limited as long as it is controlled by the protein and RNA encoded in the region containing the E2A region, the E4 region, and the VA1 RNA region, but is preferably the p5 promoter of AAV. When the first gene expression control site is the p5 promoter of AAV, it is preferably the p5 promoter of AAV of serotype 2, but is not limited thereto, and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11. The p5 promoter of AAV of serotype 2 contains the base sequence shown in SEQ ID NO: 18.

[0077] In addition, as long as the AAV p5 promoter exerts its function, it may be modified by substitution, deletion, addition, or the like in the base sequence of the wild-type p5 promoter of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In one embodiment of the present invention, p5 promoters with these mutations are also included in the p5 promoter.

[0078] When bases in the base sequence of the wild-type p5 promoter shown in SEQ ID NO: 18 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 18 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. A p5 promoter having a mutation that combines the substitution and deletion of these bases is also a p5 promoter. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 18 or to the 5'-end or 3'-end. A p5 promoter having a mutation that combines the addition, substitution, and deletion of these bases is also included in the p5 promoter. The base sequence of the mutated p5 promoter preferably exhibits 85% or more homology to the base sequence of the wild-type p5 promoter, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0079] An example of a p5 promoter modified from the p5 promoter of AAV, serotype 2, is one containing the base sequence shown in SEQ ID NO: 19. Modifications to the base sequence of this p5 promoter, such as substitutions, deletions, and additions, can also be considered as p5 promoters or their functional equivalents, so long as they exhibit the original functions of the p5 promoter.

[0080] When bases in the base sequence of the p5 promoter containing the base sequence shown in SEQ ID NO: 19 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 19 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a combination of mutations of these base substitutions and deletions can also be used as the p5 promoter or its functional equivalent. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 19 or to the 5'-end or 3'-end. A combination of mutations of these base additions, substitutions, and deletions can also be used as the p5 promoter or its functional equivalent. The base sequence of the mutated p5 promoter preferably exhibits 85% or more homology to the base sequence shown in SEQ ID NO:19, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0081] The p5 promoter or a functional equivalent thereof is usually located upstream of a base sequence encoding the Rep protein or a functional equivalent thereof of the adeno-associated virus.

[0082] A region containing a p5 promoter or a functional equivalent thereof and a base sequence encoding the Rep protein or a functional equivalent thereof of an adeno-associated virus is called a Rep region. A preferred embodiment of the Rep region includes a base sequence shown in SEQ ID NO: 20. This base sequence shown in SEQ ID NO: 20 includes the base sequence shown in SEQ ID NO: 4 downstream of the base sequence shown in SEQ ID NO: 19. That is, this base sequence shown in SEQ ID NO: 20 encodes Rep68 of serotype 2 AAV having the amino acid sequence shown in SEQ ID NO: 2 and Rep78 of serotype 2 AAV having the amino acid sequence shown in SEQ ID NO: 3.

[0083] The base sequence shown in SEQ ID NO: 20 may also be modified by substitution, deletion, addition or other such modifications as long as it exhibits the original function of the Rep region.

[0084] When bases in the base sequence of the Rep region including the base sequence shown in SEQ ID NO: 20 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 20 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a region obtained by adding a mutation that combines the substitution and deletion of these bases can also be used as the Rep region. When adding bases, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 20 or to the 5'-end or 3'-end. A region obtained by adding a mutation that combines the addition, substitution, and deletion of these bases can also be used as the Rep region. The base sequence of the mutated Rep region preferably exhibits greater than 85% homology to the base sequence shown in SEQ ID NO:20, more preferably greater than 90% homology, even more preferably greater than 95% homology, and even more preferably greater than 98% homology.

[0085] In one embodiment of the present invention, the base sequence that can be used as the base sequence containing the second gene expression control site that controls the expression of the Cap protein is not particularly limited as long as it is controlled by the protein and RNA encoded in the region containing the E2A region, the E4 region, and the VA1 RNA region, but is preferably the p40 promoter of AAV. In the case of the p40 promoter of AAV, it is preferable that it is the p40 promoter of AAV of serotype 2, but is not limited thereto, and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0086] In addition, the AAV p40 promoter may be modified by substitution, deletion, addition, or the like in the base sequence of the wild-type p40 promoter of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, so long as it exerts its function. In one embodiment of the present invention, the p40 promoter with these mutations is also included in the p40 promoter. When bases in the base sequence of the wild-type p40 promoter are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the wild-type p40 promoter are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. In addition, the p40 promoter with mutations that combine the substitution and deletion of these bases is also a p40 promoter. When bases are added, preferably 1 to 20 bases, more preferably 1 to 10 bases, and even more preferably 1 to 3 bases are added in the base sequence or to the 5'-end or 3'-end of the wild-type p40 promoter. Mutations of p40 promoters that combine addition, substitution, and deletion of these bases are also included in the p40 promoter. The base sequence of the mutated p40 promoter preferably shows 85% or more homology with the base sequence of the wild-type p40 promoter, more preferably shows 90% or more homology, even more preferably shows 95% or more homology, and even more preferably shows 98% or more homology.

[0087] The p40 promoter or a functional equivalent thereof is usually located upstream of a base sequence encoding the Cap protein or a functional equivalent thereof of the adeno-associated virus.

[0088] A region containing the p40 promoter or its functional equivalent and a base sequence encoding the Cap protein of the adeno-associated virus or its functional equivalent is called the Cap region.

[0089] A preferred embodiment of the Cap region includes one containing the nucleotide sequence shown in SEQ ID NO: 21. This Cap region contains a nucleotide sequence encoding VP1 of AAV of serotype 9 having the nucleotide sequence shown in SEQ ID NO: 7, and encodes VP1 of AAV of serotype 9 having the amino acid sequence shown in SEQ ID NO: 8. This Cap region can also contain a nucleotide sequence encoding VP1 of AAV of serotype 6 having the nucleotide sequence shown in SEQ ID NO: 5, instead of the nucleotide sequence encoding VP1 of AAV of serotype 9 having the nucleotide sequence shown in SEQ ID NO: 7. Modifications such as substitutions, deletions, and additions to these nucleotide sequences can also be used as Cap regions, so long as they exhibit the original function of the Cap region.

[0090] When the bases of the base sequence shown in SEQ ID NO: 21 in the base sequence of the Cap region are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When the bases in the base sequence shown in SEQ ID NO: 21 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a region in which a mutation is added by combining the substitution and deletion of these bases can also be used as the Cap region. When a base is added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 21 or to the 5'-end or 3'-end. A region in which a mutation is added by combining the addition, substitution, and deletion of these bases can also be used as the Cap region. The base sequence of the mutated Cap region preferably exhibits 85% or more homology with the base sequence shown in SEQ ID NO: 21, more preferably exhibits 90% or more homology, even more preferably exhibits 95% or more homology, and even more preferably exhibits 98% or more homology. The same applies to the case where the base sequence shown in SEQ ID NO:7 is replaced with the base sequence shown in SEQ ID NO:5.

[0091] In a preferred embodiment of the present invention, the Rep region may be located upstream or downstream of the Cap region. A region including the Rep region and the Cap region is called a Rep-Cap region.

[0092] A preferred embodiment of the Rep-Cap region includes a Rep region located upstream of the Cap region and containing the base sequence shown in SEQ ID NO: 22. This Rep-Cap region contains the base sequence shown in SEQ ID NO: 20 downstream of the base sequence shown in SEQ ID NO: 19, and the base sequence shown in SEQ ID NO: 21 further downstream. This Rep-Cap region contains a base sequence encoding VP1 of AAV of serotype 9 having the base sequence shown in SEQ ID NO: 7, and encodes VP1 of AAV of serotype 9 having the amino acid sequence shown in SEQ ID NO: 8. This Rep-Cap region can also contain a base sequence encoding VP1 of AAV of serotype 6 having the base sequence shown in SEQ ID NO: 5, instead of the base sequence encoding VP1 of AAV of serotype 9 having the base sequence shown in SEQ ID NO: 7. Modifications such as substitutions, deletions, and additions to these base sequences can also be used as the Rep-Cap region, so long as they exhibit the original function of the Rep-Cap region.

[0093] When bases in the base sequence of the Rep-Cap region including the base sequence shown in SEQ ID NO: 22 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 22 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a region obtained by adding a mutation that combines the substitution and deletion of these bases can also be used as the Rep-Cap region. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 22 or to the 5'-end or 3'-end. A region obtained by adding a mutation that combines the addition, substitution, and deletion of these bases can also be used as the Rep-Cap region. The base sequence of the mutated Rep-Cap region preferably exhibits 85% or more homology to the base sequence shown in SEQ ID NO:22, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0094] An enhancer sequence having the function of enhancing the expression level of the Rep protein and / or Cap protein can also be placed downstream of the Rep-Cap region. Here, there is no particular limitation on such enhancer sequence, but the AAV p5 promoter or a functional equivalent thereof can be used as the enhancer. There is no particular limitation on the AAV serotype of the p5 promoter used here, and it may be any of the AAV p5 promoters of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, but the AAV p5 promoter of serotype 2 is preferred. The AAV p5 promoter of serotype 2 contains the base sequence shown in SEQ ID NO:18.

[0095] In addition, the P5 promoter of AAV located downstream of the Rep-Cap region may be one that has been modified by substitution, deletion, addition, or the like in the base sequence of the wild-type p5 promoter of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. p5 promoters with these mutations are also included in the P5 promoter.

[0096] Here, when bases in the base sequence of the wild-type P5 promoter are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence of the wild-type p5 promoter are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. A p5 promoter that has been mutated by combining the substitution and deletion of these bases is also a P5 promoter. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence or the 5'-end or 3'-end of the wild-type P5 promoter. A P5 promoter that has been mutated by combining the addition, substitution, and deletion of these bases is also included in the P5 promoter. The base sequence of the mutated p5 promoter preferably exhibits 85% or more homology to the base sequence of the wild-type P5 promoter, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0097] An example of a p5 promoter obtained by modifying the p5 promoter of AAV serotype 2 is one containing the base sequence shown in SEQ ID NO: 19. The p5 promoter also includes those in which the base sequence of this p5 promoter has been modified by substitution, deletion, addition, or the like.

[0098] When bases in the base sequence of the p5 promoter containing the base sequence shown in SEQ ID NO: 19 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 19 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a combination of mutations of these base substitutions and deletions can also be used as the p5 promoter or its functional equivalent. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 19 or to the 5'-end or 3'-end. A combination of mutations of these base additions, substitutions, and deletions can also be used as the p5 promoter or its functional equivalent. The base sequence of the mutated p5 promoter preferably exhibits 85% or more homology to the base sequence shown in SEQ ID NO:19, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0099] When the base sequence of the AAV p5 promoter or its functional equivalent is located downstream of the Rep-Cap region, the region including the base sequence of the AAV p5 promoter or its functional equivalent can also be referred to as the Rep-Cap region. A preferred embodiment of such a Rep-Cap region includes a base sequence shown in SEQ ID NO: 23. This Rep-Cap region includes the base sequence shown in SEQ ID NO: 20 downstream of the base sequence shown in SEQ ID NO: 19, the base sequence shown in SEQ ID NO: 21 further downstream, and the base sequence shown in SEQ ID NO: 19 further downstream. Modifications such as substitutions, deletions, and additions to this base sequence can also be considered as Rep-Cap regions as long as they exhibit the original function of the Rep-Cap region.

[0100] When bases in the base sequence of the Rep-Cap region including the base sequence shown in SEQ ID NO: 23 are replaced with other bases, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. When bases in the base sequence shown in SEQ ID NO: 23 are deleted, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Furthermore, a region obtained by adding a mutation that combines the substitution and deletion of these bases can also be used as the Rep-Cap region. When bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 23 or to the 5'-end or 3'-end. A region obtained by adding a mutation that combines the addition, substitution, and deletion of these bases can also be used as the Rep-Cap region. The base sequence of the mutated Rep-Cap region preferably exhibits 85% or more homology to the base sequence shown in SEQ ID NO:23, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology.

[0101] In one embodiment of the present invention, the base sequence that can be used as the base sequence containing the third gene expression control site that controls the expression of a foreign protein is not particularly limited as long as it can express the foreign protein in a cell, tissue, or living body into which a gene encoding the foreign protein is introduced. However, those containing a promoter derived from a cytomegalovirus (optionally containing an enhancer), an SV40 early promoter, a human elongation factor-1α (EF-1α) promoter, a human ubiquitin C promoter, a retrovirus Rous sarcoma virus LTR promoter, a dihydrofolate reductase promoter, a β-actin promoter, and a phosphoglycerate kinase (PGK) promoter are preferred. As the β-actin promoter, a chicken β-actin promoter having the base sequence shown in SEQ ID NO: 38 is preferred. In addition, a promoter having the base sequence shown in SEQ ID NO: 47 in which a chicken β-actin promoter is linked downstream of a human CMV enhancer can be preferably used as the third gene expression control site.

[0102] In order to increase the expression level of a foreign protein, an intron sequence may be present between the base sequence containing the third gene expression control site and the base sequence encoding the foreign protein, or downstream of the base sequence encoding the foreign protein. For example, a chicken β-actin promoter downstream of a human CMV enhancer, and a chicken β-actin / MVM chimeric intron having the base sequence shown in SEQ ID NO: 39 downstream of that promoter, can be suitably used as the third gene expression control site. Here, MVM is an abbreviation for Minute virus of mice.

[0103] A polyadenylation sequence (polyA sequence) is placed downstream of the foreign protein. There are no particular limitations on the base sequence that can be used as the polyA sequence, so long as it can exert its original function in the cell, tissue, or living body into which the gene encoding the foreign protein is introduced. The human growth hormone polyA sequence and the bovine growth hormone polyA sequence having the base sequence shown in SEQ ID NO: 40 can be suitably used.

[0104] The nucleic acid molecule of one embodiment of the present invention can be a circular plasmid.

[0105] In one embodiment of the present invention, when the nucleic acid molecule is a circular plasmid, the Rep-Cap region, the transfer region, and the helper region may be located in any order.

[0106] In a preferred embodiment of the present invention, the transfer region is located downstream of the Rep-Cap region, and the helper region is located downstream of that. For example, the Cap region is located downstream of the Rep region, the transfer region is located downstream of that, the E2A region is located downstream of that, the E4 region is located downstream of that, and the VA1 RNA region is located downstream of that. The same applies when the nucleic acid molecule is a circular plasmid.

[0107] In a further embodiment of the present invention, the base sequence encoding the Rep protein or a functional equivalent thereof of the adeno-associated virus, the base sequence encoding the Cap protein or a functional equivalent thereof of the adeno-associated virus, the base sequence including the inverted terminal repeat (ITR) or a functional equivalent thereof of the first adeno-associated virus, the base sequence for inserting a base sequence encoding a foreign protein or / and the base sequence encoding a foreign protein, the base sequence including the inverted terminal repeat (ITR) or a functional equivalent thereof of the second adeno-associated virus, the base sequence encoding the E2A region or a functional equivalent thereof of the adenovirus, the base sequence encoding the E4 region or a functional equivalent thereof of the adenovirus, and the base sequence encoding the VA1 RNA region or a functional equivalent thereof of the adenovirus may be located in any order. The same applies when the nucleic acid molecule is a circular plasmid. A drug resistance gene may also be incorporated into any position of this nucleic acid molecule. Also, a replication origin necessary for the plasmid to replicate in a host cell may also be incorporated into any position of this nucleic acid molecule. In the case of a host cell, Coli E1 can be preferably used as the origin of replication. There is usually a polyA sequence downstream of the base sequence encoding the foreign protein. The same applies when the nucleic acid molecule is a circular plasmid.

[0108] However, the base sequence encoding the Rep protein or a functional equivalent thereof of the adeno-associated virus is preferably located on the 5' side of the base sequence encoding the Cap protein or a functional equivalent thereof of the adeno-associated virus. The base sequence of the E2A region of the adenovirus or a functional equivalent thereof is preferably located on the 5' side of the base sequence of the E4 region of the adenovirus or a functional equivalent thereof. Furthermore, the base sequence of the E4 region of the adenovirus or a functional equivalent thereof is preferably located on the 5' side of the base sequence of the VA1 RNA region of the adenovirus or a functional equivalent thereof. Furthermore, it is preferable that the base sequence of the E4 region of the adenovirus or a functional equivalent thereof is located on the 3' side of the base sequence of the E2A region of the adenovirus or a functional equivalent thereof, and further downstream of that, the base sequence of the VA1 RNA region of the adenovirus or a functional equivalent thereof is located. The same applies when the nucleic acid molecule is a circular plasmid. A drug resistance gene can also be incorporated into any position of this nucleic acid molecule. Furthermore, an origin of replication necessary for the plasmid to replicate in a host cell can also be incorporated at any position in this nucleic acid molecule. In the case of a host cell, Coli E1 is preferably used as the origin of replication. There is usually a polyA sequence downstream of the base sequence encoding the foreign protein. The same applies when the nucleic acid molecule is a circular plasmid.

[0109] As a further preferred embodiment of the present invention, a nucleic acid molecule in which a base sequence encoding a Cap protein or a functional equivalent thereof of an adeno-associated virus is located downstream of a base sequence encoding a Rep protein or a functional equivalent thereof of an adeno-associated virus, a base sequence including an inverted terminal repeat (ITR) of a first adeno-associated virus or a functional equivalent thereof further downstream, a base sequence for inserting a base sequence encoding a foreign protein further downstream or / and a base sequence encoding a foreign protein further downstream, a base sequence including an inverted terminal repeat (ITR) of a second adeno-associated virus or a functional equivalent thereof further downstream, a base sequence encoding an E2A region of an adenovirus or a functional equivalent thereof further downstream, a base sequence encoding an E4 region of an adenovirus or a functional equivalent thereof further downstream, and a base sequence encoding a VA1 RNA region of an adenovirus or a functional equivalent thereof further downstream are located. The same applies when the nucleic acid molecule is a circular plasmid. A drug resistance gene can also be incorporated into any position of this nucleic acid molecule. Furthermore, an origin of replication necessary for the plasmid to replicate in a host cell can also be incorporated at any position in this nucleic acid molecule. In the case of a host cell, Coli E1 is preferably used as the origin of replication. There is usually a polyA sequence downstream of the base sequence encoding the foreign protein. The same applies when the nucleic acid molecule is a circular plasmid.

[0110] As a further preferred embodiment of the present invention, downstream of a base sequence including a first gene expression control site that controls the expression of the Rep protein of the adeno-associated virus, a base sequence encoding a Rep protein or a functional equivalent thereof, downstream of that a base sequence including a second gene expression control site that controls the expression of the Cap protein of the adeno-associated virus, downstream of that a base sequence encoding the Cap protein of the adeno-associated virus or a functional equivalent thereof, further downstream a base sequence including an inverted terminal repeat (ITR) of a first adeno-associated virus or a functional equivalent thereof, further downstream a base sequence for inserting a base sequence encoding a foreign protein or / and a base sequence encoding a foreign protein, further downstream a base sequence including an inverted terminal repeat (ITR) of a second adeno-associated virus or a functional equivalent thereof, further downstream a base sequence encoding an E2A region of the adenovirus or a functional equivalent thereof, further downstream a base sequence encoding an E4 region of the adenovirus or a functional equivalent thereof, further downstream a base sequence encoding the VA1 region of the adenovirus, Examples of such nucleic acid molecules include those in which a base sequence encoding an RNA region or a functional equivalent thereof is located. They may further include a replication origin, and may additionally incorporate a drug resistance gene. There is usually a polyA sequence downstream of the base sequence encoding a foreign protein. The same applies when the nucleic acid molecule is a circular plasmid.

[0111] As a further preferred embodiment of the present invention, downstream of a base sequence including a first gene expression control site that controls the expression of the Rep protein of the adeno-associated virus, a base sequence encoding a Rep protein or a functional equivalent thereof, downstream of that a base sequence including a second gene expression control site that controls the expression of the Cap protein of the adeno-associated virus, downstream of that a base sequence encoding the Cap protein of the adeno-associated virus or a functional equivalent thereof, further downstream a base sequence of an AAV p5 promoter or its equivalent, further downstream a base sequence including an inverted terminal repeat (ITR) of a first adeno-associated virus or its functional equivalent, further downstream a base sequence for inserting a base sequence encoding a foreign protein or / and a base sequence encoding a foreign protein, further downstream a base sequence including an inverted terminal repeat (ITR) of a second adeno-associated virus or its functional equivalent, further downstream a base sequence encoding an E2A region of the adenovirus or its functional equivalent, further downstream a base sequence encoding an E4 region of the adenovirus or its functional equivalent, further downstream a base sequence encoding the VA1 region of the adenovirus, Examples of such nucleic acid molecules include those in which a base sequence encoding an RNA region or a functional equivalent thereof is located. The same applies when the nucleic acid molecule is a circular plasmid. A drug resistance gene can also be incorporated into any position of this nucleic acid molecule. Furthermore, an origin of replication necessary for the plasmid to replicate in a host cell can also be incorporated into any position of this nucleic acid molecule. When the host cell is used, Coli E1 can be preferably used as the origin of replication. There is usually a polyA sequence downstream of the base sequence encoding the foreign protein. The same applies when the nucleic acid molecule is a circular plasmid.

[0112] As a further preferred embodiment of the present invention, downstream of a base sequence including a first gene expression control site that controls the expression of the Rep protein of the adeno-associated virus, a base sequence encoding the Rep protein or a functional equivalent thereof, downstream of which is a base sequence including a second gene expression control site that controls the expression of the Cap protein of the adeno-associated virus, downstream of which is a base sequence encoding the Cap protein of the adeno-associated virus or a functional equivalent thereof, further downstream of which is a base sequence of the AAV p5 promoter or an equivalent thereof, further downstream of which is a replication origin, further downstream of which is a drug resistance gene, and further downstream of which is a Examples of such nucleic acid molecules include a nucleic acid molecule in which a base sequence containing a first adeno-associated virus inverted terminal repeat (ITR) or a functional equivalent thereof is located downstream, a base sequence for inserting a base sequence encoding a foreign protein further downstream and / or a base sequence encoding a foreign protein is located further downstream, a base sequence containing a second adeno-associated virus inverted terminal repeat (ITR) or a functional equivalent thereof is located further downstream, a base sequence encoding an adenovirus E2A region or a functional equivalent thereof is located further downstream, a base sequence encoding an adenovirus E4 region or a functional equivalent thereof is located further downstream, and a base sequence encoding an adenovirus VA1 RNA region or a functional equivalent thereof is located further downstream. The same applies when the nucleic acid molecule is a circular plasmid.

[0113] As a further preferred embodiment of the present invention, there is mentioned a nucleic acid molecule in which a Rep region containing a base sequence encoding the Rep protein of AAV of serotype 2, a Cap region containing a base sequence encoding the Cap protein of AAV of serotype 6 or 9 downstream thereof, a p5 promoter as an enhancer further downstream, a first ITR further downstream, a promoter derived from cytomegalovirus further downstream, a synthetic human β-globin intron further downstream, a base sequence for inserting a base sequence encoding a foreign protein further downstream / and a base sequence encoding a foreign protein further downstream, a synthetic polyA sequence further downstream, a second ITR further downstream, an adenovirus E2A region further downstream, an adenovirus E4 region further downstream, and an adenovirus VA1 RNA region further downstream are located. It may further include an origin of replication, in which case the origin of replication is preferably located downstream of the p5 promoter. A drug resistance gene may be incorporated concomitantly, in which case the drug resistance gene is preferably located upstream of the first ITR. The same applies when the nucleic acid molecule is a circular plasmid.

[0114] As a further preferred embodiment of the present invention, there is mentioned a nucleic acid molecule in which a Rep region containing a base sequence encoding the Rep protein of AAV of serotype 2, a Cap region containing a base sequence encoding the Cap protein of AAV of serotype 6 or 9 downstream thereof, a p5 promoter as an enhancer further downstream, a first ITR further downstream, a human CMV enhancer further downstream, a chicken β-actin promoter further downstream, a chicken β-actin / MVM chimeric intron further downstream, a base sequence for inserting a base sequence encoding a foreign protein further downstream or / and a base sequence encoding a foreign protein further downstream, a bovine or human growth hormone polyA sequence further downstream, a second ITR further downstream, an adenovirus E2A region further downstream, an adenovirus E4 region further downstream, and an adenovirus VA1 RNA region further downstream are located. It may further include an origin of replication, in which case the origin of replication is preferably located downstream of the p5 promoter. A drug resistance gene may be incorporated concomitantly, in which case the drug resistance gene is preferably located upstream of the first ITR. The same is true when the nucleic acid molecule is a circular plasmid.

[0115] A method for producing a recombinant adeno-associated virus virion using a nucleic acid molecule according to one embodiment of the present invention will be described below using a nucleic acid molecule comprising the base sequence shown in SEQ ID NO:24 as an example.

[0116] The nucleic acid molecule shown in SEQ ID NO:24 is a nucleic acid molecule in which a Rep region containing a base sequence encoding the Rep protein of AAV of serotype 2, a Cap region containing a base sequence encoding the Cap protein of AAV of serotype 9, a p5 promoter as an enhancer further downstream, a replication origin further downstream, an ampicillin resistance gene further downstream, a first ITR further downstream, a promoter derived from cytomegalovirus further downstream, a synthetic human β-globin intron further downstream, a base sequence for inserting a base sequence encoding a foreign protein further downstream, a synthetic polyA sequence further downstream, a second ITR further downstream, an adenovirus E2A region further downstream, an adenovirus E4 region further downstream, and an adenovirus VA1 RNA region further downstream are located. Although not shown in SEQ ID NO:24, a base sequence for inserting a base sequence encoding a foreign protein (any of the restriction enzyme sites present at 8116bp to 8147bp in the base sequence shown in SEQ ID NO:24) is assumed to have a base sequence encoding a desired protein introduced therein. The nucleic acid molecule shown in SEQ ID NO: 24 is a preferred example of a nucleic acid molecule that can be used to produce a recombinant AAV virion. The nucleic acid molecule shown in SEQ ID NO: 24 has a structure corresponding to the pHelper-ITR / CMV / GFP-R2(mod)C9 vector shown in Figure 11 with the GFP gene removed.

[0117] The nucleic acid molecule having the base sequence shown in SEQ ID NO:24 can be synthesized as a circular plasmid using general genetic engineering techniques. This circular plasmid encodes a drug resistance gene. It also has a replication origin for replication in E. coli. Therefore, this circular plasmid can be amplified by introducing it into E. coli, and can be easily prepared in large quantities.

[0118] The prepared circular plasmid is introduced into a host cell in which the E1A and E1B regions of adenovirus have been integrated into the genome. As such a host cell, HEK293 cells, which are human fetal kidney tissue-derived cells transformed with the E1A and E1B genes of adenovirus, can be suitably used.

[0119] When the circular plasmid is introduced into a host cell, the region from the first ITR to the second ITR is integrated into the host cell by non-homologous recombination. Then, single-stranded DNA is replicated from the region integrated into the host cell and packaged into a capsid to form a recombinant AAV virion. The recombinant AAV virion is released into the culture medium or accumulates within the host cell. Therefore, the recombinant AAV virion can be recovered by recovering the culture supernatant of the host cell or by recovering and destroying the host cell.

[0120] Usually, in order to produce recombinant AAV virions, three types of plasmids must be simultaneously introduced into host cells: a plasmid containing a base sequence encoding the desired protein flanked by ITRs, a plasmid containing a base sequence encoding the Rep protein and a base sequence encoding the Cap protein, and a plasmid containing the adenovirus-derived E2A, E4, and VA1 RNA sequences. The ratio of host cells into which the three types of plasmids are introduced is low, which reduces the production efficiency of recombinant AAV virions. In addition, in host cells into which the plasmid containing the base sequence encoding the desired protein flanked by ITRs is not introduced and only the other two types of plasmids are introduced, empty virus particles (empty capsids) that do not contain the rAAV genome are produced, further reducing the production efficiency.

[0121] A circular plasmid having the base sequence shown in SEQ ID NO: 24, which is one of the preferred embodiments of the present invention, has all the functions of these three types of plasmids. Therefore, by transforming a host cell with this circular plasmid, all elements necessary for the production of rAAV virions in the cell are introduced into the cell at the same time, so that not only can the production amount of rAAV virions be increased, but also the generation of empty capsids can be suppressed. Since the higher the ratio of empty capsids to the total capsids, the lower the quality of the rAAV virions, by using this circular plasmid, high-quality rAAV virions with a low ratio of empty capsids can be efficiently produced. Not only this, but also high-quality rAAV virions with a low ratio of empty capsids can be efficiently produced by transforming a host cell with an integrated rAAV plasmid. EXAMPLES

[0122] The present invention will be described in more detail below with reference to examples, but it is not intended that the present invention be limited to the examples.

[0123] Example 1: Construction of pHelper(mod) vector A DNA fragment containing the base sequence shown in SEQ ID NO:27, including the SalI site, RsrII site, replication origin (ColE1 ori), ampicillin resistance gene, BsiWI site, BstZ17I site, and BsrGI site from the 5' side, was synthesized. This DNA fragment was digested with SalI and BsrGI. The pHelper vector (Takara Bio) was digested with SalI and BsrGI, and the above restriction enzyme-treated DNA fragment was inserted into it. The resulting plasmid was designated the pHelper (mod) vector (Figure 1).

[0124] Example 2: Construction of pAAV-CMV-GFP vector and pAAV-CMV-GFP(mod) vector A DNA fragment containing the base sequence shown in SEQ ID NO:28, including an EcoRI site, an MluI site, a GFP gene (green fluorescent protein gene), a NotI site, and a BamHI site from the 5' side, was synthesized. This DNA fragment was digested with EcoRI and BamHI. A pAAV-CMV vector (Takara Bio) was digested with EcoRI and BamHI, and the above-mentioned restriction enzyme-treated DNA fragment was inserted thereinto. The resulting plasmid was designated as a pAAV-CMV-GFP vector. A multicloning site 1 containing a BsiWI site and an AvrII site and having the base sequence shown in SEQ ID NO:29 was inserted into the PciI site of the pAAV-CMV-GFP vector, and further a multicloning site 2 containing an SfoI site, a BsrGI site, an AgeI site, and a BstZ17I site and having the base sequence shown in SEQ ID NO:30 was inserted. The resulting plasmid was designated as a pAAV-CMV-GFP(mod) vector (Figure 2).

[0125] [Example 3] Construction of pR2(mod)C6 vector A DNA fragment containing the base sequence shown in SEQ ID NO:31, including the AfeI site, RsrII site, BsrGI site, AvrII site, ampicillin resistance gene site, replication origin (ColE1 ori), RsrII site, 5' portion of AAV2 Rep region (including p5 promoter), and SacII site from the 5' side, was synthesized. This DNA fragment was digested with AfeI and SacII. pRC6 vector (Takara Bio) was digested with AfeI and SacII, and the above-mentioned restricted synthetic gene was inserted into it. The obtained plasmid was named pR2(mod)C6 vector (Figure 3).

[0126] [Example 4] Construction of pR2(mod)C9 vector A DNA fragment containing the base sequence shown in SEQ ID NO: 32, including a HindIII site, the 3' portion of the AAV2 Rep region, the AAV9 Cap region, the p5 promoter, an RsrII site, and a BsrGI site from the 5' side, was synthesized. This DNA fragment was digested with HindIII and BsrGI. The pR2(mod)C6 vector was digested with HindIII and BsrGI, and the synthetic gene treated with the above restriction enzymes was inserted into it. The resulting plasmid was named pR2(mod)C9 vector (Figure 4).

[0127] Example 5: Construction of pHelper-ITR / CMV / GFP vector The pAAV-CMV-GFP(mod) vector was digested with BsiWI and BstZ17I to excise a DNA fragment containing the ITR-CMV promoter, human β-globin intron, GFP gene, synthetic polyA sequence, and ITR. The pHelper(mod) vector was digested with BsiWI and BstZ17I, and the excised DNA fragment was inserted into it. The resulting plasmid was designated the pHelper-ITR / CMV / GFP vector (Figure 5).

[0128] [Example 6] Construction of pHelper-R2(mod)C6 vector The pR2(mod)C6 vector was digested with RsrII to excise the AAV2 Rep region (including the p5 promoter) and the AAV6 Cap region-p5 promoter sequence. Separately, the pHelper(mod) vector was digested with RsrII. The excised DNA fragment was inserted into this vector to create the pHelper-R2(mod)C6 vector (Figure 6).

[0129] [Example 7] Construction of pHelper-R2(mod)C9 vector The pR2(mod)C9 vector was digested with RsrII to excise the sequence containing the p5 promoter-Rep2-AAV9 VP1-p5 promoter. The pHelper(mod) vector was digested with RsrII. The excised DNA fragment was inserted into this vector to create the pHelper-R2(mod)C9 vector (Figure 7).

[0130] [Example 8] Construction of pR2(mod)C6-ITR / CMV / GFP vector The pAAV-CMV-GFP(mod) vector was digested with AvrII and BsrGI to excise the sequence containing ITR-CMV promoter-human β-globin intron-GFP-synthetic polyA sequence-ITR. The pR2(mod)C6 vector was digested with AvrII and BsrGI. The excised DNA fragment was inserted into this vector to create the pR2(mod)C6-ITR / CMV / GFP vector (Figure 8).

[0131] Example 9: Construction of pR2(mod)C9-ITR / CMV / GFP vector The pAAV-CMV-GFP(mod) vector was digested with AvrII and BsrGI to excise a DNA fragment containing ITR-CMV promoter-human β-globin intron-GFP-synthetic polyA sequence-ITR. Separately, the pR2(mod)C9 vector was digested with AvrII and BsrGI. The excised DNA fragment was inserted into this vector to create the pR2(mod)C9-ITR / CMV / GFP vector (Figure 9).

[0132] Example 10: Construction of pHelper-ITR / CMV / GFP-R2(mod)C6 vector The pR2(mod)C6 vector was digested with RsrII to excise the AAV2 Rep region (including the p5 promoter), the AAV6 Cap region, and a sequence containing the p5 promoter that functions as an enhancer. Separately, the pHelper-ITR / CMV / GFP vector was digested with RsrII. The sequence excised above was inserted into this vector to create the pHelper-ITR / CMV / GFP-R2(mod)C6 vector (Figure 10).

[0133] Example 11: Construction of pHelper-ITR / CMV / GFP-R2(mod)C9 vector The pR2(mod)C9 vector was digested with RsrII to excise a DNA fragment containing the AAV2 Rep region (including the p5 promoter), the AAV9 Cap region, and the p5 promoter that functions as an enhancer. Separately, the pHelper-ITR / CMV / GFP vector was digested with RsrII. The excised DNA fragment was inserted into this vector to create the pHelper-ITR / CMV / GFP-R2(mod)C9 vector (Figure 11).

[0134] [Example 12] Production of rAAV virions by transient expression of three types of plasmids HEK293T cells, a cell line derived from human embryonic kidney cells expressing the SV40 large T antigen gene, were used as host cells. HEK293T cells were cultured at 1.65×10 7 The cells were seeded on 15 cm dishes at a density of 100 cells / dish and cultured in 10% FBS-containing DMEM / High Glucose medium (Thermo Fisher Scientific) at 37°C and in the presence of 5% CO2 for 16 hours. As DNA for transfection, a solution containing the following combination of plasmids (1) and (2) was prepared; (1) pHelper vector (Takara Bio), pAAV-CMV-GFP vector, and pRC6 vector (Takara Bio), (2) pHelper vector (Takara Bio), pAAV-CMV-GFP vector, and pR2(mod)C9 vector. Each solution was prepared so that the three types of plasmids were contained in equimolar concentrations and the DNA concentration was 4 μg / mL. Polyethylenimine and FBS were added to this solution to a concentration of 12 μg / mL and 1.3% (v / v), respectively, and DMEM / High Glucose medium was added to adjust the liquid volume to 30 mL to prepare a transfection solution. After removing all the culture supernatant from the cells seeded in a 15 cm dish, the entire amount of 30 mL of the transfection solution was added, and the cells were cultured at 37°C in the presence of 5% CO2 for 3 days to produce rAAV virions. Some of the rAAV virions produced in the host cells were released into the culture supernatant, and some remained within the host cells. The same applies to Examples 13 and 14.

[0135] [Example 13] Production of rAAV virions by transient expression of two types of plasmids 1.65 × 10 HEK293T cells 7 The cells were seeded on 15-cm dishes at a density of 100 cells / dish and cultured in 10% FBS-containing DMEM / High Glucose medium at 37°C in the presence of 5% CO2 for 16 hours. DNA solutions containing the following combinations of plasmids (1) to (6) were prepared for transfection: (1) pHelper-ITR / CMV / GFP vector and pRC6 vector (Takara Bio), (2) pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (3) pHelper-R2(mod)C6 vector and pAAV-CMV-GFP vector, (4) pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, (5) pR2(mod)C6-ITR / CMV / GFP vector and pHelper vector (Takara Bio), (6) pR2(mod)C9-ITR / CMV / GFP vector and pHelper (Takara Bio) vector. Each solution was prepared so that the two types of plasmids were contained in equimolar concentrations and the DNA concentration was 3.5 to 3.6 μg / mL. Polyethylenimine and FBS were added to these solutions to make the concentrations 12 μg / mL and 1.3% (v / v), respectively, and DMEM / High Glucose medium was added to adjust the volume to 30 mL to prepare the transfection solution. After removing all the culture supernatant from the cells seeded in a 15 cm dish, the entire volume of the transfection solution (30 mL) was added, and the cells were cultured at 37°C in the presence of 5% CO2 for 3 days to produce rAAV virions.

[0136] Example 14: Production of rAAV virions by transient expression of integrated rAAV plasmids 1.65 × 10 HEK293T cells 7 The cells were seeded in a 15 cm dish at a concentration of 100 cells / dish and cultured in DMEM / High Glucose medium containing 10% FBS at 37°C in the presence of 5% CO2 for 16 hours. A solution containing pHelper-ITR / CMV / GFP-R2(mod)C6 vector or pHelper-ITR / CMV / GFP-R2(mod)C9 vector at a concentration of 3.2 μg / mL was prepared as a DNA solution for transfection. Polyethylenimine and FBS were added to this solution to make the concentrations 12 μg / mL and 1.3% (v / v), respectively, and DMEM / High Glucose medium was further added to adjust the liquid volume to 30 mL to prepare a transfection solution. After removing all the culture supernatant from the cells seeded in the 15 cm dish, the entire amount of the transfection solution (30 mL) was added and cultured at 37°C in the presence of 5% CO2 for 3 days to produce rAAV6 virions and rAAV9 virions, respectively.

[0137] [Example 15] Recovery of culture supernatant After the culture in Examples 12 to 14 was completed, the cell culture solution was transferred to a 50 mL centrifuge tube and centrifuged at 3000×g for 10 minutes to recover the culture supernatant. The cells precipitated by centrifugation were used to prepare the cell lysate solution in Example 17.

[0138] [Example 16] Measurement of rAAV genome amount and total capsid amount contained in culture supernatant The viral genome content in the culture supernatant was measured using the AAVpro Titration Kit for Real Time PCR Ver.2 (Takara Bio) in accordance with the protocol attached to the kit, generally by the following method. 2 μL of 10×DNaseI Buffer, 1 μL of DNaseI, and 15 μL of pure water were added to 2 μL of the culture supernatant recovered in Example 15, mixed, and incubated at 37°C for 1 hour. DNaseI was inactivated by heat treatment at 95°C for 10 minutes, and then 20 μL of Lysis Buffer was added and heat treatment was performed at 70°C for 10 minutes. The solution after heat treatment was diluted 100 times with EASY Dilution (Takara Bio) to prepare a sample solution for real-time PCR.

[0139] To 5 μL each of the sample solution for real-time PCR and the standard plasmid solution for the calibration curve (linearized pAAV-CMV-GFP vector), 12.5 μL of TB Green Premix Ex TaqII, 0.6 μL of 10 μM forward primer (primer 1, sequence number 33), 0.6 μL of 10 μM reverse primer (primer 2, sequence number 34), 0.08 μL of ROX Reference Dye II, and 6.5 μL of water were added. The real-time PCR reaction was performed under denaturing conditions (95°C, 2 minutes) followed by 35 cycles of two-step PCR (95°C, 5 seconds → 60°C, 30 seconds). The measured values ​​of the sample were interpolated into the obtained calibration curve to determine the amount (number) of rAAV genomes contained in the sample. The region replicated in this PCR is the internal region of the ITR.

[0140] The total amount of capsids in the culture supernatant was measured using an AAV Titration ELISA Kit (PROGEN) according to the protocol attached to the kit, roughly as follows: 100 μL of the culture supernatant (specimen sample) collected in Example 15 diluted with 1× Assay Buffer was added to a plate on which mouse anti-AAV9 monoclonal antibody was immobilized, and the plate was allowed to stand at 37°C for 1 hour. 9 ~1.0×10 7 The Kit Control diluted with 1x Assay Buffer to a concentration of capsids / mL was added to the plate as a standard solution in the same manner as the sample samples and allowed to stand. After washing the plate three times with 1x Assay Buffer, biotin-labeled mouse anti-AAV9 antibody or mouse anti-AAV6 antibody was added and allowed to stand at 37°C for 1 hour. After washing the plate three times with 1x Assay Buffer, HRP-labeled streptavidin was added and allowed to stand at 37°C for 1 hour. After washing the plate three times with 1x Assay Buffer, HRP substrate was added and allowed to stand at room temperature for 15 minutes, and then sulfuric acid was added to stop the reaction. The measured values ​​of the sample were interpolated into the calibration curve obtained from the measured values ​​of the absorbance of the standard solution to determine the total amount (number) of capsids contained in the sample.

[0141] [Example 17] Preparation of cell lysate solution The cells precipitated by centrifugation of the cell culture solution in Example 15 were added with citrate buffer (32 mM sodium citrate) containing 1% TritonX-100, 4 mM MgCl2, 50 U / mL Turbonuclease (Accelagen), and 4x Protease Inhibitor Cocktail (Sigma), and left to stand at 37°C for 1.5 hours. After neutralization by adding citrate buffer (68 mM citric acid) containing 4 mM MgCl2, the mixture was centrifuged at 6000xg and 4°C for 10 minutes. A double amount of 25 mM citrate buffer (pH 3.6, 8 mM sodium citrate + 17 mM citric acid) containing 0.25% TritonX-100, 4 mM MgCl2, 12.5 U / mL Turbonuclease (Accelagen), and 1x Protease Inhibitor Cocktail (Sigma) was added to the mixture to prepare a cell lysate.

[0142] A 1 mL SP-Sepharose column (GE Healthcare) was equilibrated with 25 mM citrate buffer (pH 3.6, 8 mM sodium citrate + 17 mM citric acid) containing 4 mM MgCl2, and the cell lysate was applied to this. After elution with 50 mM citrate buffer (pH 5.18, 40.2 mM sodium citrate + 9.8 mM citric acid) containing 4 mM MgCl2 and 500 mM NaCl, 1 M Tris buffer was added to a final concentration of 50 mM to neutralize the eluate. The neutralized sample was placed in a Vivaspin-500 100K MWCO (Vivaproducts) and centrifuged at 3000 x g for 10 minutes at 4 °C, after which PBS containing 180 mM NaCl and 0.001% F68 was added and centrifuged at 3000 x g for 10 minutes at 4 °C to replace the buffer. The resulting solution was used as the cell lysate solution.

[0143] [Example 18] Measurement of rAAV genome amount and total capsid amount contained in cell lysate solution The viral genome content in the purified cell lysate solution was measured using the AAVpro Titration Kit for Real Time PCR Ver.2 (Takara Bio Inc.) in accordance with the protocol attached to the kit, generally by the following method. 2 μL of 10×DNaseI Buffer, 1 μL of DNaseI, and 15 μL of pure water were added to 2 μL of the cell lysate solution sample prepared in Example 17, mixed, and incubated at 37°C for 1 hour. DNaseI was inactivated by heat treatment at 95°C for 10 minutes, and then 20 μL of Lysis Buffer was added and heat treatment was performed at 70°C for 10 minutes. The solution after heat treatment was diluted 100 times with EASY Dilution (Takara Bio Inc.) to prepare a sample solution for real-time PCR.

[0144] To 5 μL each of the sample solution for real-time PCR and the standard plasmid solution for the calibration curve (linearized pAAV-CMV-GFP vector), 12.5 μL of TB Green Premix Ex TaqII, 0.6 μL of 10 μM forward primer (primer 1, sequence number 33), 0.6 μL of 10 μM reverse primer (primer 2, sequence number 34), 0.08 μL of ROX Reference Dye II, and 6.5 μL of water were added. The real-time PCR reaction was performed under denaturing conditions (95°C, 2 minutes) followed by 35 cycles of two-step PCR (95°C, 5 seconds → 60°C, 30 seconds). The measured values ​​of the sample were interpolated into the obtained calibration curve to determine the amount of AAV viral genome (vg) contained in the sample. Here, vg is a unit indicating the number of AAV viral genomes. The region replicated in this PCR is the internal region of the ITR.

[0145] The total amount of capsids in the cell lysate solution was measured using an AAV Titration ELISA Kit (PROGEN) according to the protocol attached to the kit, generally as follows: 100 μL of the cell lysate solution (sample) prepared in Example 17 diluted with 1× Assay Buffer was added to a plate on which mouse anti-AAV9 monoclonal antibody was immobilized, and the plate was allowed to stand at 37°C for 1 hour.9 ~1.0×10 7 The Kit Control diluted with 1x Assay Buffer to a concentration of capsids / mL was added to the plate as a standard solution in the same manner as the sample samples and allowed to stand. After washing the plate three times with 1x Assay Buffer, biotin-labeled mouse anti-AAV9 antibody or mouse anti-AAV6 antibody was added and allowed to stand at 37°C for 1 hour. After washing the plate three times with 1x Assay Buffer, HRP-labeled streptavidin was added and allowed to stand at 37°C for 1 hour. After washing the plate three times with 1x Assay Buffer, HRP substrate was added and allowed to stand at room temperature for 15 minutes, and then sulfuric acid was added to stop the reaction. The measured values ​​of the sample were interpolated into the calibration curve obtained from the measured values ​​of the absorbance of the standard solution to determine the total amount (number) of capsids contained in the sample.

[0146] [Example 19] Calculation of the abundance ratio in the total capsid of rAAV virion The amounts of recombinant AAV virions (rAAV virions) containing the rAAV genome and empty capsids were calculated from the results of measuring the amount of rAAV genome and the total amount of capsids contained in the cell lysate solution measured in Example 18. The amount of rAAV virions was determined as the amount (number) of rAAV virions, assuming that all of the rAAV genome was packaged in capsids to form virions. (Amount of rAAV virions / total amount of capsids) x 100% was calculated as the abundance ratio of rAAV virions in total capsids. The amount (number) of empty capsids was calculated by subtracting the amount (number) of rAAV virions from the measured amount (number) of total capsids.

[0147] [Example 20] Measurement of infectivity of rAAV6 virions contained in cell lysate solution 1 x 10 HEK293T cells 5The cells were seeded in a 24-well plate at a concentration of 100 cells / well and cultured in 10% FBS-containing DMEM / High Glucose medium at 37°C and 8% CO2 for 16 hours. The cell lysate solution containing the rAAV6 virions prepared in Example 18 was added to 1 mL of DMEM / High Glucose medium containing 2% FBS to prepare a transduction solution. The transduction solution contained 2x10 rAAV6 virions. 8 Pieces and 2x10 9 After removing all the culture supernatant from the 24-well plate, the transduction solution was added and cultured at 37°C in the presence of 8% CO2 for 3 days.

[0148] After 3 days, all the culture supernatants were removed from the 24-well plates, and the plates were washed with PBS and then 20 μL of TrypLE was added. TM (Enzyme solution with trypsin-like activity: Thermo Fisher Scientific) was added and left to stand at 37°C for 2 minutes. After that, the cells were suspended again in 1 mL of PBS, and 65 μL of the suspension was used to measure the percentage of GFP-positive cells using MOXI-GO II (ORFLO).

[0149] [Example 21] Measurement of infectivity of rAAV9 virions contained in cell lysate solution 1 x 10 HEK293T cells 5 The cells were seeded in a 24-well plate at a concentration of 100 cells / well and cultured in 10% FBS-containing DMEM / High Glucose medium at 37°C and 8% CO2 for 16 hours. The cell lysate solution containing the rAAV9 virions prepared in Example 18 was added to 1 mL of DMEM / High Glucose medium containing 2% FBS to prepare a transduction solution. The transduction solution contained 2x10 rAAV6 virions. 8 Pieces and 2x10 9 After removing all the culture supernatant from the 24-well plate, the transduction solution was added and cultured at 37°C in the presence of 8% CO2 for 3 days.

[0150] After 3 days, all the culture supernatants were removed from the 24-well plates, and the plates were washed with PBS and then 20 μL of TrypLE was added. TM (Enzyme solution with trypsin-like activity: Thermo Fisher Scientific) was added and left to stand at 37°C for 2 minutes. After that, the cells were suspended again in 1 mL of PBS, and 65 μL of the suspension was used to measure the percentage of GFP-positive cells using MOXI-GO II (ORFLO).

[0151] [Example 22] Calculation of Transducing Unit The Transducing Unit was calculated using the following formula. Here, the Transducing Unit means the number of GFP-positive cells (the number of cells infected with rAAV virions) obtained when the entire volume of the cell lysate solution is used to infect host cells. ([Total number of cells in the well] x [Proportion of GFP-positive cells (%)] / 100) x ([Total volume of rAAV virion sample (mL)] / [Volume of rAAV virion sample added to each well (mL)])

[0152] [Example 23] Results (measurement of rAAV genome amount and total capsid amount contained in culture supernatant) The measurement results of the amount of rAAV genome contained in the culture supernatant obtained in Example 15 are shown by black bars in Figure 12. In the figure, (1) shows the results when two types of plasmids were introduced: pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when two types of plasmids were introduced: pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids were introduced: pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector. The amount of rAAV genome was 2.6 × 10 11 vg,7.2×10 11 vg,11.1×10 11 (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced, and the amount of rAAV genome was 15.6 × 10 11In contrast, (4) shows the results when the pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced, and the amount of rAAV genome was 35.0 × 10 11 It was vg.

[0153] The results of measuring the total capsid amount are shown by the white bars in Figure 12. In the figure, (1) shows the results when two types of plasmids were introduced: pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when two types of plasmids were introduced: pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids were introduced: pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector. The total capsid amount was 2.6 × 10 12 Capsids, 5.6×10 12 Capsids, 5.9×10 12 (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced, and the total amount of capsids was 3.6 × 10 12 In contrast, (4)% represents the result of transfection with pHelper-ITR / CMV / GFP-R2(mod)C9 vector, with a total capsid amount of 6.6 × 10 12 Both the rAAV genome amount and the total capsid amount showed the highest expression levels when the integrative rAAV plasmid, i.e., pHelper-ITR / CMV / GFP-R2(mod)C9 vector, was introduced (Figure 12).

[0154] [Example 24] Results (measurement of the amount of rAAV contained in the cell lysate solution) The measurement results of the amount of rAAV genome contained in the cell lysate solution prepared in Example 17 are shown by black bars in Figure 13. In the figure, (1) shows the results when two types of plasmids were introduced: pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when two types of plasmids were introduced: pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids were introduced: pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector. The amount of rAAV genome was 2.5 × 10 11 vg,23.1×10 11 vg,22.0×10 11 (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced, and the amount of rAAV genome was 6.8 × 10 11 In contrast, (4) shows the results when the pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced, and the amount of rAAV genome was 29.7 × 10 11 vg (Figure 13).

[0155] The results of measuring the total capsid amount are shown by the white bars in Figure 13. In the figure, (1) shows the results when two types of plasmids were introduced: pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when two types of plasmids were introduced: pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids were introduced: pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector. The total capsid amount was 3.3 × 10 12 Capsids, 5.3×10 12 Capsids, 5.3×10 12 (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced, and the amount of rAAV genome was 3.8 × 10 12 In contrast, (4) shows the results when the pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced, and the amount of rAAV genome was 6.1 × 1012 Both the rAAV genome amount and the total capsid amount showed the highest expression levels when the integrated rAAV plasmid, i.e., pHelper-ITR / CMV / GFP-R2(mod)C9 vector, was introduced (Figure 13).

[0156] [Example 25] Results (abundance ratio in total capsids of rAAV virions) Theoretically, when there are no empty capsids and all capsids are rAAV virions containing rAAV, the ratio of the rAAV genome amount to the total capsid amount is 1:1, as calculated from the rAAV genome amount and total capsid amount in the cell lysate solution calculated in Example 17. The ratio of the rAAV genome amount to the total capsid amount is shown in Figure 14. In the figure, (1) shows the results when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, were introduced, and the ratios were 7.5%, 43.5%, and 41.5%, respectively. (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced, and the proportion of empty capsids was 17.8%. In contrast, (4) shows the results when the pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced, and the proportion of empty capsids was 48.6% (Figure 14). These results showed that the proportion of empty capsids was low when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, were introduced, and when the pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced.

[0157] [Example 26] Results (measurement of infectivity of rAAV6 virions contained in cell lysate solution) The results of measuring the infectivity of rAAV6 virions contained in the cell lysate solution prepared in Example 17 are shown in Figure 15. In the figure, the black bar indicates 2 x 10 8 The figures show the results when cells were infected with rAAV6 virions. In the figure, (1) shows the results when two types of plasmids, pHelper-ITR / CMV / GFP vector and pRC6 vector, (2) shows the results when two types of plasmids, pHelper-R2(mod)C6 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids, pR2(mod)C6-ITR / CMV / GFP vector and pHelper vector, were introduced, and the percentage of GFP-positive cells was 1.5%, 9.3%, and 5%, respectively. (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pRC6 vector, were introduced, and the percentage of GFP-positive cells was 0.6%, and (4) shows the results when pHelper-ITR / CMV / GFP-R2(mod)C6 vector was introduced, and the percentage of GFP-positive cells was 11.2%.

[0158] In addition, in Figure 15, the white bars are 2×10 9 The figures show the results when cells were infected with rAAV6 virions. In the figure, (1) shows the results when two types of plasmids, pHelper-ITR / CMV / GFP vector and pRC6 vector, (2) shows the results when two types of plasmids, pHelper-R2(mod)C6 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids, pR2(mod)C6-ITR / CMV / GFP vector and pHelper vector, were introduced, and the percentage of GFP-positive cells was 23.2%, 70.5%, and 53.6%, respectively. (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pRC6 vector, were introduced, and the percentage of GFP-positive cells was 12.6%, and (4) shows the results when pHelper-ITR / CMV / GFP-R2(mod)C6 vector was introduced, and the percentage of GFP-positive cells was 71.1%.

[0159] These results indicate that a highly infectious rAAV6 virion solution can be obtained by using two types of plasmids, the pHelper-R2(mod)C6 vector and the pAAV-CMV-GFP vector, or the pR2(mod)C6-ITR / CMV / GFP vector and the pHelper vector, or by using the pHelper-ITR / CMV / GFP-R2(mod)C6 vector.

[0160] [Example 27] Results (measurement of infectivity of rAAV9 virions contained in cell lysate solution) The results of measuring the infectivity of rAAV9 virions contained in the cell lysate solution prepared in Example 17 are shown in Figure 16. In the figure, the black bar indicates 2 x 10 8 The figures show the results when cells were infected with rAAV9 virions. In the figure, (1) shows the results when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, were introduced. The percentage of GFP-positive cells was 0.8%, 0.3%, and 2%, respectively. (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced. The percentage of GFP-positive cells was 3.5%, and (4) shows the results when pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced. The percentage of GFP-positive cells was 7.3%.

[0161] In addition, in Figure 16, the white bars are 2×10 9The figures show the results when cells were infected with rAAV9 virions. In the figure, (1) shows the results when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when two types of plasmids, pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids, pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector, were introduced. The percentage of GFP-positive cells was 4.6%, 1%, and 11.4%, respectively. (5) shows the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector, were introduced. The percentage of GFP-positive cells was 21.7%, and (4) shows the results when pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced. The percentage of GFP-positive cells was 50.3%.

[0162] These results indicate that the pHelper-ITR / CMV / GFP-R2(mod)C9 vector can be used to obtain a rAAV9 virion solution with high infectivity.

[0163] [Example 28] Results (calculation of Transducing Unit) FIG. 17 shows the results of measuring the infectivity of rAAV6 virions contained in the cell lysate solution prepared in Example 17. In the figure, (1) shows the results when two types of plasmids, the pHelper-ITR / CMV / GFP vector and the pRC6 vector, (2) shows the results when two types of plasmids, the pHelper-R2(mod)C6 vector and the pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids, the pR2(mod)C6-ITR / CMV / GFP vector and the pHelper vector, were introduced. In addition, (5) shows the results when three types of plasmids, the pHelper vector, the pAAV-CMV-GFP vector, and the pRC6 vector, were introduced. In addition, (4) shows the results when the pHelper-ITR / CMV / GFP-R2(mod)C6 vector was introduced.

[0164] FIG. 18 shows the results of measuring the infectivity of rAAV9 virions contained in the cell lysate solution prepared in Example 17. In the figure, (1) shows the results when two types of plasmids were introduced: pHelper-ITR / CMV / GFP vector and pR2(mod)C9 vector, (2) shows the results when two types of plasmids were introduced: pHelper-R2(mod)C9 vector and pAAV-CMV-GFP vector, and (3) shows the results when two types of plasmids were introduced: pR2(mod)C9-ITR / CMV / GFP vector and pHelper vector. (5) shows the results when three types of plasmids were introduced: pHelper vector, pAAV-CMV-GFP vector, and pR2(mod)C9 vector. (4) shows the results when the pHelper-ITR / CMV / GFP-R2(mod)C9 vector was introduced.

[0165] These results indicate that more infectious rAAV6 or rAAV9 virions can be produced by transfecting host cells with the integrative rAAV plasmids, i.e., pHelper-ITR / CMV / GFP-R2(mod)C6 vector or pHelper-ITR / CMV / GFP-R2(mod)C9 vector, respectively.

[0166] Example 29: Construction of pAAV-CBA(BAM)-I2S vector From the experimental results for two types of vectors, pHelper-ITR / CMV / GFP-R2(mod)C6 vector or pHelper-ITR / CMV / GFP-R2(mod)C9 vector up to Example 28, it was found that highly infectious rAAV6 virions or rAAV9 virions can be produced by using the integrated rAAV plasmid. The high infectiousness of the integrated rAAV plasmid is considered to be due to its high presence in the total capsid of rAAV virions. Therefore, in order to further verify the results, integrated rAAV plasmids with different structures were constructed, and the presence ratio in the total capsid of rAAV virions was confirmed by the following experiment.

[0167] A DNA fragment containing the base sequence shown in SEQ ID NO: 35, including the human CMV enhancer, chicken β-actin promoter, chicken β-actin / MVM chimeric intron, human I2S gene, and bovine growth hormone polyA sequence, was synthesized from the 5' side. This DNA fragment was used as a template to perform a PCR reaction using primer 7 (SEQ ID NO: 48) and primer 8 (SEQ ID NO: 49). The pAAV-CMV-GFP(mod) vector constructed in Example 2 was digested with restriction enzymes (HindIII / BglII), mixed with the DNA fragment obtained by the PCR reaction, and the two were fused using an in fusion HD cloning Kit (Takara Bio). The resulting plasmid was designated as the pAAV-CBA(BAM)-I2S vector (Figure 19). Here, the human I2S gene encodes a human IDS containing the amino acid sequence shown in SEQ ID NO: 36 (same as in Example 30). In addition, the human CMV enhancer includes the base sequence shown in sequence number 37, the chicken beta-actin promoter includes the base sequence shown in sequence number 38, the chicken beta-actin / MVM chimeric intron includes the base sequence shown in sequence number 39, and the bovine growth hormone polyA sequence includes the base sequence shown in sequence number 40.

[0168] Example 30: Construction of pHelper-ITR / CBA(BAM) / I2S-R2(mod)C9 vector A DNA fragment having the base sequence shown in SEQ ID NO: 50, including ITR, human CMV enhancer, chicken β-actin promoter, chicken β-actin / MVM chimeric intron, bovine growth hormone polyA sequence, and ITR from the 5' side, was synthesized. This DNA fragment was digested with BsiWI and BstZ17I and inserted between the BsiWI site and the BstZ17I site of pHelper(mod) constructed in Example 1. The obtained plasmid was designated as pHelper-ITR / CBA(BAM) vector.

[0169] The pHelper-ITR / CBA(BAM) vector was digested with RsrII, and a DNA fragment containing the AAV9 Cap region obtained by digesting pR2(mod)C9 constructed in Example 4 with RsrII was inserted into it. The resulting plasmid was named pHelper-ITR / CBA(BAM) / -R2(mod)C9 vector.

[0170] A DNA fragment having the base sequence shown in SEQ ID NO: 51 containing the human IDS gene was synthesized. This DNA fragment was digested with PacI and NotI and inserted into the pHelper-ITR / CBA(BAM) / -R2(mod)C9 vector digested with PacI and NotI. The resulting plasmid was designated as the pHelper-ITR / CBA(BAM) / I2S-R2(mod)C9 vector (Figure 20).

[0171] [Example 31] Production of rAAV virions by transient expression of three types of plasmids (2) HEK293T cells, 2.5 × 10 6 The cells were seeded in a T-25 flask at a concentration of 100 cells / flask and cultured in 10% FBS-containing MEM medium (Sigma) at 37°C in the presence of 5% CO2 for 16 hours. A solution containing pHelper vector (Takara Bio), pAAV-CBA(BAM)-I2S vector, and pR2(mod)C9 vector was prepared as DNA for transfection. This solution was prepared so that the DNA concentration was 2.9-3.9 μg / mL and the three types of plasmids were contained in equimolar concentrations. Polyethylenimine was added to this solution at a concentration ratio of DNA (μg):polyethylenimine (μg) = 1:3, and MEM medium was further added to adjust the liquid volume to 5 mL to prepare the transfection solution. After removing all the culture supernatant from the cells seeded in the T-25 flask, the entire amount of 5 mL of the transfection solution was added, and the cells were cultured at 37°C in the presence of 5% CO2 for 5 days to produce rAAV virions. The rAAV virions produced in the host cells are partly released into the culture supernatant and partly retained within the host cells.

[0172] [Example 32] Production of rAAV virions by transient expression of integrated plasmid (2) HEK293T cells, 2.5 × 10 6 The cells were seeded in a T-25 flask at a concentration of 100 cells / flask and cultured in 10% FBS-containing MEM medium (Sigma) at 37°C and 5% CO2 for 16 hours. A solution containing 2.9-3.9 μg / mL of pHelper-ITR / CBA(BAM) / I2S-R2(mod)C9 vector was prepared as DNA for transfection. Polyethylenimine was added to this solution so that the concentration ratio was DNA (μg):polyethylenimine (μL) = 1:3, and MEM medium was further added to adjust the liquid volume to 5 mL to prepare the transfection solution. After removing all the culture supernatant from the cells seeded in the T-25 flask, the entire 5 mL of transfection solution was added and cultured at 37°C and 5% CO2 for 5 days to produce rAAV virions.

[0173] [Example 33] Recovery of culture supernatant After the culture in Examples 31 and 32 was completed, the cell culture medium was transferred to a 15 mL centrifuge tube and centrifuged at 4° C. and 5,800×g for 5 minutes to recover the culture supernatant.

[0174] [Example 34] Preparation of rAAV virion solution from culture supernatant The culture supernatant collected in Example 33 was treated with benzonase, an endonuclease. TM (Merck brand) was added to a concentration of 14 U / mL, mixed, and incubated at 37°C for 2 hours. TM CaptureSelect TMAAV9 Affinity Resin (Thermo Fisher Scientific, Inc., 0.15 mL) was equilibrated with binding buffer (containing 20 mM Tris-HCl, 150 mM NaCl, and 4 mM MgCl2, pH 7.5), and the endonuclease-treated culture supernatant was loaded onto the column. After washing the column with the binding buffer, the solution containing rAAV virions was eluted with 0.5 mL elution buffer (containing 0.1 M citric acid, pH 2.06). The obtained eluate was transferred to a PD MiniTrap column pre-equilibrated with PBS containing 150 mM NaCl and 0.001% F68. TM The rAAV virions were bound to the column through a G-25 (GE Healthcare) column. The rAAV virions were then eluted with 1 mL of PBS containing 150 mM NaCl and 0.001% F68. TM The solution was concentrated by centrifugation at 2000×g for 3 minutes at 4° C. using a Turbo 15 (100K MWCO, PES, Sartorius). The resulting solution was used as a rAAV virion solution.

[0175] [Example 35] Measurement of rAAV genome amount and total capsid amount contained in rAAV virion solution The viral genome content in the rAAV virion solution obtained in Example 34 was measured by real-time PCR using the AAVpro Titration Kit for Real Time PCR Ver.2 (Takara Bio Inc.) in accordance with the protocol attached to the kit, generally in the following manner. 2 μL of 10×DNaseI Buffer, 1 μL of DNaseI, and 15 μL of pure water were added to 2 μL of the rAAV virion solution prepared in Example 34, mixed, and incubated at 37°C for 30 minutes. DNaseI was inactivated by heat treatment at 95°C for 10 minutes, and then 20 μL of Lysis Buffer was added and heat treatment was performed at 70°C for 10 minutes. The solution after heat treatment was diluted 50-fold with EASY Dilution (Takara Bio Inc.) to prepare a sample solution for real-time PCR. The sample solution for real-time PCR and 2×10 2 ~2×10 712.5 μL of TB Green Premix Ex TaqII, 0.6 μL of 10 μM forward primer (primer 1, sequence number 33), 0.6 μL of 10 μM reverse primer (primer 2, sequence number 34), 0.08 μL of ROX Reference Dye II, and 6.5 μL of water were added to 5 μL of each of the standard curve kit control solutions diluted with EASY Dilution (Takara Bio) to a concentration of genome copy number / μL. The real-time PCR reaction was performed under denaturing conditions (95°C, 2 minutes) followed by 35 cycles of two-step PCR (95°C, 5 seconds → 60°C, 30 seconds). The measured values ​​of the sample were interpolated into the obtained standard curve to determine the amount (number) of rAAV genomes contained in the sample. The region replicated in this PCR is the internal region of the ITR.

[0176] In addition to the above real-time PCR method, the viral genome content in the rAAV virion solution was also measured by droplet digital PCR. 1 μL of DNase I (Takara Bio), 2 μL of 10× DNase I Buffer (Takara Bio), and 15 μL of water containing 0.05% F-68 were added to 2 μL of the rAAV virion solution, and incubated at 37°C for 30 minutes to digest the DNA outside the rAAV virion.

[0177] The rAAV virion solution treated with DNaseI was appropriately diluted with TE buffer containing 0.05% F-68 to prepare a sample solution for droplet digital PCR. A FAM-labeled 20x primer / probe mix was prepared containing 1.0 μM forward primer (primer 3, sequence number 41), 1.0 μM reverse primer (primer 4, sequence number 42), and 0.25 μM probe (probe 1, sequence number 43 modified with FAM as a reporter dye at the 5' end and BHQ1 as a quencher dye at the 3' end). A HEX-labeled 20x primer / probe mix was also prepared containing 1.0 μM forward primer (primer 5, sequence number 44), 1.0 μM reverse primer (primer 6, sequence number 45), and 0.25 μM probe (probe 2, sequence number 46 modified with HEX as a reporter dye at the 5' end and BHQ1 as a quencher dye at the 3' end).

[0178] A PCR reaction solution was prepared by adding 10 μL of ddPCR Supermix for probes (no dUTP) (BioRad), 1 μL of FAM-labeled 20× primer / probe mix, 1 μL of HEX-labeled 20× primer / probe mix, 2 μL of 5M betaine solution (Sigma), and 5 μL of water containing 0.05% F-68 to 1 μL of droplet digital PCR sample solution. A suspension (droplet) of 20 μL of PCR reaction solution and 70 μL of Droplet Generator Oil for Probes (BioRad) was prepared using a droplet generator (BioRad). PCR reaction was performed using this droplet. The PCR reaction was performed under denaturing conditions (95°C, 10 minutes), followed by 40 cycles of 3-step PCR (95°C, 30 seconds → 60°C, 1 minute → 72°C, 15 seconds), followed by inactivation of the PCR enzyme (98°C, 10 minutes). The droplets were analyzed using the QX200 Droplet Digital PCR System (BioRad Laboratories), and the amount (number) of rAAV genome was determined based on the FAM / HEX double positive droplets. The regions replicated in this PCR were the bovine growth hormone polyA and the internal region of the ITR.

[0179] The total amount of capsids in the culture supernatant was measured using an AAV Titration ELISA Kit (PROGEN) according to the protocol attached to the kit, roughly as follows: 100 μL of the rAAV virion solution prepared in Example 34, diluted with 1× Assay Buffer, was added to a plate on which mouse anti-AAV9 monoclonal antibody was immobilized, and the plate was allowed to stand at 37°C for 1 hour. 7 ~1.3×10 9A known amount of AAV9 empty capsid diluted with 1x Assay Buffer to a concentration of capsids / mL was added to the plate as a standard solution in the same manner as the sample. After washing the plate three times with 1x Assay Buffer, a biotin-labeled mouse anti-AAV9 antibody was added and left to stand at 37°C for 1 hour. After washing the plate three times with 1x Assay Buffer, HRP-labeled streptavidin was added and left to stand at 37°C for 1 hour. After washing the plate three times with 1x Assay Buffer, HRP substrate was added and left to stand at room temperature for 15 minutes, and then sulfuric acid was added to stop the reaction. The measured values ​​of the sample were interpolated into the calibration curve obtained from the measured values ​​of the absorbance of the standard solution to determine the total amount (number) of capsids contained in the sample.

[0180] [Example 36] Results (measurement of rAAV genome amount and total capsid amount contained in rAAV virion solution) The measurement results of the rAAV genome amount contained in the rAAV virion solution prepared in Example 34 are shown in Table 1.

[0181] [Table 1]

[0182] Theoretically, if there are no empty capsids and all capsids are rAAV virions containing the rAAV genome, the ratio of the amount of rAAV genome to the amount of total capsids is 1:1. Assuming that all rAAV genomes are packaged into capsids to form virions, the amount of rAAV genome (vg) was taken as the amount (number) of rAAV virions. The amount of rAAV genome determined by real-time PCR was used to calculate the ratio of the amount of rAAV virions in the total capsids, which was 12.7% when three types of plasmids were used and 62.2% when the integrated rAAV plasmid was used. That is, it was verified that the use of the integrated rAAV plasmid can increase the ratio of rAAV virions in the total capsids, as in the case of the pHelper-ITR / CMV / GFP-R2(mod)C6 vector and the pHelper-ITR / CMV / GFP-R2(mod)C9 vector. In addition, if the amount of rAAV virions (vg) is the amount (number) of rAAV virions, the use of the integrated rAAV plasmid can produce 8 times more rAAV virions than the use of three types of plasmids, so the use of the integrated rAAV plasmid for the production of rAAV virions can significantly increase the production efficiency of rAAV virions. The same conclusion can be obtained from the results of quantifying the amount of rAAV genome by the auxiliary droplet digital PCR method. [Industrial Applicability]

[0183] According to one embodiment of the present invention, rAAV virions packaged with a nucleic acid sequence including a base sequence encoding a foreign protein that can be used for gene therapy, etc. can be efficiently produced, and such rAAV virions can be steadily supplied to medical institutions. [Sequence List Free Text]

[0184] SEQ ID NO: 1: Nucleotide sequence encoding the Rep protein of AAV serotype 2, wild type SEQ ID NO:2: Amino acid sequence of Rep68 of AAV serotype 2, wild type SEQ ID NO: 3: Amino acid sequence of Rep78 of AAV serotype 2, wild type SEQ ID NO: 4: A preferred example of a base sequence encoding the Rep protein of AAV serotype 2, a synthetic sequence SEQ ID NO: 5: Nucleotide sequence encoding VP1 of AAV serotype 6, wild type SEQ ID NO: 6: Amino acid sequence of VP1 of AAV serotype 6, wild type SEQ ID NO: 7: Nucleotide sequence encoding VP1 of AAV serotype 9, wild type SEQ ID NO: 8: Amino acid sequence of VP1 of AAV serotype 9, wild type SEQ ID NO: 9: Nucleotide sequence of the first inverted terminal repeat (first ITR) of AAV serotype 2, wild type SEQ ID NO: 10: Nucleotide sequence of the first inverted terminal repeat (second ITR) of AAV serotype 2, wild type SEQ ID NO: 11: A preferred example of the base sequence of the first inverted terminal repeat (first ITR), synthetic sequence SEQ ID NO: 12: A preferred example of the base sequence of the second inverted terminal repeat (second ITR), synthetic sequence SEQ ID NO: 15: A suitable example of the base sequence of the E4 region, a synthetic sequence SEQ ID NO: 17: A suitable example of the base sequence of the helper region, a synthetic sequence SEQ ID NO: 19: A preferred example of a base sequence contained in the first gene expression control section, a synthetic sequence SEQ ID NO: 20: A suitable example of the base sequence of the Rep region, a synthetic sequence SEQ ID NO: 21: A suitable example of the base sequence of the Cap region, a synthetic sequence SEQ ID NO: 22: A suitable example of the base sequence of the Rep-Cap region, a synthetic sequence SEQ ID NO: 23: A preferred example of the base sequence of the Rep-Cap region (2), synthetic sequence SEQ ID NO: 24: A preferred example of a base sequence for a nucleic acid molecule used to generate recombinant AAV virions, a synthetic sequence SEQ ID NO: 25: Synthetic human β-globin intron base sequence, synthetic sequence SEQ ID NO: 26: Nucleotide sequence of synthetic polyadenylation signal sequence, synthetic sequence SEQ ID NO: 27: Base sequence including SalI site, RsrII site, ColE1 ori, ampicillin resistance gene, BsiWI site, BstZ17I site, and BsrGI site, synthetic sequence SEQ ID NO: 28: Base sequence including EcoRI site, MluI site, GFP gene, NotI site, and BamHI site, synthetic sequence SEQ ID NO: 29: Nucleotide sequence of multicloning site 1, synthetic sequence SEQ ID NO: 30: Nucleotide sequence of multicloning site 2, synthetic sequence SEQ ID NO: 31: A base sequence including an AfeI site, an RsrII site, a BsrGI site, an AvrII site, an ampicillin resistance gene, a ColE1 ori, an RsrII site, a 5' portion of the Rep region of serotype 2 (including the p5 promoter), and a SacII site, a synthetic sequence SEQ ID NO: 32: A base sequence including a HindIII site, the 3' portion of the AAV Rep region of serotype 2, the Cap region of serotype 9, the p5 promoter, the RsrII site, and the BsrGI site, and a synthetic sequence SEQ ID NO: 33: forward primer, base sequence of primer 1, synthetic sequence SEQ ID NO: 34: Reverse primer, base sequence of primer 2, synthetic sequence SEQ ID NO: 35: A base sequence including a human CMV enhancer, a chicken β-actin promoter, a chicken β-actin / MVM chimeric intron, a human I2S gene, and a bovine growth hormone polyA sequence, a synthetic sequence SEQ ID NO: 36: Amino acid sequence of human IDS SEQ ID NO: 37: Nucleotide sequence of human CMV enhancer SEQ ID NO: 38: Nucleotide sequence of chicken β-actin promoter SEQ ID NO: 39: Base sequence of chicken β-actin / MVM chimeric intron, synthetic sequence SEQ ID NO: 40: Nucleotide sequence of bovine growth hormone polyA sequence SEQ ID NO: 41: Base sequence of primer 3, synthetic sequence SEQ ID NO: 42: Base sequence of primer 4, synthetic sequence SEQ ID NO: 43: Base sequence of probe 1, synthetic sequence SEQ ID NO: 44: Base sequence of primer 5, synthetic sequence SEQ ID NO: 45: Base sequence of primer 6, synthetic sequence SEQ ID NO: 46: Base sequence of probe 2, synthetic sequence SEQ ID NO: 47: A base sequence containing a human CMV enhancer and a chicken β-actin promoter, a synthetic sequence SEQ ID NO: 48: Base sequence of primer 7, synthetic sequence SEQ ID NO: 49: Base sequence of primer 8, synthetic sequence SEQ ID NO: 50: A base sequence containing a human CMV enhancer, a chicken β-actin promoter, a chicken β-actin / MVM chimeric intron, and a bovine growth hormone polyA sequence, a synthetic sequence SEQ ID NO: 51: Base sequence containing human IDS gene, synthetic sequence

Claims

1. A nucleic acid molecule comprising at least the following base sequence: A base sequence including, from upstream to downstream, a base sequence including a P5 promoter of the adeno-associated virus, a base sequence encoding a Rep protein of adeno-associated virus serotype 2, a base sequence including a P40 promoter of the adeno-associated virus, a base sequence encoding a Cap protein of adeno-associated virus serotype 9, a base sequence having an enhancer function for increasing the expression levels of the Rep protein and the Cap protein, a base sequence including an inverted terminal repeat (first ITR) of a first adeno-associated virus, a base sequence including a gene expression control site for controlling the expression of a foreign protein, a base sequence encoding the foreign protein, and a base sequence including an inverted terminal repeat (second ITR) of a second adeno-associated virus, in this order; and A base sequence including, from upstream to downstream, a base sequence including an adenovirus E2A region, a base sequence including an adenovirus E4 region, and a base sequence including an adenovirus VA1 RNA region.

2. The nucleic acid molecule of claim 1, further comprising a base sequence comprising a drug resistance gene.

3. The nucleic acid molecule of claim 2, which comprises a base sequence containing a drug resistance gene downstream of the base sequence encoding the Cap protein and upstream of the base sequence containing the first ITR.

4. The nucleic acid molecule of any one of claims 1 to 3, wherein the Rep protein comprises Rep68 or / and Rep78.

5. The nucleic acid molecule of any one of claims 1 to 4, wherein the Cap protein comprises VP1.

6. The nucleic acid molecule of any one of claims 1 to 5, wherein the adeno-associated virus serotype of said first ITR and said second ITR is 2.

7. The nucleic acid molecule of any one of claims 2 to 6, wherein the drug resistance gene confers resistance to a drug corresponding to the drug resistance gene to a prokaryotic cell.

8. the gene expression control site is selected from the group consisting of a promoter derived from a cytomegalovirus, an SV40 early promoter, a human elongation factor-1 alpha (EF-1α) promoter, a human ubiquitin C promoter, a β-actin promoter, a promoter comprising the base sequence shown in SEQ ID NO:47, and a promoter comprising the base sequence shown in SEQ ID NO:39 downstream of the promoter comprising the base sequence shown in SEQ ID NO:47, A nucleic acid molecule according to any one of claims 1 to 7.

9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the base sequence having an enhancer function is the P5 promoter of an adeno-associated virus or a functional equivalent thereof.

10. The nucleic acid molecule of any one of claims 1 to 9, wherein the foreign protein is human.

11. 10. The nucleic acid molecule of any one of claims 1 to 9, wherein the foreign protein is selected from the group consisting of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody.

12. The foreign protein may be growth hormone, lysosomal enzyme, somatomedin, insulin, glucagon, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of antibody and other protein, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle stimulating hormone (FSH), gonadotropin releasing hormone (GnRH), gonadotropin, DNase, thyroid stimulating hormone (TSH), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF). , glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), an enzyme having activity of degrading beta-amyloid, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and GLP-1 receptor agonist, A nucleic acid molecule according to any one of claims 1 to 10.

13. The foreign protein is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2, A nucleic acid molecule according to any one of claims 1 to 10.

14. The nucleic acid molecule of any one of claims 1 to 11, wherein the foreign protein is selected from the group consisting of an anti-IL-6 antibody, an anti-beta amyloid antibody, an anti-BACE antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-HER2 antibody, an anti-PCSK9 antibody, an anti-TNF-α antibody, and an antibody having affinity for a protein present on the surface of a vascular endothelial cell.

15. The nucleic acid molecule of claim 14, wherein the protein present on the surface of the vascular endothelial cell is selected from the group consisting of transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.

16. the foreign protein is a fusion protein of an antibody and another protein, The antibody is selected from the group consisting of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody; and The other proteins are growth hormone, lysosomal enzymes, cytokines, lymphokines, blood coagulation factors, antibodies, fusion proteins of antibodies with other proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone, DNase1, thyroid-stimulating hormone (TSH), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cells, a cellular neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), and an enzyme having an activity of degrading beta amyloid, A nucleic acid molecule according to any one of claims 1 to 9.

17. the foreign protein is a fusion protein of an antibody and another protein, The antibody is selected from the group consisting of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody; and The other protein is a lysosomal enzyme, and the lysosomal enzyme is α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, CLN1, and CLN2, A nucleic acid molecule according to any one of claims 1 to 9.

18. 18. The nucleic acid molecule of claim 16 or 17, wherein the other protein is human.

19. The antibody has affinity for a protein present on the surface of a vascular endothelial cell. A nucleic acid molecule according to any one of claims 16 to 18.

20. The nucleic acid molecule of claim 19, wherein the protein present on the surface of the vascular endothelial cell is selected from the group consisting of transferrin receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transporter 1, organic anion transporter, monocarboxylate transporter, low-density lipoprotein receptor-related protein 1, low-density lipoprotein receptor-related protein 8, and membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.

21. 21. The nucleic acid molecule of any one of claims 1 to 20, which is a circular plasmid.

22. A method for producing a recombinant AAV particle, comprising the step of introducing a nucleic acid molecule according to any one of claims 1 to 21 into a host cell.

23. The method of claim 22, wherein the genome of the host cell comprises a base sequence including the E1A and E1B regions of an adenovirus.

24. The method of claim 23, wherein the host cell is a HEK293 cell.

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