JP2020188763A - Nucleic acid molecule used for producing recombinant aav virion - Google Patents

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

The present invention relates to the field of recombinant adeno-associated virus (rAAV) virions that can be used for gene therapy in one embodiment thereof, and more specifically, uses nucleic acid molecules that can be used to produce rAAV virions and the nucleic acid molecules. Regarding the manufacturing method of the rAAV virion that was there.

Adeno-associated virus (AAV) belongs to the Parvovirus family, which is the smallest class of linear single-stranded DNA viruses in nature, and its viral genome is about 4.7 kb. AAV has no envelope and forms icosahedron particles with a diameter of about 22 nm. AAV is also classified into the dependent virus genus, which is a deficient virus that cannot propagate by single infection, and is co-infected with a helper virus that assists AAV replication in order for efficient replication in animal cells. is required. Helper viruses that assist in AAV replication include, for example, adenovirus or herpesvirus. In the absence of helper virus, no replication of AAV is observed and the AAV genome is integrated into the host genome.

When wild-type AAV alone infects host human cells, the viral genome is site-specifically integrated into chromosome 19 via reverse terminal repeats (ITRs) present at both ends of the viral genome. The genes of the viral genome incorporated into the host cell genome are rarely expressed, but when the cell is infected with a helper virus, AAV is excised from the host genome and replication of the infectious virus is initiated. When the helper virus is adenovirus, the genes responsible for the helper action are E1A, E1B, E2A, VA1 and E4. Here, in the case where the host cell is HEK293 cell, which is a cell derived from human fetal kidney tissue transformed by adenovirus E1A and E1B, the E1A and E1B genes are originally expressed in the host cell.

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 intervene in the integration of the viral genome into chromosomes. The cap gene is responsible for the production of three capsid proteins (VP1, VP2 and VP3).

In the wild-type AAV genome, ITRs are present at both ends, and the rep gene and cap gene are present between the ITRs. Recombinant AAV virions (rAAV virions) are capsids in which the region containing the rep gene and cap gene is replaced with a gene encoding a foreign protein.

rAAV virions are used in gene therapy as vectors to deliver exogenous genes to cells and have the ability to provide treatment options for previously incurable diseases.

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

International Publication No. 95/34670 International Publication No. 97/06272 International Publication No. 97/17458

Maria S. et al., HUMAN GENE THERAPY METHODS, 28, 101-108 (2017)

An object of the present invention is to efficiently produce rAAV virions in which a nucleic acid sequence containing a base sequence encoding a foreign protein, which can be used for gene therapy or the like, is packaged in an AAV capsid in one embodiment thereof. The present invention relates to a nucleic acid molecule and a method for producing such rAAV virion.

In the research aimed at the above objectives, as a result of diligent studies, the present inventors have found that the nucleotide sequence encoding the Rep protein of the adeno-associated virus, the nucleotide sequence encoding the Cap protein of the adeno-associated virus, and the first adeno-associated virus. Includes a base sequence containing the reverse terminal repeat (ITR) of the virus, a base sequence containing the reverse terminal repeat (ITR) of the second adeno-associated virus, a base sequence containing the E2A region of the adenovirus, and the E4 region of the adenovirus. A nucleic acid molecule containing a base sequence, a base sequence containing the VA1 RNA region of adenovirus, and a base sequence encoding a foreign protein between the first and second ITRs of the nucleic acid molecule is introduced into a host cell. As a result, we found that rAAV virions could be produced efficiently, and completed the present invention. That is, the present invention includes the following.
1. 1. Nucleic acid molecule containing at least the following base sequence;
(A) Nucleotide sequence encoding the Rep protein of adeno-associated virus or its functional equivalent,
(B) Nucleotide sequence encoding cap protein of adeno-associated virus or its functional equivalent,
(C) Nucleotide sequence containing the reverse terminal repeat (ITR) of the first adeno-associated virus or its functional equivalent,
(D) Nucleotide sequence containing the reverse terminal repeat (ITR) of the second adeno-associated virus or its functional equivalent,
(E) A base sequence for inserting a base sequence encoding a foreign protein and / and a base sequence encoding a foreign protein located between the first ITR and the second ITR,
(F) Nucleotide sequence containing the E2A region of adenovirus or its functional equivalent,
(G) A base sequence containing the E4 region of adenovirus or its functional equivalent, and (h) a base sequence containing the VA1 RNA region of adenovirus or its functional equivalent.
2. 2. A base sequence containing a first gene expression control site that controls the expression of the Rep protein,
The nucleic acid of 1 above, further comprising a base sequence containing a second gene expression control site that controls the expression of the Cap protein, and a base sequence containing a third gene expression control site that controls the expression of the foreign protein. molecule.
3. 3. The nucleic acid molecule of 1 or 2 above, which further comprises a base sequence containing a drug resistance gene.
4. The base sequence encoding the Rep protein is encoded downstream of the base sequence containing the first gene expression control site, the base sequence containing the second gene expression control site is further downstream, and the Cap protein is further downstream. The nucleic acid molecule of the above 2 or 3 which contains the base sequence.
5. A base for inserting a base sequence containing the third gene expression control site downstream of the base sequence containing the first reverse terminal repeat (ITR), and a base sequence encoding the foreign protein further downstream. The nucleic acid molecule according to any one of 2 to 4 above, which comprises a sequence and / and a base sequence encoding the foreign protein, and a base sequence further downstream containing the second reverse terminal repeat (ITR).
6. Downstream of the base sequence of the E2A region of the adenovirus or its functional equivalent, the base sequence of the E4 region of the adenovirus or its functional equivalent, and further downstream of the base sequence of the adenovirus VA1 RNA region or its functional equivalent The nucleic acid molecule according to any one of 2 to 5 above, which comprises the base sequence of.
7. Downstream of the base sequence encoding the Cap protein is a base sequence containing the first reverse terminal repeat (ITR), further downstream is a base sequence containing the third gene expression control site, and further downstream is the foreign substance. It contains a base sequence for inserting a base sequence encoding a protein and / and a base sequence encoding the foreign protein, and a base sequence further downstream containing the second reverse terminal repeat (ITR). 4 nucleic acid molecules.
8. Downstream of the base sequence containing the second reverse terminal repeat (ITR) is the base sequence of the E2A region of the adenovirus or its functional equivalent, and further downstream is the E4 region of the adenovirus or its functional equivalent. The nucleic acid molecule of 7 above, which further contains the nucleotide sequence of the adenovirus VA1 RNA region or its functional equivalent.
9. The nucleic acid molecule according to any one of 1 to 8 above, which further comprises a base sequence containing a drug resistance gene.
10. 9. The nucleic acid molecule according to 9 above, 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.
11. The nucleic acid molecule according to any one of 1 to 10 above, which contains a base sequence having an enhancer function for increasing the expression level of Rep protein and Cap protein downstream of the base sequence encoding the Cap protein.
12. 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, any of the above 1 to 11. Nucleic acid molecule.
13. The above 12 nucleic acid molecules, wherein the Rep protein comprises Rep68 and / and Rep78.
14. 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, any of the above 1 to 13. Nucleic acid molecule.
15. The above 14 nucleic acid molecules, wherein the Cap protein contains VP1.
16. The nucleic acid molecule according to any one of 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 above 16 nucleic acid molecules whose first gene expression control site is the p5 promoter of adeno-associated virus.
18. The nucleic acid molecule according to any one of 2 to 17 above, wherein the second gene expression control site is controlled by VA1 RNA of adenovirus or a functional equivalent thereof.
19. The above 18 nucleic acid molecules whose second gene expression control site is the p40 promoter of adeno-associated virus.
20. 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. A nucleic acid molecule of any of 19 to 19.
21. The nucleic acid molecule according to any one of 1 to 19 above, wherein the adeno-associated virus serotype of the ITR is 2.
22. The 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. The serotypes of the adenovirus 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 to 30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdHu49, AdHu50, AdC6, The nucleic acid molecule according to any one of 1 to 22 above, which is selected from the group consisting of serotype, dog Ad2 type, sheep Ad, and porcine Ad3 type.
24. The functional equivalent of the E2A region of the adenovirus is derived from any of the groups selected from the group consisting of herpes simplex virus, vaccinia virus, cytomegalovirus, and pseudorabies virus, any of the above 1 to 23. The nucleic acid molecule.
25. Any of the above 1 to 24, wherein the functional equivalent of the E4 region of the adenovirus is derived from any of the groups selected from the group consisting of herpes simplex virus, vaccinia virus, cytomegalovirus, and pseudorabies. Nucleic acid molecule.
26. The functional equivalent of the VA1 RNA region of the adenovirus is derived from one selected from the group consisting of herpes simplex virus, vaccinia virus, cytomegalovirus, and pseudorabies virus, 1 to 25 above. Any nucleic acid molecule.
27. The nucleic acid molecule of any of 2 to 26 above, wherein the E2A region of the adenovirus is regulated by E1B of the adenovirus or its functional equivalent.
28. The nucleic acid molecule of any of 2 to 27 above, wherein the E4 region of the adenovirus is regulated by E1B of the adenovirus or its functional equivalent.
29. The nucleic acid molecule according to any one of 9 to 28 above, wherein the drug resistance gene imparts resistance to a drug corresponding to the drug resistance gene to prokaryotic cells.
30. The third gene expression control site is indicated by a promoter derived from cytomegalovirus, an SV40 early promoter, a human elongation factor-1alpha (EF-1α) promoter, a human ubiquitin C promoter, and a β-actin promoter, SEQ ID NO: 47. Any of the above 2 to 29, which is selected from the group consisting of a promoter containing the base sequence shown in the above, and a promoter containing the base sequence shown in SEQ ID NO: 47 downstream of the promoter containing the base sequence shown in SEQ ID NO: 47. That nucleic acid molecule.
31. The nucleic acid molecule according to any one of 11 to 30 above, wherein the base sequence having the enhancer function is the p5 promoter of adeno-associated virus or a functional equivalent thereof.
32. The nucleic acid molecule according to any one of 1 to 31 above, wherein the foreign protein is of human origin.
33. The nucleic acid molecule according to any one of 1 to 3 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 proteins are growth hormone, lysosome enzyme, somatomedin, insulin, glucagon, lysosome enzyme, cytokine, phosphokine, blood coagulation factor, antibody, fusion protein of antibody and other proteins, granulocyte macrophage colony stimulator (GM- CSF), granulocyte colony stimulator (G-CSF), macrophage colony stimulator (M-CSF), erythropoetin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicular stimulator (FSH), gonads Stimulant hormone releasing hormone (GnRH), gonadotropin, DNasel, thyroid stimulating hormone (TSH), nerve growth factor (NGF), hairy neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3 , Neurotrophin 4/5, Neurotrophin 6, Neurotrophin 6, Neurogulin 1, Activin, Basic fibroblast growth factor (bFGF), Fibroblast growth factor 2 (FGF2), Epithelial cell growth factor (EGF), Vascular endothelium Has the activity of degrading growth factors (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), beta amyloid. The nucleic acid molecule of any of 1-32 above, which is selected from the group consisting of enzymes, etanercepts, pegbisomants, metrereptin, avatacepts, ashotases, and GLP-1 receptor agonists.
35. The foreign protein is a lysosomal enzyme, and the lysosomal enzyme is α-L-izronidase, isulonic acid-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activated protein, β-hexosaminidase A, β. -Hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartyl glucosaminidase, α-N- Acetylgalactosaminidase, acidic sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetylCoAα-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, Any of the above 1-32, which is selected from the group consisting of acidic ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, trypeptidylpeptidase-1, hyaluronidase-1, CLN1 and CLN2. The nucleic acid molecule.
36. The foreign proteins are anti-IL-6 antibody, anti-beta amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-HER2 antibody, anti-PCSK9 antibody, anti-TNF-α antibody. , And any of the above 1-33 nucleic acid molecules selected from the group consisting of antibodies having an affinity for proteins present on the surface of vascular endothelial cells.
37. The proteins present on the surface of the vascular endothelial cells are transferase receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transport carrier 1, A group consisting of organic anion transporters, monocarboxylic acid transporters, low-density lipoprotein receptor-related proteins 1, low-density lipoprotein receptor-related proteins 8, and membrane-bound precursors of heparin-binding epithelial growth factor-like growth factors. 36 nucleic acid molecules above, which are selected from.
38. The foreign protein is a fusion protein of an antibody and another protein, and the antibody is selected from the group consisting of mouse antibody, humanized antibody, human-mouse chimeric antibody, and human antibody, and , The other proteins are growth hormone, lysosome enzyme, cytokine, neurophocaine, blood coagulation factor, antibody, fusion protein of antibody and other protein, granulocyte macrophage colony stimulator (GM-CSF), granulocyte colony stimulator (G-CSF), macrophage colony stimulator (M-CSF), erythropoetin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicular stimulator, DNasel, thyroid stimulator (TSH), neurogrowth factor (NGF), Hairy Neurotrophic Factor (CNTF), Glia Cellline Neurotrophic Factor (GDNF), Neurotrophin 3, Neurotrophin 4/5, Neurotrophin 6, Neurotrophin 1, Actibin, Basic Fiber Sprout growth factor (bFGF), fibroblast growth factor 2 (FGF2), epithelial cell growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1 , PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), and an enzyme having an activity of degrading beta-amyloid, which is selected from the group consisting of any of the above 1-31 nucleic acids. molecule.
39. The foreign protein is a fusion protein of an antibody and another protein, and the antibody is selected from the group consisting of mouse antibody, humanized antibody, human-mouse chimeric antibody, and human antibody, and The other protein is a lysosome enzyme, and the lysosome enzyme is α-L-isronidase, isulonic acid-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activated 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-acetyl Galactosaminidase, acidic sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetylCoAα-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acidic Any of the above 1-31 selected from the group consisting of ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, trypeptidylpeptidase-1, hyaluronidase-1, CLN1 and CLN2. Nucleic acid molecule.
40. The nucleic acid molecule of 38 or 39 above, wherein the other protein is of human origin.
41. The nucleic acid molecule according to any one of 38 to 40 above, wherein the antibody has an affinity for a protein present on the surface of vascular endothelial cells.
42. The proteins present on the surface of the vascular endothelial cells are transferase receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transport carrier 1, A group consisting of organic anion transporters, monocarboxylic acid transporters, low-density lipoprotein receptor-related proteins 1, low-density lipoprotein receptor-related proteins 8, and membrane-bound precursors of heparin-binding epithelial growth factor-like growth factors. The 41 nucleic acid molecules described above, which are selected from.
43. The nucleic acid molecule according to any one of 1 to 42 above, which is a cyclic plasmid.
44. A method for producing a recombinant AAV virion, which comprises a step of introducing any of the above 1 to 43 nucleic acid molecules into a host cell.
45. 44. The production method according to 44 above, wherein the genome of the host cell contains a base sequence containing an E1A region and an E1B region of adenovirus.
46. The production method according to the above 45, wherein the host cell is a HEK293 cell.

According to one embodiment of the present invention, rAAV virions in which a nucleic acid sequence containing a base sequence encoding a foreign protein, which can be used for gene therapy or the like, is packaged can be efficiently produced, so that rAAV virions can be inexpensively produced. It will be possible to supply it, and the medical expenses required for gene therapy can be reduced.

Schematic diagram showing the structure of the pHelper (mod) vector. Schematic diagram showing the structure of the pAAV-CMV-GFP (mod) vector. Schematic diagram showing the structure of the pR2 (mod) C6 vector. Schematic diagram showing the structure of the pR2 (mod) C9 vector. Schematic diagram showing the structure of the pHelper-ITR / CMV / GFP vector. Schematic diagram showing the structure of the pHelper-R2 (mod) C6 vector. Schematic diagram showing the structure of the pHelper-R2 (mod) C9 vector. Schematic diagram showing the structure of the pR2 (mod) C6-ITR / CMV / GFP vector. Schematic diagram showing the structure of the pR2 (mod) C9-ITR / CMV / GFP vector. Schematic diagram showing the structure of the pHelper-ITR / CMV / GFP-R2 (mod) C6 vector. Schematic diagram showing the structure of the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector. FIG. 12 is a diagram showing the results of measurement of the amount of rAAV genome and the total amount of capsid contained in the culture supernatant obtained in Example 16. The vertical axis shows the amount of rAAV genome (x10 ¹¹ ) or the total amount of capsid (x10 ¹¹ ). The black bar indicates the amount of rAAV genome, and the white bar indicates the total amount of capsid. From left to right, (1): when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2 (mod) C9 vector, are introduced, (2): pHelper-R2 (mod) C9 vector and pAAV-CMV- When two types of GFP vector plasmids are introduced, (3): pR2 (mod) C9-ITR / CMV / When two types of vectors, GFP vector and pHelper vector, are introduced (4): pHelper-ITR / CMV Measurement results for each of the / GFP-R2 (mod) C9 vector introduced and (5): pHelper vector, pAAV-CMV-GFP vector and pR2 (mod) C9 vector introduced. Is shown. The measurement results are shown only for rAAV9. FIG. 13 is a diagram showing the results of measurement of the amount of rAAV genome and the total amount of capsid contained in the purified cell lysate solution obtained in Example 18. The vertical axis shows the amount of rAAV genome (x10 ¹¹ ) or the total amount of capsid (x10 ¹¹ ). The black bar indicates the amount of rAAV genome, and the white bar indicates the total amount of capsid. From left to right, (1): when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2 (mod) C9 vector, are introduced, (2): pHelper-R2 (mod) C9 vector and pAAV-CMV- When two types of GFP vector plasmids are introduced, (3): pR2 (mod) C9-ITR / CMV / When two types of vectors, GFP vector and pHelper vector, are introduced (4): pHelper-ITR / CMV Measurement results for each of the introduced / GFP-R2 (mod) C9 vector and (5): pHelper vector, pAAV-CMV-GFP vector, and pR2 (mod) C9 vector introduced. Shown. The measurement results are shown only for rAAV9. FIG. 14 is a diagram showing the result of calculation of the abundance ratio of the rAAV virion obtained in Example 19 in the total capsid. The vertical axis shows the abundance ratio (%). From left to right, (1): when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2 (mod) C9 vector, are introduced, (2): pHelper-R2 (mod) C9 vector and pAAV-CMV- When two types of GFP vector plasmids are introduced, (3): pR2 (mod) C9-ITR / CMV / When two types of vectors, GFP vector and pHelper vector, are introduced (4): pHelper-ITR / CMV Values for each of the introduced / GFP-R2 (mod) C9 vector and (5): pHelper vector, pAAV-CMV-GFP vector and pR2 (mod) C9 vector introduced. Shown. The measurement results are shown only for rAAV9. FIG. 15 is a diagram showing the results of infectivity measurement of rAAV6 virion purified from the cell lysate solution obtained in Example 20. The vertical axis shows the proportion of GFP-positive cells, that is, the infection efficiency (%) of rAAV6 virions. From left to right, (1): When two types of plasmids, pHelper-ITR / CMV / GFP vector and pRC6 vector, are introduced, (2): pHelper-R2 (mod) C6 vector and pAAV-CMV-GFP vector 2 When two types of plasmids are introduced (3): pR2 (mod) C6-ITR / CMV / GFP vector and two types of plasmids, pHelper vector, (4): pHelper-ITR / CMV / GFP-R2 The black bar was infected per well when (mod) C6 vector was introduced and (5): when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector and pRC6 vector, were introduced. When the amount of rAAV genome is 2x10 ⁸ vg (that is, the amount of rAAV virion is 2x10 ⁸ ), the white bar is the case where the amount of infected rAAV genome is 2x10 ⁹ vg (that is, the amount of rAAV virion is 2x10 ⁹ ) Shown. FIG. 16 is a diagram showing the results of infectivity measurement of rAAV9 virion purified from the cell lysate solution obtained in Example 21. The vertical axis shows the proportion of GFP-positive cells, that is, the infection efficiency (%) of rAAV9 virions. From left to right, when two types of plasmids (1): pHelper-ITR / CMV / GFP vector and pR2 (mod) C9 vector are introduced, (2): pHelper-R2 (mod) C9 vector and pAAV-CMV- When two types of plasmids of GFP vector are introduced, (3): When two types of plasmids, pR2 (mod) C9-ITR / CMV / GFP vector and pHelper vector, are introduced, (4): Integrated rAAV plasmid (pHelper) -ITR / CMV / GFP-R2 (mod) C9 vector) is introduced, and (5): pHelper vector, pAAV-CMV-GFP vector and pR2 (mod) C9 vector are introduced. For each, the black bar shows the amount of rAAV genome infected per well at 2x10 ⁹ vg (that is, the amount of rAAV virion is 2x10 ⁹ ), and the white bar shows the amount of rAAV genome infected per well at 1x10 ¹⁰ vg. (That is, the amount of rAAV virion is 2x10 ¹⁰ pieces). FIG. 17 is a diagram showing the calculation result of the Transducing Unit of the rAAV6 virion obtained in Example 22. The vertical axis shows the value of Transducing Unit (x10 ⁵ ). From left to right, (1): When two types of plasmids, pHelper-ITR / CMV / GFP vector and pRC6 vector, are introduced, (2): pHelper-R2 (mod) C6 vector and pAAV-CMV-GFP vector 2 When two types of plasmids are introduced, (3): pR2 (mod) C6-ITR / CMV / GFP vector and pHelper vector, (4): Integrated rAAV plasmid (pHelper-ITR / CMV) / GFP-R2 (mod) C6 vector) is introduced, and (5): The value of Transducing Unit is calculated for each of the three types of plasmids, pHelper vector, pAAV-CMV-GFP vector and pRC6 vector. Shown. FIG. 18 is a diagram showing the calculation result of the Transducing Unit of the rAAV9 virion obtained in Example 22. The vertical axis shows the value of Transducing Unit (x10 ⁵ ). From left to right, (1): when two types of plasmids, pHelper-ITR / CMV / GFP vector and pR2 (mod) C9 vector, are introduced, (2): pHelper-R2 (mod) C9 vector and pAAV-CMV- When two types of GFP vector are introduced, (3): When two types of plasmids, pR2 (mod) C9-ITR / CMV / GFP vector and pHelper vector, are introduced, (4): Integrated rAAV plasmid (pHelper) -ITR / CMV / GFP-R2 (mod) C9 vector) is introduced, and (5): pHelper vector, pAAV-CMV-GFP vector and pR2 (mod) C9 vector are introduced. The value of the Transducing Unit is shown for each. Schematic diagram showing the structure of the pAAV-CBA (BAM) -I2S vector. Schematic diagram showing the structure of the pHelper-ITR / CBA (BAM) / I2S-R2 (mod) C9 vector.

The production of recombinant adeno-associated virus (rAAV) virions used to introduce foreign genes into cells, tissues, or organisms is usually (1) the first reverse end derived from a virus such as AAV. A plasmid (plasmid) having a structure containing a base sequence containing a repeat (ITR), a base sequence containing a second reverse terminal repeat (ITR), and a gene encoding a desired protein placed between these two ITRs. 1), (2) Encode the AAV Rep gene having the function necessary for integrating the base sequence of the region (including the ITR sequence) sandwiched between the ITR sequences into the genome of the host cell, and the plasmid protein of AAV. Three types of plasmids are used: a plasmid containing the gene (plasmid 2) and (3) a plasmid containing the E2A region, E4 region, and VA1 RNA region of adenovirus (plasmid 3).

In general, for the production of 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 integrated into the genome. .. Then, the base sequence containing the first reverse terminal repeat (ITR), the base sequence containing the second reverse terminal repeat (ITR), and the gene encoding the desired protein placed between these two ITRs are obtained. The region containing is integrated 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. This recombinant adeno-associated virus (rAAV) virion is infectious and can be used to introduce foreign genes into cells, tissues, or living organisms.

In one embodiment of the present invention, the components of the three types of plasmids 1 to 3 described above, which are necessary for the production of recombinant adeno-associated virus (rAAV) virions, that is, (a) Rep protein of adeno-associated virus. Or a base sequence encoding its functional equivalent, (b) a base sequence encoding the Cap protein of adeno-associated virus or its functional equivalent, (c) a base sequence containing the first reverse terminal repeat (ITR). , (D) A base sequence containing the second reverse terminal repeat (ITR), (e) To insert a base sequence encoding a foreign protein located between the first ITR and the second ITR. Nucleotide sequence and / and the base sequence encoding the foreign protein, (f) the base sequence encoding the E2A protein of adenovirus or its functional equivalent, (g) the E4 protein of adenovirus or its functional equivalent. It relates to a nucleic acid molecule containing a base sequence encoding and (h) a base sequence encoding VA1 RNA of adenovirus or a functional equivalent thereof.

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

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

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

In one embodiment of the present invention, the base sequence encoding the Rep protein of adeno-associated virus means at least the base sequence encoding Rep68 and Rep78, or a base sequence obtained by adding a mutation to the base sequence. The Rep protein is preferably of AAV of serotype 2, but is not limited to this, and is of serotype 1,3,4,5,6,7,8,9,10 or 11. It may be any of the above. The nucleotide sequence encoding the AAV Rep protein of wild-type serotype 2 has the nucleotide sequence shown in SEQ ID NO: 1. Wild-type serotype 2 AAV Rep68 and Rep78 have the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

In addition, Rep68 is replaced with the amino acid sequence of the wild-type Rep68 of AAV of any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 as long as it exerts its function. , Deletion, addition, etc. may be modified. In the present invention, Rep68 to which these mutations are added is also included in Rep68.

When substituting an amino acid in the amino acid sequence of wild-type Rep68 with another amino acid, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. .. When deleting an amino acid in the amino acid sequence of wild-type Rep68, 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 a mutation that combines substitutions and deletions of these amino acids is also Rep68. When adding amino acids, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence of the wild-type Rep68 or at the N-terminal or C-terminal. Rep68 with mutations that combine additions, substitutions and deletions of these amino acids is also included in Rep68. The mutated Rep68 amino acid sequence preferably exhibits 85% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology to the wild-type Rep68 amino acid sequence. It shows homology, and even more preferably 98% or more homology.

In addition, Rep78 is replaced with the amino acid sequence of the wild-type Rep78 of AAV of any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 as long as it exerts its function. , Deletion, addition, etc. may be modified. In the present invention, Rep78 to which these mutations are added is also included in Rep78.

When substituting an amino acid in the amino acid sequence of wild-type Rep78 with another amino acid, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. .. When deleting an amino acid in the amino acid sequence of wild-type Rep78, 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 substitutions and deletions of these amino acids is also Rep78. When adding amino acids, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence of the wild-type Rep78 or at the N-terminal or C-terminal. Rep78 with mutations that combine additions, substitutions and deletions of these amino acids is also included in Rep78. The mutated Rep78 amino acid sequence preferably exhibits 85% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology to the wild-type Rep78 amino acid sequence. It shows homology, and even more preferably 98% or more homology.

Functional equivalents of AAV Rep proteins are those that can be functionally used in place of Rep68 in Rep68 and can be functionally used in place of Rep78 in Rep78. Say something.

As long as the nucleotide sequence shown in SEQ ID NO: 1 encodes functional Rep68 and Rep78, modifications such as substitution, deletion, and addition can be added. When the base in the base sequence shown by SEQ ID NO: 1 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. is there. When deleting a base in the base sequence shown by SEQ ID NO: 1, 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, a mutation that combines these base substitutions and deletions can also be used as the base sequence encoding the Rep protein. 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: 1 or at the 5'end or 3'end. Add. Mutations that combine additions, substitutions, and deletions of these bases can also be used as nucleic acid molecules encoding Rep proteins. The mutated base sequence shows preferably 85% or more homology, more preferably 90% or more homology, and further preferably 95% or more homology with the base sequence shown in SEQ ID NO: 1. It shows sex, and even more preferably 98% or more homology. However, when these mutations are added, it is preferable that the start codon of the gene encoding Rep68 and Rep78 of the Rep protein is ACG.

Examples of the nucleic acid molecule encoding the Rep protein in the preferred embodiment of the present invention include those containing the nucleotide 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 start codon of the gene encoding Rep68 and Rep78 of the Rep protein is ACG. Modifications such as substitutions, deletions, and additions to this base sequence are also nucleic acid molecules that encode the Rep protein as long as they encode the Rep protein.

In one embodiment of the present invention, the base sequence encoding the Cap protein of the adeno-associated virus is at least the base sequence encoding VP1 which is one of the proteins constituting the capsid of AAV, or the base sequence obtained by adding a mutation thereof. Refers to a base sequence containing. VP1 is preferably of AAV of serotype 9, but is not limited to that of serotype 1,2,3,4,5,6,7,8,10 or 11. It may be any of them. The nucleotide sequence encoding VP1 of AAV of wild-type serotype 6 has the nucleotide sequence shown in SEQ ID NO: 5. Wild-type serotype 6 AAV VP1 has the amino acid sequence set forth in SEQ ID NO: 6. The nucleotide sequence encoding VP1 of AAV of wild-type serotype 9 has the nucleotide sequence shown in SEQ ID NO: 7. Wild-type serotype 9 AAV VP1 has the amino acid sequence set forth in SEQ ID NO: 8.

In addition, VP1 is replaced with the amino acid sequence of wild-type VP1 of AAV of any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 as long as it exerts its function. , Deletion, addition, etc. may be modified. In one embodiment of the present invention, VP1 to which these mutations are added is also included in VP1.

Among the amino acid sequences of wild-type VP1, for example, the amino acid sequence of VP1 of wild-type serotype 6 AAV shown by SEQ ID NO: 6 or the amino acid sequence of wild-type serotype 9 AAV VP1 shown by SEQ ID NO: 9. When the amino acid of 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. When deleting an amino acid in the amino acid sequence of wild-type VP1, 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 substitutions and deletions of these amino acids is also VP1. When adding amino acids, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence of wild-type VP1 or to the N-terminal or C-terminal. VP1 with mutations that combine additions, substitutions and deletions of these amino acids is also included in VP1. The mutated VP1 amino acid sequence preferably exhibits 85% or more homology, more preferably 90% or more homology, and even more preferably 95% or more homology to the wild-type VP1 amino acid sequence. It shows homology, and even more preferably 98% or more homology.

Functional equivalents of AAV VP1 are those that can be functionally used in place of VP1.

Examples of the nucleic acid molecule encoding the Cap protein in a preferred embodiment of the present invention include those containing the base sequence represented by SEQ ID NO: 5 or 7. These nucleic acid molecules are the base sequence encoding VP1 of wild-type serotype 6 AAV and the base sequence encoding wild-type serotype 9 AAV VP1, respectively. Modifications such as substitutions, deletions, and additions to these base sequences are also nucleic acid molecules encoding the Cap protein as long as they encode functional VP1.

When the base in the base sequence represented by SEQ ID NO: 5 or 7 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. It is an individual. When deleting a base in the base sequence represented by SEQ ID NO: 5 or 7, the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. is there. In addition, a mutation that combines these base substitutions and deletions can also be used as the base sequence encoding the Cap protein. When a base is added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 in the base sequence represented by SEQ ID NO: 5 or 7 or at the 5'end or 3'end. Add a base. Mutations that combine additions, substitutions and deletions of these bases can also be used as nucleic acid molecules encoding Cap proteins. The mutated base sequence shows preferably 85% or more homology with the base sequence shown by SEQ ID NO: 5 or 7, more preferably 90% or more homology, and further preferably 95% or more. It shows the homology of 98% or more, and more preferably 98% or more.

In one embodiment of the present invention, the term reverse end repeat (ITR) of adeno-associated virus refers to a base sequence essential for the gene of adeno-associated virus to be integrated into the genome sequence of a host cell by illegitimate recombination. Say that. In one embodiment of the invention, there are two adeno-associated virus reverse end repeats (ITRs) in the nucleic acid molecule, the first adeno-associated virus reverse end repeats (ITR) and the second, respectively. It is called reverse end repeat (ITR) of adeno-associated virus. Here, when a gene encoding a foreign protein is placed between two ITRs, the ITR located on the 5'side is called the reverse end repeat (ITR) of the first adeno-associated virus, 3'. The ITR located on the side is called the reverse terminal repeat (ITR) of the second adeno-associated virus.

Reverse end repeats (ITRs) are preferably, but not limited to, those of serotype 2 AAV, with serotypes 1,3,4,5,6,7,8, It may be any of 9, 10 or 11. In the reverse end repeat (ITR) of serotype 2 AAV, the first ITR contains the base sequence set forth in SEQ ID NO: 9 and the second ITR contains the base sequence set forth in SEQ ID NO: 10.

In addition, reverse end repeat (ITR) is a wild-type AAV of any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, as long as it exerts its function. The ITR base sequence may be modified by substitution, deletion, addition, or the like. ITRs with these mutations are also included in ITRs.

When substituting a base in the base sequence of a wild-type ITR with another base, the number of bases to be substituted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. .. When deleting a base in the ITR base sequence, 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, ITRs with mutations that combine substitutions and deletions of these bases are also ITRs. When adding bases, 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 ITR or to the 5'end or 3'end. To do. Mutations that combine the addition, substitution, and deletion of these bases are also included in the ITR. The mutated ITR nucleotide sequence preferably exhibits 85% or more homology with the wild-type ITR nucleotide sequence, more preferably 90% or more homology, and even more preferably 95% or more. It shows homology, and even more preferably 98% or more.

Functional equivalents of AAV ITRs are those that can be functionally used in place of AAV ITRs. Also, an ITR artificially constructed based on the AAV ITR is a functional equivalent of the AAV ITR as long as it can replace the AAV ITR.

As such an artificially constructed first reverse terminal repeat (first ITR), those having the base sequence shown by SEQ ID NO: 11 can be mentioned. The base sequence represented by SEQ ID NO: 11 with substitutions, deletions, and mutations is also a functional equivalent of AAV ITR as long as it can be functionally used in place of AAV ITR. included. When the base in the base sequence shown by 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. is there. When deleting a base in the base sequence shown by SEQ ID NO: 11, 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, the ITR with mutations that combine substitutions and deletions of these bases is also the first ITR. When a base is added to the base sequence shown by SEQ ID NO: 11, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 in the base sequence or at the 5'end or 3'end. Add a number of bases. Mutations that combine the addition, substitution, and deletion of these bases are also included in the first ITR. The base sequence of the mutated ITR shows preferably 85% or more homology with the base sequence shown in SEQ ID NO: 11, more preferably 90% or more homology, and further preferably 95% or more. It shows the homology of 98% or more, and more preferably 98% or more.

In addition, as such an artificially constructed second reverse terminal repeat (second ITR), one having the base sequence shown by SEQ ID NO: 12 can be mentioned. The base sequence represented by SEQ ID NO: 12 with substitutions, deletions, and mutations is also a functional equivalent of AAV ITR as long as it can be functionally used in place of AAV ITR. included. When the base in the base sequence shown by 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. is there. When deleting a base in the base sequence shown by SEQ ID NO: 12, the number of bases to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The ITR with mutations that combine the substitutions and deletions of these bases is also the second ITR. When a base is added to the base sequence shown by SEQ ID NO: 12, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 are added to the base sequence or at the 5'end or 3'end. Add a number of bases. Mutations that combine the addition, substitution, and deletion of these bases are also included in the second ITR. The base sequence of the mutated ITR shows preferably 85% or more homology with the base sequence shown in SEQ ID NO: 12, more preferably 90% or more homology, and further preferably 95% or more. It shows the homology of 98% or more, and more preferably 98% or more.

In one embodiment of the invention, a foreign protein is encoded between the reverse adeno-associated virus reverse end repeat (ITR) and the second adeno-associated virus reverse end repeat (ITR). There is a base sequence for inserting a base sequence and / and a base sequence encoding a foreign protein.

In one embodiment of the present invention, the base sequence for inserting a base sequence encoding a foreign protein means a base sequence containing a base sequence that can be specifically cleaved with a restriction enzyme. This includes so-called multi-cloning sites. This base sequence can be cleaved with a restriction enzyme, and a nucleic acid molecule encoding a desired foreign protein can be inserted at this cleavage site.

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 species, the species is not particularly limited and may be either a prokaryotic cell or a protein encoded in the genome of a eukaryotic cell. Here, eukaryotic cells are, for example, fungi, yeasts, insects, protozoans, amphibians, reptiles, birds, mammals, and plants. When the species is a mammal, for example, it is a human, a primate other than a human, a domestic animal such as a cow, a horse, a pig, or a sheep, or a pet animal such as a cat or a dog. In addition, the foreign protein may be a wild-type protein derived from a specific species with mutations such as substitution, deletion, or addition added to the amino acid sequence. In addition, the foreign protein may be an artificial protein containing an amino acid sequence that does not exist in nature.

When the 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. ~ 3 pieces. When deleting an amino acid in the amino acid sequence of a wild-type protein, 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 that combine substitutions and deletions of these amino acids can also be added. When adding amino acids, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence of the wild-type protein or at the N-terminal or C-terminal. Mutations that combine additions, substitutions and deletions of these amino acids can also be added. The amino acid sequence of the mutated protein preferably exhibits 85% or more homology, more preferably 90% or more homology, and even more preferably 95%, with the amino acid sequence of the corresponding wild-type protein. It shows a homology of% or more, and even more preferably 98% or more.

In one embodiment of the present invention, the foreign protein is not particularly limited, and examples thereof include proteins that are partially or wholly functionally deficient in a genetic disease. Examples of such genetic diseases include lysosome disease, cystic fibrosis, and hemophilia. Other foreign proteins include growth hormone, somatomedin, insulin, glucagon, lysosome enzyme, cytokine, phosphokine, blood coagulation factor, antibody, fusion protein of antibody and other proteins, granulocyte macrophage colony stimulator (GM-CSF). , Granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoetin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicular stimulating hormone (FSH), gonad stimulating hormone Release hormone (GnRH), gonadotropin, DNasel, thyroid stimulating hormone (TSH), nerve growth factor (NGF), hairy neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neuro Trophin 4/5, Neurotrophin 6, Neurotrophin 1, Actibin, Basic fibroblast growth factor (bFGF), Fibroblast growth factor 2 (FGF2), Epithelial cell growth factor (EGF), Vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor growth factor α receptor (TNF-α receptor), enzyme having activity to decompose beta amyloid, Examples include etanercept, pegbisomant, metrereptin, avatacept, ashotase, and GLP-1 receptor agonists.

Examples of foreign proteins include mouse antibodies, humanized antibodies, human-mouse chimeric antibodies, and human antibodies. Furthermore, as foreign proteins, anti-IL-6 antibody, anti-beta amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-HER2 antibody, anti-PCSK9 antibody, and anti-TNF- An α antibody can be exemplified.

When the foreign protein is a lysosomal enzyme, the genes of the foreign protein include α-L-isuronidase, isulonic acid-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activated protein, and β-hex. Sosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartyl Glucosaminidase, α-N-acetylglucosaminidase, acidic sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl CoAα-glucosaminide N-acetyltransferase, N-acetylglucosamine Examples thereof include -6-sulfatase sulfate, acidic ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, trypeptidylpeptidase-1, hyalronidase-1, CLN1 and CLN2.

The foreign protein may be a fusion protein of an antibody and another protein. Such fusion proteins include the antibody being either a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, or a human antibody, and other proteins are growth hormones, lysosome enzymes, cytokines, neurophokines, blood coagulation factors. , Antibodies, fusion proteins of antibodies to other proteins, granulocyte macrophage colony stimulator (GM-CSF), granulocyte colony stimulator (G-CSF), macrophage colony stimulator (M-CSF), erythropoetin, darbepoetin, Tissue plasminogen activator (t-PA), thrombomodulin, follicular stimulating hormone, DNasel, thyroid stimulating hormone (TSH), nerve growth factor (NGF), hairy neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), Neurotrophin 3, Neurotrophin 4/5, Neurotrophin 6, Neurotrophin 6, Neurogulin 1, Actibin, Basic fibroblast growth factor (bFGF), Fibroblast growth factor 2 (FGF2), Epithelial cells Growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), And any of the enzymes having the activity of degrading beta-amyloid can be exemplified.

The foreign protein may be a fusion protein of an antibody and a lysosomal enzyme. Such fusion proteins include the antibody being either a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, or a human antibody, and the lysosomal enzyme is α-L-isronidase, isulonate-2-sulfatase, gluco. Celebrosidase, β-galactosidase, GM2 activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosulfatase, Saposin C, arylsulfatase A, α-L-fucosidase, aspartyl glucosaminidase, α-N-acetylgalactosaminidase, acidic sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetyl Glucosaminidase, acetyl CoAα-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acidic ceramidase, amilo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, trypeptidyl peptidase-1, hyalronidase-1 , CLN1 and CLN2 can be exemplified.

When the foreign protein is a fusion protein of an antibody and another protein or a fusion protein of an antibody and a lysosome enzyme, the antibody has a specific affinity for, for example, a protein present on the surface of vascular endothelial cells. It is an antibody. Such proteins include transferase receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transport carrier 1, organic anion transporter, monocarboxylic. Examples thereof include acid transporters, low-density lipoprotein receptor-related proteins 1, low-density lipoprotein receptor-related proteins 8, and membrane-bound precursors of heparin-binding epithelial growth factor-like growth factors. Further, OATP-F can be exemplified as an organic anion transporter, and MCT-8 can be exemplified as a monocarboxylic acid transporter.

Nucleotide sequence containing the reverse end repeat (ITR) of the first adeno-associated virus or its functional equivalent, the base sequence containing the reverse end repeat (ITR) of the second adeno-associated virus or its functional equivalent, And the region between the first ITR and the second ITR that contains the base sequence for inserting the base sequence encoding the foreign protein and / and the base sequence encoding the foreign protein is called the transfer region. ..

Adenovirus provides the functions required for the AAV genome to be replicated and packaged in capsids to form viral virions in host cells. This function is exerted by the E1 region, E2A region, E4 region, and VA1 RNA region of the adenovirus genome.

In one embodiment of the invention, the term recombinant AAV virion (rAAV virion) refers to a nucleic acid molecule in which the AAV capsid protein (including its functional equivalent) is modified in the wild-type AAV genome. For example, a packaged rAAV genome. Here, the term rAAV genome (recombinant AAV genome) refers to a nucleic acid molecule in which the wild-type AAV genome has been modified. The rAAV genome packaged in the rAAV virion is a single-stranded DNA. Such nucleic acid molecules include, from the 5'end side, a base sequence containing the reverse end repeat (ITR) of the first adeno-associated virus or its functional equivalent, and a region containing a base sequence encoding a foreign protein. And there is single-stranded DNA containing the reverse terminal repeat (ITR) of the second adeno-associated virus. However, the nucleic acid molecule that is not packaged in the rAAV virion can also be called the rAAV genome. Therefore, the rAAV genome may be single-stranded DNA or double-stranded DNA.

In one embodiment of the present invention, the amount of rAAV genome means the number of rAAV genomes, and the unit thereof is vg.

In one embodiment of the invention, an empty capsid is a particle composed of a capsid protein in which the rAAV genome is not packaged. The total capsid is a general term for empty capsids and rAAV virions, and the total capsid amount is the sum of the numbers of empty capsids and rAAV virions.

In recombinant AAV virions, when the capsid protein is derived from serum type 6 AAV, it can be called recombinant AAV6 virion (rAAV6 virion), and when the capsid protein is derived from serum type 9 AAV. It can be called a recombinant AAV9 virion (rAAV9 virion). The same applies to AAV of other serotypes.

Furthermore, such nucleic acid molecules include a base sequence containing the reverse terminal repeat (ITR) of the first adeno-associated virus from the 5'end to the side or its functional equivalent, and gene expression that controls the expression of the foreign protein. There is a single-stranded DNA containing a base sequence containing a control site, a region containing a base sequence encoding a foreign protein, and a reverse terminal repeat (ITR) of the first adeno-associated virus.

Furthermore, such nucleic acid molecules include a base sequence containing the reverse terminal repeat (ITR) of the first adeno-associated virus from the 5'end to the side or its functional equivalent, and gene expression that controls the expression of the foreign protein. There is a single-stranded DNA containing a base sequence containing a control site, an intron sequence, a region containing a base sequence encoding a foreign protein, a poly A sequence, and a reverse terminal repeat (ITR) of the first adeno-associated virus. Here, the base sequence that can be used as the intron sequence is not particularly limited, but a synthetic human β-globin intron in which a mutation is introduced into the human β-globin gene, for example, an intron containing the base sequence shown in SEQ ID NO: 25 is preferably used. Can be. The base sequence that can be used as the polyadenylation signal sequence (poly A sequence) is not particularly limited, but the base sequence of the synthetic polyadenylation signal sequence (synthetic poly A sequence) including the base sequence shown by SEQ ID NO: 26. There is.

In order for recombinant AAV virions to be formed in host cells, the function of the E1 region of adenovirus is required. In general, recombinant AAV virions are made in host cells that have all or part of the E1 region. As such cells, HEK293 cells are known. The genome of HEK293 cells contains at least the E1A and E1B coding regions.

When using host cells that have all or part of the E1 region, the regions of the adenovirus genome required for the AAV genome to replicate and be packaged in capsids to form viral virions are the E2A region, E4. The region and the VA1 RNA region. These regions encode the proteins and RNA required for AAV replication. For the functionality provided by the E4 region, the E4 34kD protein encoded by the open reading frame 6 (ORF6) of the E4 region is required for AAV replication.

In one embodiment of the invention, the E2A region has a serotype of 2,1,5,6,19,3,11,7 as long as it exerts the original function required for AAV replication. , 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, sheep Ad, or porcine adenovirus. May be good. The E2A region of adenovirus, which is serotype 2, is one of the preferred ones in the present invention. The E2A region of adenovirus of serotype 2 contains the nucleotide sequence shown in SEQ ID NO: 13.

In addition, as the E2A region, as long as the region exerts its original function, the E2A region of the wild-type adenovirus that has been modified such as substitution, deletion, or addition can be used. In one embodiment of the present invention, the E2A region to which these mutations have been added is also included in the E2A region.

When substituting a base in the base sequence of the wild-type E2A region 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. is there. When deleting a base in the base sequence of the E2A region, 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 E2A region to which mutations that combine substitutions and deletions of these bases have been added is also the E2A region. When adding bases, 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 E2A region or at the 5'end or 3'end. Add. The E2A region is also included in the E2A region, which is mutated by combining additions, substitutions and deletions of these bases. The nucleotide sequence of the mutated E2A region preferably shows 85% or more homology with the wild-type E2A nucleotide sequence, more preferably 90% or more homology, and further preferably 95% or more. It shows the homology of 98% or more, and more preferably 98% or more.

In one embodiment of the invention, the E4 region has a serotype of 2,1,5,6,19,3,11,7 as long as it exerts the original function required for AAV replication. , 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, sheep Ad, or porcine adenovirus. May be good. The E4 region of adenovirus, which is serotype 2, is one of the preferred ones in the present invention. The E4 region of adenovirus, which is serotype 2, contains the nucleotide sequence shown in SEQ ID NO: 14.

In addition, as the E4 region, as long as the region originally exhibits the function, a wild-type adenovirus E4 region modified by substitution, deletion, addition, or the like can be used. In one embodiment of the present invention, the E4 region to which these mutations have been added is also included in the E4 region.

When substituting a base in the base sequence of the wild-type E4 region 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. is there. When deleting a base in the base sequence of the E4 region, 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 E4 region to which mutations that combine substitutions and deletions of these bases have been added is also the E4 region. When adding bases, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the wild-type E4 base sequence or to the 5'end or 3'end. To do. E4 with mutations that combine additions, substitutions and deletions of these bases is also included in E4. The mutated E4 base sequence shows preferably 85% or more homology with the wild type E4 base sequence, more preferably 90% or more homology, and even more preferably 95% or more. It shows homology, and even more preferably 98% or more homology.

As a preferable example of the E4 region, there is one containing the base sequence shown by SEQ ID NO: 15. A nucleotide sequence represented by SEQ ID NO: 15 that has been modified by substitution, deletion, addition, or the like can also be used as the E4 region as long as it exhibits the function originally possessed by the region.

When the base in the base sequence shown by SEQ ID NO: 15 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. is there. When deleting a base in the base sequence of the E4 region, 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 that combine substitutions and deletions of these bases can also be added. When adding bases, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases are added to the base sequence shown by SEQ ID NO: 15 or at the 5'end or 3'end. Add. Mutations that combine additions, substitutions and deletions of these bases can also be added. The mutated base sequence shows preferably 85% or more homology, more preferably 90% or more homology, and further preferably 95% or more homology with the base sequence shown by SEQ ID NO: 15. It shows sex, and even more preferably 98% or more homology. The E4 region is also the one to which these mutations are added.

In one embodiment of the invention, the VA1 RNA region has a serotype of 2,1,5,6,19,3,11, as long as it exerts the original function required for AAV replication. 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, Dog Ad2, Sheep Ad3, Dog Ad3, Sheep Ad3, You may. The VA1 RNA region of adenovirus of serotype 2 is one of the preferred ones in the present invention. The VA1 RNA region of adenovirus of serotype 2 contains the nucleotide sequence shown by SEQ ID NO: 16.

In addition, as the VA1 RNA region, as long as the region exerts its original function, the VA1 RNA region of the wild-type adenovirus that has been modified by substitution, deletion, addition, etc. may be used. it can. 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.

When substituting a base in the base sequence of the wild-type VA1 RNA region 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. Is. When deleting a base in the base sequence of the VA1 RNA region, 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 VA1 RNA region to which mutations that combine substitutions and deletions of these bases have been added is also the VA1 RNA region. When adding bases, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 bases in the base sequence of the wild-type VA1 RNA region or at the 5'end or 3'end. Is added. The VA1 RNA region to which mutations combining additions, substitutions and deletions of these bases have been added is also included in the VA1 RNA region. The nucleotide sequence of the mutated VA1 RNA region preferably shows 85% or more homology with the wild-type VA1 RNA region base sequence, more preferably 90% or more homology, and even more preferably. It shows 95% or more homology, and even more preferably 98% or more homology.

In one preferred embodiment of the invention, the E2A region, E4 region and VA1 RNA region may be located in any order. The base sequence containing the E2A region, E4 region, and VA1 RNA region is called the helper region.

A preferred embodiment of the helper region includes a base sequence represented by 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. Modifications such as substitutions, deletions, and additions to this base sequence can also be used as helper regions as long as each region of the E2A region, E4 region, and VA1 RNA region exerts its original function.

When the base in the base sequence of the helper region containing the base sequence shown by SEQ ID NO: 17 is replaced with another base, the number of bases to be replaced is preferably 1 to 60, more preferably 1 to 30, and further. The number is preferably 1 to 10. When deleting a base in the base sequence of the helper region containing the base sequence shown by SEQ ID NO: 17, the number of bases to be deleted is preferably 1 to 60, more preferably 1 to 30, and even more preferably. 1 to 10 pieces. In addition, mutations that combine substitutions and deletions of these bases can also be used as helper regions. When a base is added, preferably 1 to 60, more preferably 1 to 30, and further preferably 1 to 60 in the base sequence of the helper region containing the base sequence shown by SEQ ID NO: 17 or at the 5'end or 3'end. Add 1 to 10 bases. Mutations that combine additions, substitutions, and deletions of these bases can also be used as helper regions. The nucleotide sequence of the mutated helper region preferably shows 85% or more homology with the nucleotide sequence of the helper region containing the nucleotide sequence shown in SEQ ID NO: 17, and more preferably 90% or more homology. More preferably, it exhibits 95% or more homology, and even more preferably 98% or more homology.

The above rules apply to modifications in each of the E2A region, E4 region, and VA1 RNA region included in the helper region.

In one embodiment of the present invention, a base sequence that can be used as a base sequence containing a first gene expression control site that controls the expression of Rep protein includes a region containing an E2A region, an E4 region, and a VA1 RNA region. As long as it is regulated by the protein and RNA encoded by the above, it is not particularly limited, but is preferably the p5 promoter of AAV. When the first gene expression control site is the p5 promoter of AAV, it is preferable that it is the p5 promoter of AAV which is serotype 2, but it is not limited to this, and serotypes 1, 3, 4, 5 , 6, 7, 8, 9, 10 or 11 may be used. The p5 promoter of serotype 2 AAV contains the nucleotide sequence set forth in SEQ ID NO: 18.

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

When the base in the base sequence of the wild-type p5 promoter shown in SEQ ID NO: 18 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably. Is 1 to 3. When deleting a base in the base sequence shown by SEQ ID NO: 18, 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 p5 promoter with mutations that combine substitutions and deletions of these bases is also a p5 promoter. 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 by SEQ ID NO: 18 or at the 5'end or 3'end. Add. Mutations that combine the addition, substitution, and deletion of these bases are also included in the p5 promoter. The nucleotide sequence of the mutated p5 promoter preferably shows 85% or more homology with the wild-type p5 promoter nucleotide sequence, more preferably 90% or more homology, and further preferably 95%. It shows the above homology, and even more preferably 98% or more.

Examples of the p5 promoter obtained by modifying the p5 promoter of AAV, which is serotype 2, include those containing the nucleotide sequence shown in SEQ ID NO: 19. A modification such as substitution, deletion, or addition to the base sequence of the p5 promoter may be a p5 promoter or a functional equivalent thereof as long as it exhibits the original function of the p5 promoter.

When the base in the base sequence of the p5 promoter containing the base sequence shown in SEQ ID NO: 19 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and further. The number is preferably 1 to 3. When deleting a base in the base sequence shown by SEQ ID NO: 19, 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, a p5 promoter or a functional equivalent thereof can also be used by adding a mutation that combines substitutions and deletions of these bases. 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: 19 or at the 5'end or 3'end. Add. Mutations that combine additions, substitutions and deletions of these bases can also be used as the p5 promoter or its functional equivalent. The base sequence of the mutated p5 promoter preferably shows 85% or more homology with the base sequence shown in SEQ ID NO: 19, more preferably 90% or more homology, and further preferably 95%. It shows the above homology, and even more preferably 98% or more.

The p5 promoter or its functional equivalent is usually located upstream of the nucleotide sequence encoding the adeno-associated virus Rep protein or its functional equivalent.

The region containing the p5 promoter or its functional equivalent and the base sequence encoding the Rep protein of the adeno-associated virus or its functional equivalent is called the Rep region. A preferred embodiment of the Rep region includes one containing the base sequence shown in SEQ ID NO: 20. The base sequence shown by SEQ ID NO: 20 includes the base sequence shown by SEQ ID NO: 4 downstream of the base sequence shown by SEQ ID NO: 19. That is, the nucleotide sequence shown by SEQ ID NO: 20 includes Rep68 of AAV of serotype 2 having the amino acid sequence shown by SEQ ID NO: 2 and Rep78 of AAV of serotype 2 having the amino acid sequence shown by SEQ ID NO: 3. To code.

A nucleotide sequence represented by SEQ ID NO: 20 that has been modified by substitution, deletion, addition, or the like can also be used as the Rep region as long as it exhibits the original function of the Rep region.

When the base in the base sequence of the Rep region containing the base sequence shown by SEQ ID NO: 20 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and further. The number is preferably 1 to 3. When deleting a base in the base sequence shown by SEQ ID NO: 20, 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, a mutation that combines substitutions and deletions of these bases can also be used as the Rep 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 by SEQ ID NO: 20 or at the 5'end or 3'end. Add. Mutations that combine the addition, substitution, and deletion of these bases can also be used as the Rep region. The nucleotide sequence of the mutated Rep region preferably shows 85% or more homology with the nucleotide sequence shown in SEQ ID NO: 20, more preferably 90% or more homology, and further preferably 95%. It shows the above homology, and even more preferably 98% or more.

In one embodiment of the present invention, the base sequence that can be used as a base sequence containing a second gene expression control site that controls the expression of Cap protein includes a region containing an E2A region, an E4 region, and a VA1 RNA region. As long as it is regulated by the protein and RNA encoded by the above, it is not particularly limited, but is preferably the p40 promoter of AAV. When it is the p40 promoter of AAV, it is preferable that it is the p40 promoter of AAV which is serotype 2, but it is not limited to this, and serotypes 1,3,4,5,6,7,8,9 , 10 or 11.

In addition, the p40 promoter of AAV is a wild-type AAV of any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, as long as it exerts its function. The base sequence of the p40 promoter may be modified by substitution, deletion, addition, or the like. In one embodiment of the present invention, the p40 promoter to which these mutations are added is also included in the p40 promoter. When substituting a base in the base sequence of the wild-type p40 promoter 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. is there. When deleting a base in the base sequence of the wild-type p40 promoter, 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 substitutions and deletions of these bases is also a p40 promoter. 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 of the wild-type p40 promoter or at the 5'end or 3'end. Add. Mutations that combine the addition, substitution and deletion of these bases are also included in the p40 promoter. The nucleotide sequence of the mutated p40 promoter preferably shows 85% or more homology with the wild-type p40 promoter nucleotide sequence, more preferably 90% or more homology, and further preferably 95%. It shows the above homology, and even more preferably 98% or more.

The p40 promoter or its functional equivalent is usually located upstream of the nucleotide sequence encoding the adeno-associated virus Cap protein or its functional equivalent.

The region containing the p40 promoter or its functional equivalent and the base sequence encoding the adeno-associated virus Cap protein or its functional equivalent is called the Cap region.

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

When the base of the base sequence represented by SEQ ID NO: 21 in the base sequence of the Cap region is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably. Is 1 to 3. When deleting a base in the base sequence shown by SEQ ID NO: 21, 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, a mutation that combines substitutions and deletions of these bases can also be used as the Cap region. 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 by SEQ ID NO: 21 or at the 5'end or 3'end. Add. Mutations that combine the addition, substitution, and deletion of these bases can also be used as the Cap region. The nucleotide sequence of the mutated Cap region preferably exhibits 85% or more homology with the nucleotide sequence shown in SEQ ID NO: 21, more preferably 90% or more homology, and further preferably 95%. It shows the above homology, and even more preferably 98% or more. 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.

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

A preferred embodiment of the Rep-Cap region includes one in which the Rep region is located upstream of the Cap region and contains the base sequence shown by SEQ ID NO: 22. This Rep-Cap region contains the base sequence shown by SEQ ID NO: 20 downstream of the base sequence shown by SEQ ID NO: 19, and the base sequence shown by SEQ ID NO: 21 further downstream. This Rep-Cap region contains a base sequence encoding VP1 of serotype 9 AAV having the base sequence shown by SEQ ID NO: 7, and VP1 of serotype 9 AAV having the amino acid sequence shown by SEQ ID NO: 8. Code. This Rep-Cap region is replaced with the base sequence encoding VP1 of AAV of serotype 9 having the base sequence shown by SEQ ID NO: 7, and VP1 of AAV of serotype 6 having the base sequence shown by SEQ ID NO: 5. It can also contain a base sequence encoding. Modifications such as substitutions, deletions, and additions to these base sequences can also be used as the Rep-Cap region as long as they exhibit the original functions of the Rep-Cap region.

When the base in the base sequence of the Rep-Cap region containing the base sequence shown by SEQ ID NO: 22 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10. , More preferably 1 to 3. When deleting a base in the base sequence shown by SEQ ID NO: 22, 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, a mutation that combines substitutions and deletions of these bases can also be used as the Rep-Cap region. 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 by SEQ ID NO: 22 or at the 5'end or 3'end. Add. Mutations that combine the addition, substitution, and deletion of these bases can also be used as the Rep-Cap region. The nucleotide sequence of the mutated Rep-Cap region preferably exhibits 85% or more homology with the nucleotide sequence shown in SEQ ID NO: 22, more preferably 90% or more homology, and even more preferably. It shows 95% or more homology, and even more preferably 98% or more homology.

An enhancer sequence having a function of increasing the expression level of Rep protein and / and Cap protein can also be placed downstream of the Rep-Cap region. Here, the enhancer sequence is not particularly limited, but the p5 promoter of AAV or its functional equivalent can be used as the enhancer. The AAV serotype of the p5 promoter used here is not particularly limited, and is any AAV p5 promoter of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. It may be, but preferably a serotype 2 AAV p5 promoter. The p5 promoter of serotype 2 AAV contains the nucleotide sequence set forth in SEQ ID NO: 18.

In addition, the P5 promoter of AAV located downstream of the Rep-Cap region is a wild-type AAV of any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. The base sequence of the p5 promoter may be modified by substitution, deletion, addition, or the like. The p5 promoter with these mutations is also included in the P5 promoter.

Here, when the base in the base sequence of the wild-type P5 promoter is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 1. There are three. When deleting a base in the base sequence of the wild-type p5 promoter, 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 p5 promoter with mutations that combine substitutions and deletions of these bases is also a P5 promoter. 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 of the wild-type P5 promoter or at the 5'end or 3'end. Add. Mutations that combine the addition, substitution, and deletion of these bases are also included in the P5 promoter. The nucleotide sequence of the mutated p5 promoter preferably shows 85% or more homology with the wild-type P5 promoter nucleotide sequence, more preferably 90% or more homology, and further preferably 95%. It shows the above homology, and even more preferably 98% or more.

Examples of the p5 promoter modified from the p5 promoter of serotype 2 AAV include those containing the nucleotide sequence shown in SEQ ID NO: 19. Modifications such as substitutions, deletions, and additions to the base sequence of this p5 promoter are also included in the p5 promoter.

When the base in the base sequence of the p5 promoter containing the base sequence shown in SEQ ID NO: 19 is replaced with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and further. The number is preferably 1 to 3. When deleting a base in the base sequence shown by SEQ ID NO: 19, 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, a p5 promoter or a functional equivalent thereof can also be used by adding a mutation that combines substitutions and deletions of these bases. 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: 19 or at the 5'end or 3'end. Add. Mutations that combine additions, substitutions and deletions of these bases can also be used as the p5 promoter or its functional equivalent. The base sequence of the mutated p5 promoter preferably shows 85% or more homology with the base sequence shown in SEQ ID NO: 19, more preferably 90% or more homology, and further preferably 95%. It shows the above homology, and even more preferably 98% or more.

When the nucleotide sequence of the AAV p5 promoter or its functional equivalent is located downstream of the Rep-Cap region, the region including the nucleotide sequence of this AAV p5 promoter or its functional equivalent is the Rep-Cap region. You can also say that. A preferred embodiment of such a Rep-Cap region includes one containing the base sequence shown in SEQ ID NO: 23. In this Rep-Cap region, the base sequence shown by SEQ ID NO: 20 is downstream of the base sequence shown by SEQ ID NO: 19, the base sequence shown by SEQ ID NO: 21 is further downstream, and the base sequence shown by SEQ ID NO: 19 is further downstream. including. Even if this base sequence is modified by substitution, deletion, addition, etc., it can be regarded as a Rep-Cap region as long as it exhibits the original function of the Rep-Cap region.

When substituting a base in the base sequence of the Rep-Cap region containing the base sequence shown by SEQ ID NO: 23 with another base, the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10. , More preferably 1 to 3. When deleting a base in the base sequence shown by SEQ ID NO: 23, 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, a mutation that combines substitutions and deletions of these bases can also be used as the Rep-Cap region. 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 by SEQ ID NO: 23 or at the 5'end or 3'end. Add. Mutations that combine the addition, substitution, and deletion of these bases can also be used as the Rep-Cap region. The nucleotide sequence of the mutated Rep-Cap region preferably shows 85% or more homology with the nucleotide sequence shown in SEQ ID NO: 23, more preferably 90% or more homology, and even more preferably. It shows 95% or more homology, and even more preferably 98% or more homology.

In one embodiment of the present invention, a gene encoding this foreign protein is introduced into a base sequence that can be used as a base sequence containing a third gene expression control site that controls the expression of the foreign protein. As long as this foreign protein can be expressed in the above-mentioned cells, tissues or living body, there is no particular limitation, but a promoter derived from cytomegalovirus (including an enhancer optionally), an SV40 initial promoter, Preferably those containing human elongation factor-1α (EF-1α) promoter, human ubiquitin C promoter, retrovirus Laus sarcoma virus LTR promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter Is. As the β-actin promoter, the chicken β-actin promoter having the nucleotide sequence shown in SEQ ID NO: 38 is suitable. In addition, a promoter having the nucleotide sequence shown by SEQ ID NO: 47 to which the chicken β-actin promoter is bound downstream of the human CMV enhancer can be suitably used as a third gene expression control site.

In order to increase the expression level of the foreign protein, the intron sequence is located 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. May exist. For example, a chicken β-actin promoter downstream of a human CMV enhancer and a chicken β-actin / MVM chimeric intron having the nucleotide sequence shown by SEQ ID NO: 39 bound downstream thereof are suitable as a third gene expression control site. Available. Here, MVM is an abbreviation for Minute virus of mice.

A polyadenylation sequence (polyA sequence) is placed downstream of the foreign protein. The base sequence that can be used as a polyA sequence is not particularly limited as 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. A growth hormone polyA sequence having a human growth hormone polyA sequence and the nucleotide sequence represented by SEQ ID NO: 40 can be preferably used.

The nucleic acid molecule according to one embodiment of the present invention can be a circular plasmid.

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

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 thereof. For example, the Cap region is located downstream of the Rep region, the transfer region is located downstream of it, the E2A region is located downstream of it, the E4 region is located downstream of it, and the VA1 RNA region is located downstream of it. The same applies when the nucleic acid molecule is a circular plasmid.

In a further embodiment of the present invention, a nucleotide sequence encoding the Rep protein of an adeno-associated virus or a functional equivalent thereof, a nucleotide sequence encoding a Cap protein of an adeno-associated virus or a functional equivalent thereof, a first adenocon. A base sequence containing the reverse terminal repeat (ITR) of the concomitant virus or its functional equivalent, a base sequence for inserting a base sequence encoding a foreign protein, and / and a base sequence encoding a foreign protein, second Nucleotide sequence containing the reverse terminal repeat (ITR) of adeno-associated virus or its functional equivalent, the nucleotide sequence encoding the E2A region of adenovirus or its functional equivalent, the E4 region of adenovirus or its functional equivalent The nucleotide sequence encoding the substance and the nucleotide sequence encoding the VA1 RNA region of adenovirus or its functional equivalent may be located in any order. The same applies when the nucleic acid molecule is a circular plasmid. A drug resistance gene can also be incorporated at any position on this nucleic acid molecule. In addition, the replication origin required for the plasmid to replicate in the host cell can be incorporated at any position of this nucleic acid molecule. Coli E1 can be preferably used as a replication origin in the case of a host cell. Downstream of the base sequence encoding a foreign protein is usually the polyA sequence. The same applies when the nucleic acid molecule is a circular plasmid.

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

As a further preferred embodiment of the present invention, a base sequence encoding the Cap protein of the adeno-associated virus or its functional equivalent is downstream of the base sequence encoding the Rep protein of the adeno-associated virus or its functional equivalent. Further downstream, a base sequence containing the reverse terminal repeat (ITR) of the first adeno-associated virus or its functional equivalent, and further downstream, a base sequence for inserting a base sequence encoding a foreign protein and / and a foreign base. Nucleotide sequence encoding the protein of, further downstream contains the reverse terminal repeat (ITR) of the second adeno-associated virus or its functional equivalent, and further downstream is the E2A region of adenovirus or its functional equivalent. Nucleotide sequence encoding the nucleotide sequence, the nucleotide sequence encoding the E4 region of adenovirus or its functional equivalent further downstream, and the nucleotide sequence encoding the VA1 RNA region of adenovirus or its functional equivalent further downstream. Includes molecules. The same applies when the nucleic acid molecule is a circular plasmid. A drug resistance gene can also be incorporated at any position on this nucleic acid molecule. In addition, the replication origin required for the plasmid to replicate in the host cell can be incorporated at any position of this nucleic acid molecule. Coli E1 can be preferably used as a replication origin in the case of a host cell. Downstream of the base sequence encoding a foreign protein is usually the polyA sequence. The same applies when the nucleic acid molecule is a circular plasmid.

As a further preferred embodiment of the present invention, a base sequence encoding the Rep protein or a functional equivalent thereof is downstream of the base sequence containing the first gene expression control site that controls the expression of the Rep protein of the adeno-associated virus. , Nucleotide sequence containing a second gene expression control site that controls the expression of Cap protein of adeno-associated virus downstream of it, Nucleotide sequence encoding Cap protein of Adeno-associated virus or its functional equivalent further downstream A base sequence containing the reverse terminal repeat (ITR) of the first adeno-associated virus or its functional equivalent, and a base sequence for inserting a base sequence encoding a foreign protein further downstream and / or a foreign protein. The nucleotide sequence encoding the nucleotide sequence containing the reverse terminal repeat (ITR) of the second adeno-associated virus or its functional equivalent further downstream, and the E2A region of the adenovirus or its functional equivalent further downstream. Nucleotide sequence is located further downstream, the base sequence encoding the E4 region of adenovirus or its functional equivalent, and further downstream is the base sequence encoding the VA1 RNA region of adenovirus or its functional equivalent. Can be mentioned. It may further include a replication origin and may optionally incorporate a drug resistance gene. Downstream of the base sequence encoding a foreign protein is usually the polyA sequence. The same applies when the nucleic acid molecule is a circular plasmid.

As a further preferred embodiment of the present invention, a base sequence encoding the Rep protein or a functional equivalent thereof is downstream of the base sequence containing the first gene expression control site that controls the expression of the Rep protein of the adeno-associated virus. , Nucleotide sequence containing a second gene expression control site that controls the expression of Cap protein of adeno-associated virus downstream of it, Nucleotide sequence encoding Cap protein of Adeno-associated virus or its functional equivalent further downstream It encodes the base sequence of the p5 promoter of AAV or its equivalent, the base sequence containing the reverse terminal repeat (ITR) of the first adeno-associated virus or its functional equivalent further downstream, and the foreign protein further downstream. A base sequence for inserting a base sequence and / or a base sequence encoding a foreign protein, and a base sequence further downstream containing the reverse terminal repeat (ITR) of the second adeno-associated virus or its functional equivalent. The base sequence encoding the E2A region of adenovirus or its functional equivalent is downstream, the base sequence encoding the E4 region of adenovirus or its functional equivalent is further downstream, and the VA1 RNA region of adenovirus or its functional equivalent is further downstream. Examples thereof include nucleic acid molecules in which a base sequence encoding a functional equivalent is located. The same applies when the nucleic acid molecule is a circular plasmid. A drug resistance gene can also be incorporated at any position on this nucleic acid molecule. In addition, the replication origin required for the plasmid to replicate in the host cell can be incorporated at any position of this nucleic acid molecule. Coli E1 can be preferably used as a replication origin in the case of a host cell. Downstream of the base sequence encoding a foreign protein is usually the polyA sequence. The same applies when the nucleic acid molecule is a circular plasmid.

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

As a further preferred embodiment of the present invention, a Cap region containing a base sequence encoding the AAV Rep protein of serum type 2 and a Cap containing a base sequence encoding the Cap protein of AAV of serum type 6 or 9 downstream thereof. Insert the p5 promoter as an enhancer further downstream, the first ITR further downstream, the cytomegalovirus-derived promoter further downstream, the synthetic human β-globin intron further downstream, and the base sequence encoding a foreign protein further downstream. Nucleotide sequence / and a base sequence encoding a foreign protein, a synthetic polyA sequence further downstream, a second ITR further downstream, an adenovirus E2A region further downstream, an adenovirus E4 region further downstream, and further. Nucleic acid molecules in which the VA1 RNA region of adenovirus is located downstream can be mentioned. It may also include a replication origin, in which case the replication origin is preferably located downstream of the p5 promoter. A drug resistance gene may be incorporated incidentally, 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.

As a further preferred embodiment of the present invention, a Cap region containing a base sequence encoding the AAV Rep protein of serum type 2 and a Cap containing a base sequence encoding the Cap protein of AAV of serum type 6 or 9 downstream thereof. Region, p5 promoter as enhancer further downstream, first ITR further downstream, human CMV enhancer further downstream, chicken β-actin promoter further downstream, chicken β-actin / MVM chimeric intron further downstream, foreign protein further downstream The base sequence for inserting the base sequence encoding the above and / and the base sequence encoding the foreign protein, the bovine or human growth hormone polyA sequence further downstream, the second ITR further downstream, and the adenovirus E2A further downstream. Nucleic acid molecules in which the E4 region of adenovirus is located further downstream and the VA1 RNA region of adenovirus are located further downstream. It may also include a replication origin, in which case the replication origin is preferably located downstream of the p5 promoter. A drug resistance gene may be incorporated incidentally, 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.

A method for producing a recombinant adeno-associated virus virion using the nucleic acid molecule of one embodiment of the present invention will be described below by taking a nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO: 24 as an example.

The nucleic acid molecule represented by SEQ ID NO: 24 is the Rep region containing the nucleotide sequence encoding the AAV Rep protein of serum type 2, the Cap region containing the nucleotide sequence encoding the AAV Cap protein of serum type 9 downstream thereof, and further. The p5 promoter as an enhancer downstream, the replication initiation site further downstream, the ampicillin resistance gene further downstream, the first ITR further downstream, the cytomegalovirus-derived promoter further downstream, the synthetic human β-globin intron further downstream, and further downstream. Nucleotide sequence for inserting a base sequence encoding a foreign protein, a synthetic polyA sequence further downstream, a second ITR further downstream, an adenovirus E2A region further downstream, and an adenovirus E4 region further downstream. It is a nucleic acid molecule in which the VA1 RNA region of adenovirus is located further downstream. Although not shown in SEQ ID NO: 24, a nucleotide sequence for inserting a nucleotide sequence encoding a foreign protein (any of the restriction enzyme sites present at 8116 bp to 8147 bp in the nucleotide sequence shown in SEQ ID NO: 24). It is assumed that the base sequence encoding the desired protein has been introduced into. The nucleic acid molecule represented by SEQ ID NO: 24 is a suitable example of a nucleic acid molecule that can be used to generate recombinant AAV virions. 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 FIG. 11 excluding the GFP gene.

The nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 24 can be synthesized as a cyclic plasmid using a general genetic engineering technique. The drug resistance gene is encoded in this circular plasmid. It also has a replication origin for replication in E. coli. Therefore, this circular plasmid can be amplified by introduction into Escherichia coli and can be easily prepared in large quantities.

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 host cells, HEK293 cells, which are cells derived from human fetal kidney tissue transformed with the E1A gene and E1B gene of adenovirus, can be preferably used.

When the cyclic plasmid is introduced into the host cell, the region from the first ITR to the second ITR is integrated into the host cell by illegitimate recombination. Single-stranded DNA is then replicated from the region integrated into this host cell and packaged in capsids to form recombinant AAV virions. Recombinant AAV virions are released into the culture medium or accumulate in the host cell. Therefore, recombinant AAV virions can be recovered by recovering the culture supernatant of the host cell or by recovering and destroying the host cell.

Normally, in order to produce a recombinant AAV virion, a plasmid containing a base sequence encoding a desired protein sandwiched between ITRs, a plasmid containing a base sequence encoding a Rep protein, and a plasmid containing a base sequence encoding a Cap protein, and It is necessary to simultaneously introduce three types of plasmids containing adenovirus-derived E2A, E4, and VA1 RNA sequences into host cells. Among the host cells, the proportion of cells into which the three plasmids are introduced is low, which reduces the efficiency of recombinant AAV virions. In addition, among the three types of plasmids, the host cell in which the plasmid containing the nucleotide sequence encoding the desired protein sandwiched between the ITRs was not introduced and only the other two types of plasmids were introduced contained the rAAV genome. Since no empty virus particles (empty capsids) will be produced, the production efficiency will be further reduced.

The cyclic plasmid having the nucleotide sequence shown by SEQ ID NO: 24, which is one of the preferred examples of the present invention, has all the functions of these three types of plasmids. Therefore, by transforming the host cell with this cyclic plasmid, all the elements necessary for the intracellular production of rAAV virion are simultaneously introduced into the cell, and the production amount of rAAV virion is increased. Not only can it be caused, but the generation of empty capsids can also be suppressed. The higher the ratio of empty capsids to the total capsids, the lower the quality of rAAV virions. Therefore, by using this cyclic plasmid, high-quality rAAV virions with a low ratio of empty capsids can be efficiently produced. Not limited to this, by transforming host cells with an integrated rAAV plasmid, high-quality rAAV virions with a low proportion of empty capsids can be efficiently produced.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not intended to be limited to Examples.

[Example 1] Construction of pHelper (mod) vector
From the 5'side, a DNA fragment containing the nucleotide sequence shown by SEQ ID NO: 27 including SalI site, RsrII site, origin of replication (ColE1 ori), ampicillin resistance gene, BsiWI site, BstZ17I site, and BsrGI site was synthesized. This DNA fragment was digested with SalI and BsrGI. The pHelper vector (Takara Bio Inc.) was digested with SalI and BsrGI, and the above restriction enzyme-treated DNA fragment was inserted into this. The obtained plasmid was used as a pHelper (mod) vector (Fig. 1).

[Example 2] Construction of pAAV-CMV-GFP vector and pAAV-CMV-GFP (mod) vector
From the 5'side, a DNA fragment containing the nucleotide sequence shown by SEQ ID NO: 28 including the EcoRI site, MluI site, GFP gene (green fluorescent protein gene), NotI site, and BamHI site was synthesized. This DNA fragment was digested with EcoRI and BamHI. The pAAV-CMV vector (Takara Bio Inc.) was digested with EcoRI and BamHI, and the above restriction enzyme-treated DNA fragment was inserted into this vector. The obtained plasmid was used as a pAAV-CMV-GFP vector. The multicloning site 1 containing the BsiWI site and the AvrII site, which has the nucleotide sequence shown by SEQ ID NO: 29, is inserted into the PciI site of the pAAV-CMV-GFP vector, and further, the SfoI site and the nucleotide sequence shown by SEQ ID NO: 30 are inserted. A multi-cloning site 2 containing the BsrGI site, AgeI site, and BstZ17I site was inserted. The obtained plasmid was used as a pAAV-CMV-GFP (mod) vector (Fig. 2).

[Example 3] Construction of pR2 (mod) C6 vector
From the 5'side, AfeI site, RsrII site, BsrGI site, AvrII site, ampicillin resistance gene site, origin of replication (ColE1 ori), RsrII site, 5'part of AAV2 Rep region (including p5 promoter), and SacII site. A DNA fragment containing the nucleotide sequence represented by SEQ ID NO: 31 containing the above was synthesized. This DNA fragment was digested with Afe I and Sac II. The pRC6 vector (Takara Bio Inc.) was digested with AfeI and SacII, and the above-mentioned restricted synthetic gene was inserted into this. The obtained plasmid was used as a pR2 (mod) C6 vector (Fig. 3).

[Example 4] Construction of pR2 (mod) C9 vector
From the 5'side, a DNA fragment containing the nucleotide sequence shown by SEQ ID NO: 32 including the HindIII site, the 3'part of the AAV2 Rep region, the AAV9 Cap region, the p5 promoter, the RsrII site, and the BsrGI site was synthesized. This DNA fragment was digested with HindIII and BsrGI. The pR2 (mod) C6 vector was digested with HindIII and BsrGI, and the above restriction enzyme-treated synthetic gene was inserted into this. The obtained plasmid was used as a pR2 (mod) C9 vector (Fig. 4).

[Example 5] Construction of pHelper-ITR / CMV / GFP vector
The pAAV-CMV-GFP (mod) vector was digested with BsiWI and BstZ17I, and a DNA fragment containing the ITR-CMV promoter, human β-globin intron, GFP gene, synthetic poly A sequence, and ITR was excised. The pHelper (mod) vector was digested with BsiWI and BstZ17I, and the excised DNA fragment was inserted into it. The obtained plasmid was used as a pHelper-ITR / CMV / GFP vector (Fig. 5).

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

[Example 7] Construction of pHelper-R2 (mod) C9 vector
The pR2 (mod) C9 vector was digested with RsrII, and the sequence containing the p5 promoter-Rep2-AAV9 VP1-p5 promoter was excised. The pHelper (mod) vector was digested with RsrII. The excised DNA fragment was inserted into this and used as a pHelper-R2 (mod) C9 vector (Fig. 7).

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

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

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

[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 to prepare a pHelper-ITR / CMV / GFP-R2 (mod) C9 vector (Fig. 11).

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

[Example 13] Production of rAAV virions by transient expression of two types of plasmids
HEK293T cells were seeded in a 15 cm dish at a concentration of 1.65 × 10 ⁷ cells / dish and cultured in DMEM / High Glucose medium containing 10% FBS at 37 ° C. in the presence of 5% CO ₂ for 16 hours. As a DNA solution for transfection, a solution containing the plasmid of the combination of (1) to (6) below was prepared;
(1) pHelper-ITR / CMV / GFP vector and pRC6 vector (Takara Bio Inc.),
(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 Inc.),
(6) pR2 (mod) C9-ITR / CMV / GFP vector and pHelper (Takara Bio Inc.) vector.
Each solution was prepared so that the DNA concentration was 3.5 to 3.6 μg / mL, which contained two types of plasmids at equimolar concentrations. Polyethylenimine and FBS were added to this solution so that the concentrations were 12 μg / mL and 1.3% (v / v), respectively, and DMEM / High Glucose medium was added to adjust the liquid volume to 30 mL. Was used as a transfection solution. After removing all the culture supernatants of the cells seeded on the 15 cm dish, add the entire amount of 30 mL of the transfection solution and incubate for 3 days at 37 ° C. in the presence of 5% CO ₂ to produce rAAV virions. It was.

[Example 14] Production of rAAV virions by transient expression of integrated rAAV plasmid
HEK293T cells were seeded in a 15 cm dish at a concentration of 1.65 × 10 ⁷ cells / dish and cultured in DMEM / High Glucose medium containing 10% FBS at 37 ° C. in the presence of 5% CO ₂ for 16 hours. Prepare 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 as a DNA solution for transfection. did. Polyethylenimine and FBS were added to this solution so that the concentrations were 12 μg / mL and 1.3% (v / v), respectively, and DMEM / High Glucose medium was added to adjust the liquid volume to 30 mL. Was used as a transfection solution. After removing all the culture supernatants of the cells seeded on the 15 cm dish, add the entire amount of 30 mL of the transfection solution, and incubate for 3 days at 37 ° C. in the presence of 5% CO ₂ , with rAAV6 virion and rAAV9 virion. Was produced respectively.

[Example 15] Recovery of culture supernatant After completion of the culture of Examples 12 to 14, 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 for preparing the cell lysate solution of Example 17.

[Example 16] Measurement of rAAV genome amount and total capsid amount contained in culture supernatant The virus genome content in the culture supernatant is a kit using AAVpro Titration Kit for Real Time PCR Ver.2 (Takara Bio). According to the protocol attached to, the measurement was performed by the following method. To 2 μL of the culture supernatant collected in Example 15, 2 μL of 10 × DNase I Buffer, 1 μL of DNase I, and 15 μL of pure water were added, mixed, and incubated at 37 ° C. for 1 hour. The DNase I 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 heat-treated solution was diluted 100-fold with EASY Dilution (Takara Bio Inc.) to prepare a sample solution for real-time PCR.

12.5 μL of TB Green Premix Ex TaqII and 0.6 μL of 10 μM forward primer in 5 μL of each of the sample solution for real-time PCR and the standard plasmid solution for calibration line (straightened pAAV-CMV-GFP vector). Primer 1, SEQ ID NO: 33), 0.6 μL 10 μM reverse primer (Primer 2, SEQ ID NO: 34), 0.08 μL ROX Reference Dye II, and 6.5 μL water were added. For the real-time PCR reaction, 35 cycles of 2-step PCR (95 ° C, 5 seconds → 60 ° C, 30 seconds) were performed after denaturation conditions (95 ° C, 2 minutes). The measured value of the sample was interpolated into the obtained calibration curve to determine the amount (number) of rAAV genome contained in the sample. The region replicated in this PCR is the internal region of the ITR.

The total amount of capsid in the culture supernatant was measured by the following method using the AAV Titration ELISA Kit (PROGEN) according to the protocol attached to the kit. 100 μL of the culture supernatant (sample sample) collected in Example 15 diluted with 1 × Assay Buffer was added to a plate on which the mouse anti-AAV9 monoclonal antibody was immobilized, and the mixture was allowed to stand at 37 ° C. for 1 hour. Kit Control diluted with 1 × Assay Buffer to a concentration of 1.3 × 10 ^{9 to} 1.0 × 10 ⁷ capsids / mL was added as a standard solution to the plate in the same manner as the sample sample and allowed to stand. The plate was washed 3 times with 1 × Assay Buffer, biotin-labeled mouse anti-AAV9 antibody or mouse anti-AAV6 antibody was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. The plate was washed 3 times with 1 × Assay Buffer, HRP-labeled streptavidin was added, and the plate was allowed to stand at 37 ° C. for 1 hour. The plate was washed 3 times with 1 × Assay Buffer, HRP substrate was added, and the mixture was allowed to stand at room temperature for 15 minutes, and then sulfuric acid was added to stop the reaction. The measured value of the sample was inserted into the calibration curve obtained from the measured value of the absorbance of the standard solution, and the total amount (number) of capsids contained in the sample was determined.

[Example 17] Preparation of cell lysate solution In the cells in which the cell culture solution was centrifuged and precipitated in Example 15, 1% TritonX-100, 4 mM MgCl ₂ , 50 U / mL Turbonuclease (Accelagen), and 4 × A citrate buffer (32 mM sodium citrate) containing Protease Inhibitor Cocktail (Sigma) was added, and the cells were allowed to stand at 37 ° C. for 1.5 hours. After neutralization by adding 4 mM MgCl ₂ containing citric acid buffer (68 mM citric acid), the mixture was centrifuged at 6000 × g at 4 ° C. for 10 minutes. 25 mM citric acid buffer (pH 3.6, 8 mM citric acid) containing 0.25% TritonX-100, 4 mM MgCl ₂ , 12.5 U / mL Turbonuclease (Accelagen), and 1 × Protease Inhibitor Cocktail (Sigma). (Sodium + 17 mM citric acid) was added in a double amount to prepare a cell lysate.

An SP-Sepharose 1 mL column (GE Healthcare) was equilibrated with 25 mM citric acid buffer (pH 3.6, 8 mM sodium citrate + 17 mM citric acid) containing 4 mM MgCl ₂ , and cell lysate was added thereto. Was offered. After elution with 50 mM citric acid buffer (pH 5.18, 40.2 mM sodium citrate + 9.8 mM citric acid) containing 4 mM MgCl ₂ , 500 mM NaCl, 1 M Tris buffer so that the final concentration is 50 mM. Was added to neutralize the eluate. Place the neutralized sample in Vivaspin-500 100K MWCO (Vivaproducts), centrifuge at 3000 × g at 4 ° C for 10 minutes, add 180 mM NaCl, 0.001% F68-containing PBS, and add further 3000 × g at 4 ° C for 10 minutes. The buffer was replaced by centrifugation. The obtained solution was used as a cell lysate solution.

[Example 18] Measurement of rAAV genome amount and total capsid amount contained in cell lysate solution The virus genome content in the purified cell lysate solution is AAVpro Titration Kit for Real Time PCR Ver.2 (Takara Bio). According to the protocol attached to the kit, the measurement was performed by the following method. To the 2 μL cell lysate solution sample prepared in Example 17, 2 μL of 10 × DNase I Buffer, 1 μL of DNase I, and 15 μL of pure water were added, mixed, and incubated at 37 ° C. for 1 hour. The DNase I 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-fold with EASY Dilution (Takara Bio Inc.) to prepare a sample solution for real-time PCR.

12.5 μL of TB Green Premix Ex TaqII and 0.6 μL of 10 μM forward primer in 5 μL of each of the sample solution for real-time PCR and the standard plasmid solution for calibration lines (straightened pAAV-CMV-GFP vector). Primer 1, SEQ ID NO: 33), 0.6 μL 10 μM reverse primer (Primer 2, SEQ ID NO: 34), 0.08 μL ROX Reference Dye II, and 6.5 μL water were added. For the real-time PCR reaction, 35 cycles of 2-step PCR (95 ° C, 5 seconds → 60 ° C, 30 seconds) were performed after denaturation conditions (95 ° C, 2 minutes). The measured values of the sample were interpolated into the obtained calibration curve to determine the amount of AAV virus genome (vg) contained in the sample. Here, vg is a unit indicating the number of AAV virus genomes. The region replicated in this PCR is the internal region of the ITR.

The total amount of capsid in the cell lysate solution was measured using the AAV Titration ELISA Kit (PROGEN) by the following method according to the protocol attached to the kit. 100 μL of the cell lysate solution (sample sample) prepared in Example 17 diluted with 1 × Assay Buffer was added to a plate on which the mouse anti-AAV9 monoclonal antibody was immobilized, and the mixture was allowed to stand at 37 ° C. for 1 hour. Kit Control diluted with 1 × Assay Buffer to a concentration of 1.3 × 10 ^{9 to} 1.0 × 10 ⁷ capsids / mL was added as a standard solution to the plate in the same manner as the sample sample and allowed to stand. The plate was washed 3 times with 1 × Assay Buffer, biotin-labeled mouse anti-AAV9 antibody or mouse anti-AAV6 antibody was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. The plate was washed 3 times with 1 × Assay Buffer, HRP-labeled streptavidin was added, and the plate was allowed to stand at 37 ° C. for 1 hour. The plate was washed 3 times with 1 × Assay Buffer, HRP substrate was added, and the mixture was allowed to stand at room temperature for 15 minutes, and then sulfuric acid was added to stop the reaction. The measured value of the sample was inserted into the calibration curve obtained from the measured value of the absorbance of the standard solution, and the total amount (number) of capsids contained in the sample was determined.

[Example 19] Calculation of abundance ratio of rAAV virion in total capsid From the measurement result of the amount of rAAV genome contained in the cell lysate solution measured in Example 18 and the measurement result of the total amount of capsid, recombination containing the rAAV genome The amounts of AAV virion (rAAV virion) and empty capsid were calculated respectively. The amount of rAAV virions was taken as the amount of rAAV genomes (vg) as the amount (number) of rAAV virions, assuming that the entire rAAV genome was packaged in capsids to form virions. (Amount of rAAV virions / total amount of capsids) X100% was calculated as the abundance ratio of rAAV virions in the total capsids. The empty capsid amount (number) is calculated by subtracting the amount (number) of rAAV virions from the measured value of the total capsid amount (number).

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

After 3 days, all the culture supernatants of the 24-well plate were removed, washed with PBS, 20 μL of TrypLE ^TM (enzyme solution having trypsin-like activity: Thermo Fisher Scientific) was added, and the mixture was allowed to stand at 37 ° C. for 2 minutes. Then, it was suspended again in 1 mL of PBS, and 65 μL of this was used to measure the proportion of GFP-positive cells with MOXI-GO II (ORFLO).

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

After 3 days, all the culture supernatants of the 24-well plate were removed, washed with PBS, 20 μL of TrypLE ^TM (enzyme solution having trypsin-like activity: Thermo Fisher Scientific) was added, and the mixture was allowed to stand at 37 ° C. for 2 minutes. Then, it was suspended again in 1 mL of PBS, and 65 μL of this was used to measure the proportion of GFP-positive cells with MOXI-GO II (ORFLO).

[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 virion) obtained when host cells are infected with the total amount of the cell lysate solution. ([Total number of cells in well] x [Percentage of GFP-positive cells (%)] / 100) x ([Total amount of rAAV virion sample (mL)] / [Amount of rAAV virion sample put into each well (mL)])

[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 FIG. , (1) is pHelper-ITR / CMV / GFP vector and pR2 (mod) C9 vector, (2) is pHelper-R2 (mod) C9 vector and pAAV-CMV-GFP vector, (3) is pR2 (mod) ) The results when two types of plasmids, C9-ITR / CMV / GFP vector and pHelper vector, are introduced, and the rAAV genome amounts are 2.6 × 10 ¹¹ vg, 7.2 × 10 ¹¹ vg, 11.1 × 10 ¹¹ vg, respectively. Met. (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 ¹¹ vg. On the other hand, (4) shows the result when the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced, and the amount of rAAV genome was 35.0 × 10 ¹¹ vg.

The measurement results of the total capsid amount are shown by white bars in FIG. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and the pR2 (mod) C9 vector, (2) is the pHelper-R2 (mod) C9 vector and the pAAV-CMV-GFP vector, and (3) is the pR2 (mod). The results when two types of plasmids, C9-ITR / CMV / GFP vector and pHelper vector, were introduced, and the total capsid amount was 2.6 × 10 ¹² capsids, 5.6 × 10 ¹² capsids, and 5.9 × 10 ¹² capsids, respectively. It was. (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 capsid amount was 3.6 × 10 ¹² capsids. On the other hand, (4)% showed the results when the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced, and the total capsid amount was 6.6 × 10 ¹² capsids. Both the rAAV genome amount and the total capsid amount showed the highest expression level when the integrated rAAV plasmid, that is, the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced (Fig. 12).

[Example 24] Result (Measurement of amount of rAAV contained in 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 FIG. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and the pR2 (mod) C9 vector, (2) is the pHelper-R2 (mod) C9 vector and the pAAV-CMV-GFP vector, and (3) is the pR2 (mod). The results when two types of plasmids, C9-ITR / CMV / GFP vector and pHelper vector, were introduced, and the rAAV genome amounts were 2.5 × 10 ¹¹ vg, 23.1 × 10 ¹¹ vg, and 22.0 × 10 ¹¹ vg, respectively. It was. (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 vg. On the other hand, (4) shows the result when the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced, and the rAAV genome amount was 29.7 × 10 ¹¹ vg (Fig. 13).

The measurement results of the total capsid amount are shown by white bars in FIG. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and the pR2 (mod) C9 vector, (2) is the pHelper-R2 (mod) C9 vector and the pAAV-CMV-GFP vector, and (3) is the pR2 (mod). The results when two types of plasmids, C9-ITR / CMV / GFP vector and pHelper vector, were introduced, and the total capsid amount was 3.3 × 10 ¹² capsids, 5.3 × 10 ¹² capsids, and 5.3 × 10 ¹² capsids, respectively. It was. (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 capsids. On the other hand, (4) shows the result when the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced, and the amount of rAAV genome was 6.1 × 10 ¹² caps ids. Both the results of rAAV genome amount and total capsid amount were the highest expression levels when the integrated rAAV plasmid, that is, the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced (Fig. 13).

[Example 25] Results (Ratio of rAAV virions in total capsid)
In theory, when there is no empty capsid and all capsids are rAAV virions containing rAAV, the rAAV genome amount and total capsid amount obtained from the rAAV genome amount and total capsid amount of the cell lysate solution calculated in Example 17 The abundance ratio of the amount of capsid is 1: 1. The abundance ratio of rAAV virions (rAAV virions) in the total capsid is shown in FIG. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and the pR2 (mod) C9 vector, (2) is the pHelper-R2 (mod) C9 vector and the pAAV-CMV-GFP vector, and (3) is the pR2 (mod). The results when two types of plasmids, C9-ITR / CMV / GFP vector and pHelper vector, were introduced were shown, and the abundance 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 abundance ratio was 17.8%. On the other hand, (4) shows the result when the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced, and the abundance ratio was 48.6% (Fig. 14). From this result, 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 pHelper-ITR. It was shown that the proportion of empty capsids was low when the / CMV / GFP-R2 (mod) C9 vector was introduced.

[Example 26] Results (Measurement of infectivity of rAAV6 virion contained in cell lysate solution)
The infectivity measurement result of rAAV6 virion contained in the cell lysate solution prepared in Example 17 is shown in FIG. Black bars in the figure show the results when 2 × 10 ⁸ rAAV6 virions were infected. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and the pRC6 vector, (2) is the pHelper-R2 (mod) C6 vector and the pAAV-CMV-GFP vector, and (3) is the pR2 (mod) C6-ITR. The results when two types of plasmids, the / CMV / GFP vector and the pHelper vector, were introduced were shown, and the proportions of GFP-positive cells were 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. The ratio of GFP-positive cells was 0.6%, and (4) was pHelper-ITR / CMV. The results when the / GFP-R2 (mod) C6 vector was introduced were shown, and the proportion of GFP-positive cells was 11.2%.

Moreover, in FIG. 15, the white bar shows the result when 2 × 10 ⁹ rAAV6 virions were infected. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and pRC6 vector, (2) is the pHelper-R2 (mod) C6 vector and pAAV-CMV-GFP vector, and (3) is the pR2 (mod) C6-ITR /. The results when two types of plasmids, CMV / GFP vector and pHelper vector, were introduced were shown, and the proportions of GFP-positive cells were 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. The ratio of GFP-positive cells was 12.6%, and (4) was pHelper-ITR / CMV. The results when the / GFP-R2 (mod) C6 vector was introduced were shown, and the proportion of GFP-positive cells was 71.1%.

For these results, pHelper-R2 (mod) C6 vector and pAAV-CMV-GFP vector, or pR2 (mod) C6-ITR / CMV / GFP vector and pHelper vector, two types of plasmids, or pHelper- It is shown that the rAAV6 virion solution with high infectivity can be obtained by using the ITR / CMV / GFP-R2 (mod) C6 vector.

[Example 27] Results (Measurement of infectivity of rAAV9 virion contained in cell lysate solution)
The infectivity measurement result of rAAV9 virion contained in the cell lysate solution prepared in Example 17 is shown in FIG. Black bars in the figure show the results when 2 × 10 ⁸ rAAV9 virions were infected. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and the pR2 (mod) C9 vector, (2) is the pHelper-R2 (mod) C9 vector and the pAAV-CMV-GFP vector, and (3) is the pR2 (mod). The results when two types of plasmids, C9-ITR / CMV / GFP vector and pHelper vector, were introduced were shown, and the proportions of GFP-positive cells were 0.8%, 0.3%, and 2%, respectively. (5) The results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector and pR2 (mod) C9 vector, are introduced, the ratio of GFP-positive cells is 3.5%, and (4) is pHelper-. The results when the ITR / CMV / GFP-R2 (mod) C9 vector was introduced were shown, and the proportion of GFP-positive cells was 7.3%.

Moreover, in FIG. 16, the white bar shows the result when 2 × 10 ⁹ rAAV9 virions were infected. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and the pR2 (mod) C9 vector, (2) is the pHelper-R2 (mod) C9 vector and the pAAV-CMV-GFP vector, and (3) is the pR2 (mod). The results when two types of plasmids, C9-ITR / CMV / GFP vector and pHelper vector, were introduced were shown, and the proportions of GFP-positive cells were 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 proportion of GFP-positive cells was 21.7%, and (4) was pHelper. The results when the -ITR / CMV / GFP-R2 (mod) C9 vector was introduced were shown, and the proportion of GFP-positive cells was 50.3%.

These results indicate that a highly infectious rAAV9 virion solution can be obtained by using the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector.

[Example 28] Result (Calculation of Transducing Unit)
The infectivity measurement result of rAAV6 virion contained in the cell lysate solution prepared in Example 17 is shown in FIG. In the figure, (1) is the pHelper-ITR / CMV / GFP vector and pRC6 vector, (2) is the pHelper-R2 (mod) C6 vector and pAAV-CMV-GFP vector, and (3) is the pR2 (mod) C6-ITR. The results when two types of plasmids, the / CMV / GFP vector and the pHelper vector, are introduced are shown. 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.

FIG. 18 shows the measurement results of the infectivity of rAAV9 virion contained in the cell lysate solution prepared in Example 17. In the figure, (1) is pHelper-ITR / CMV / GFP vector and pR2 (mod) C9 vector, (2) is pHelper-R2 (mod) C9 vector and pAAV-CMV-GFP vector, (3). Shows the results when two types of plasmids, pR2 (mod) C9-ITR / CMV / GFP vector and pHelper vector, are introduced. In addition, (5) the results when three types of plasmids, pHelper vector, pAAV-CMV-GFP vector, and pR2 (mod) C9 vector, are introduced are shown. In addition, (4) shows the results when the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector was introduced.

These results should be introduced into host cells using an integrated rAAV plasmid, namely the pHelper-ITR / CMV / GFP-R2 (mod) C6 vector or the pHelper-ITR / CMV / GFP-R2 (mod) C9 vector. This indicates that the more infectious rAAV6 virion or rAAV9 virion can be produced, respectively.

[Example 29] Construction of pAAV-CBA (BAM) -I2S vector pHelper-ITR / CMV / GFP-R2 (mod) C6 vector or pHelper-ITR / CMV / GFP-R2 (mod) C9 vector up to Example 28 From the experimental results of these two types of vectors, it was found that highly infectious rAAV6 virion or rAAV9 virion can be produced by using the integrated rAAV plasmid. The high infectivity of this integrated rAAV plasmid is considered to be due to the high abundance ratio of rAAV virions in the total capsid. Therefore, in order to further verify the results, an integrated rAAV plasmid having a different structure was constructed, and the abundance ratio of rAAV virions in the total capsid was confirmed by the following experiment.

From the 5'side, a DNA fragment containing the nucleotide sequence shown by SEQ ID NO: 35 containing 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 was synthesized. A PCR reaction was carried out using this DNA fragment as a template and 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 a restriction enzyme (HindIII / BglII), and the DNA fragment obtained by the above PCR reaction was mixed with this, and both were combined with the in fusion HD cloning Kit (in fusion HD cloning Kit ( It was fused using Takara Bio). The obtained plasmid was used as a pAAV-CBA (BAM) -I2S vector (Fig. 19). Here, the human I2S gene encodes a human IDS containing the amino acid sequence set forth in SEQ ID NO: 36 (same as in Example 30). The human CMV enhancer has the nucleotide sequence shown by SEQ ID NO: 37, the chicken β-actin promoter has the nucleotide sequence shown by SEQ ID NO: 38, the chicken β-actin / MVM chimeric intron has the nucleotide sequence shown by SEQ ID NO: 39, and bovine growth hormone. The polyA sequence contains the base sequence shown by SEQ ID NO: 40.

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

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 the pHelper-ITR / CBA (BAM) vector. The obtained plasmid was used as a pHelper-ITR / CBA (BAM) /-R2 (mod) C9 vector.

A DNA fragment having the nucleotide sequence shown by 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 obtained plasmid was used as a pHelper-ITR / CBA (BAM) / I2S-R2 (mod) C9 vector (Fig. 20).

[Example 31] Production of rAAV virions by transient expression of three types of plasmids (2)
HEK293T cells were seeded in T-25 flasks at a concentration of 2.5 × 10 ⁶ cells / flask and cultured in MEM medium containing 10% FBS (Sigma) at 37 ° C. in the presence of 5% CO ₂ for 16 hours. As DNA for transfection, a solution containing pHelper vector (Takara Bio Inc.), pAAV-CBA (BAM) -I2S vector, and pR2 (mod) C9 vector was prepared. This solution was prepared so that the DNA concentration was 2.9 to 3.9 μg / mL, which contained three types of plasmids at equimolar concentrations. Polyethyleneimine was added to this solution so that the concentration ratio was DNA (μg): polyethyleneimine (μg) = 1: 3, and MEM medium was further added to adjust the liquid volume to 5 mL for transfection. It was used as a solution. After removing all the culture supernatants of the cells seeded in the T-25 flask, add the entire amount of 5 mL of the transfection solution and incubate for 5 days at 37 ° C. in the presence of 5% CO ₂ to produce rAAV virions. I let you. Part of the rAAV virion produced in the host cell is released into the culture supernatant, and part remains in the host cell. The same applies to Example 32.

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

[Example 33] Recovery of culture supernatant After completion of the culture of Examples 31 and 32, the cell culture solution was transferred to a 15 mL centrifuge tube and centrifuged at 4 ° C. and 5800 × g for 5 minutes to recover the culture supernatant.

The culture supernatant was recovered in Preparative Example 33 of rAAV virions solution from supernatants Example 34] culture, an endonuclease Benzonase ^TM a (Merck name) was added to a concentration of 14 U / mL They were mixed and incubated at 37 ° C for 2 hours. POROS ^TM CaptureSelect ^TM AAV9 Affinity Resin (Thermo Fisher Scientific) 0.15 mL, which is a column filled with a resin having a specific affinity for AAV9, is contained in a binding buffer (20 mM Tris-HCl, 150 mM NaCl, 4 mM MgCl _2). , PH 7.5), and the culture supernatant treated with endonuclease was loaded onto the equilibrium. After washing the column with binding buffer, the solution containing rAAV virion was eluted through 0.5 mL elution buffer (containing 0.1 M citric acid, pH 2.06). The resulting eluate was passed through PD MiniTrap ^TM G-25 (GE Healthcare) pre-equilibrium with PBS containing 150 mM NaCl and 0.001% F68 to bind rAAV virions to the column. The rAAV virion was then eluted with 1 mL PBS containing 150 mM NaCl and 0.001% F68. This was concentrated by centrifugation at 4 ° C. and 2000 × g for 3 minutes using a Vivaspin ^TM turbo 15 (100K MWCO, PES, Sartorius). The obtained solution was used as a rAAV virion solution.

[Example 35] Measurement of rAAV genome amount and total capsid amount contained in rAAV virion solution AAVpro Titration Kit for Real Time PCR Ver.2 (Takara) is used to measure the virus genome content in the rAAV virion solution obtained in Example 34. Using Bio (Bio), real-time PCR measurement was performed by the following method according to the protocol attached to the kit. To 2 μL of rAAV virion solution prepared in Example 34, 2 μL of 10 × DNase I Buffer, 1 μL of DNase I, and 15 μL of pure water were added and mixed, and incubated at 37 ° C. for 30 minutes. The DNase I 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. 12.5 μL TB in 5 μL each of the sample solution for real-time PCR and the Kit control solution for calibration lines diluted with EASY Dilution (Takara Bio) to a concentration of 2 × 10 ² to 2 × 10 ⁷ genome copies / μL. Green Premix Ex TaqII, 0.6 μL 10 μM forward primer (primer 1, SEQ ID NO: 33), 0.6 μL 10 μM reverse primer (primer 2, SEQ ID NO: 34), 0.08 μL ROX Reference Dye II, and 6.5 μL Water was added. For the real-time PCR reaction, 35 cycles of 2-step PCR (95 ° C, 5 seconds → 60 ° C, 30 seconds) were performed after denaturation conditions (95 ° C, 2 minutes). The measured value of the sample was interpolated into the obtained calibration curve to determine the amount (number) of rAAV genome contained in the sample. The region replicated in this PCR is the internal region of the ITR.

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

The DNase I-treated rAAV virion solution was appropriately diluted with a TE buffer containing 0.05% F-68 to prepare a sample solution for droplet digital PCR. 1.0 μM forward primer (primer 3, SEQ ID NO: 41), 1.0 μM reverse primer (primer 4, SEQ ID NO: 42), and 0.25 μM probe (probe 1, SEQ ID NO: 43 with FAM as a reporter dye at the 5'end. A FAM-labeled 20 × primer / probe mix containing BHQ1 as a quencher dye at the 3'end (each modified) was prepared. In addition, 1.0 μM forward primer (primer 5, SEQ ID NO: 44), 1.0 μM reverse primer (primer 6, SEQ ID NO: 45), and 0.25 μM probe (probe 2, HEX as a reporter dye at the 5'end of SEQ ID NO: 46). , A HEX-labeled 20 × primer / probe mix containing BHQ1 as a quencher dye at the 3'end) was prepared.

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

The total amount of capsid in the culture supernatant was measured by the following method using the AAV Titration ELISA Kit (PROGEN) according to the protocol attached to the kit. 100 μL of the rAAV virion solution prepared in Example 34 diluted with 1 × Assay Buffer was added to a plate on which the mouse anti-AAV9 monoclonal antibody was immobilized, and the mixture was allowed to stand at 37 ° C. for 1 hour. A known amount of AAV9 empty capsid diluted with 1 × Assay Buffer to a concentration of 1.0 × 10 ^{7 to} 1.3 × 10 ⁹ capsids / mL was added to a plate as a standard solution in the same manner as the sample sample and allowed to stand. The plate was washed 3 times with 1 × Assay Buffer, biotin-labeled mouse anti-AAV9 antibody was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. The plate was washed 3 times with 1 × Assay Buffer, HRP-labeled streptavidin was added, and the plate was allowed to stand at 37 ° C. for 1 hour. The plate was washed 3 times with 1 × Assay Buffer, HRP substrate was added, and the mixture was allowed to stand at room temperature for 15 minutes, and then sulfuric acid was added to stop the reaction. The measured value of the sample was inserted into the calibration curve obtained from the measured value of the absorbance of the standard solution, and the total amount (number) of capsids contained in the sample was determined.

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

Theoretically, if there are no empty capsids and all capsids are rAAV virions containing the rAAV genome, the abundance ratio of rAAV genome to total capsid is 1: 1. The amount of rAAV genome (vg) was defined as the amount (number) of rAAV virions, assuming that all rAAV genomes were packaged in capsids to form virions. (Amount (number) of rAAV virions / total capsid amount (number)) X100 (%) is used as the abundance ratio of rAAV virions in the total capsids, and the abundance ratio is calculated using the amount of rAAV genome obtained by real-time PCR. Then, it was 12.7% when three types of plasmids were used, and 62.2% when the integrated rAAV plasmid was used. That is, pHelper-ITR / CBA (BAM) / I2S-R2 (mod) C9 as well as pHelper-ITR / CMV / GFP-R2 (mod) C6 vector and pHelper-ITR / CMV / GFP-R2 (mod) C9 vector. In the case of vectors as well, it was verified that the abundance ratio of rAAV virions in the total capsid can be increased by using the integrated rAAV plasmid. In addition, assuming that the amount of rAAV virions (vg) is the amount (number) of rAAV virions, using the integrated rAAV plasmid can obtain 8 times or more of the rAAV virions compared to the case of using three types of plasmids. Therefore, by using the integrated rAAV plasmid for the production of rAAV virions, the production efficiency of rAAV virions can be significantly increased. Similar conclusions can be obtained from the results of quantification of the amount of rAAV genome by the supplementary droplet digital PCR method.

According to one embodiment of the present invention, it is possible to efficiently produce an rAAV virion in which a nucleic acid sequence containing a base sequence encoding a foreign protein, which can be used for gene therapy or the like, is packaged, and thus the rAAV virion can be medically treated. It can be stably supplied to the engine.

SEQ ID NO: 1: Nucleotide sequence encoding Rep protein of AAV of serum type 2, Wild type SEQ ID NO: 2: Amino acid sequence of Rep68 of AAV of serum type 2, Wild type SEQ ID NO: 3: Amino acid of Rep78 of AAV of serum type 2 Sequence, wild type SEQ ID NO: 4: A preferable example of the base sequence encoding the Rep protein of AAV of serum type 2, synthetic sequence No. 5: the base sequence encoding VP1 of AAV of serum type 6, wild type SEQ ID NO: 6: AAV VP1 amino acid sequence of serum type 6, wild type SEQ ID NO: 7: nucleotide sequence encoding VP1 of AAV of serum type 9, wild type SEQ ID NO: 8: amino acid sequence of VP1 of AAV of serum type 9, wild type sequence No. 9: Nucleotide sequence of the first reverse terminal repeat (first ITR) of AAV of serum type 2, Wild type SEQ ID NO: 10: First reverse terminal repeat of AAV of serum type 2 (second ITR) ), Wild type SEQ ID NO: 11: A suitable example of the base sequence of the first reverse end repeat (first ITR), Synthetic sequence SEQ ID NO: 12: Second reverse end repeat (second ITR) ), Synthetic sequence SEQ ID NO: 15: A preferred example of the base sequence of the E4 region, Synthetic sequence SEQ ID NO: 17: A preferred example of the base sequence of the helper region, Synthetic sequence SEQ ID NO: 19: 1st A preferred example of the base sequence contained in the gene expression control unit, synthetic sequence SEQ ID NO: 20: a suitable example of the base sequence of the Rep region, synthetic sequence SEQ ID NO: 21: a preferred example of the base sequence of the Cap region, synthetic sequence SEQ ID NO: 22: A suitable example of the base sequence of the Rep-Cap region, Synthetic sequence SEQ ID NO: 23: A suitable example of the base sequence of the Rep-Cap region (2), Synthetic sequence SEQ ID NO: 24: To prepare a recombinant AAV virion A preferred example of the nucleotide sequence of the nucleic acid molecule used, Synthetic SEQ ID NO: 25: Nucleotide sequence of synthetic human β-globin intron, Nucleotide sequence 26: Nucleotide sequence of synthetic polyadenylation signal sequence, Nucleotide sequence No. 27: Nucleotide sequence including SalI site, RsrII site, ColE1 ori, ampicillin resistance gene, BsiWI site, BstZ17I site, and BsrGI site, synthetic sequence SEQ ID NO: 28: EcoRI site, MluI site, GFP gene, NotI site, and BamHI site Nucleotide sequence, Nucleotide sequence SEQ ID NO: 29: Nucleotide sequence of multicloning site 1, Nucleotide sequence SEQ ID NO: 30: Nucleotide sequence of multicloning site 2, Nucleotide sequence SEQ ID NO: 31: AfeI site, RsrII site, BsrGI Site, AvrII site, Ampicillin resistance gene, ColE1 ori, RsrII site, 5'part of Rep region of serum type 2 (including p5 promoter), and nucleotide sequence containing SacII site, Synthetic sequence SEQ ID NO: 32: HindIII site, serum Nucleotide sequence containing 3'part of Rep region of AAV of type 2, Cap region of serum type 9, p5 promoter, RsrII site, and BsrGI site, Nucleotide sequence SEQ ID NO: 33: Forward primer, Nucleotide sequence of primer 1, Nucleotide sequence SEQ ID NO: 34: Nucleotide sequence of reverse primer, primer 2, Synthesis SEQ ID NO: 35: Contains human CMV enhancer, chicken β-actin promoter, chicken β-actin / MVM chimeric intron, human I2S gene, and bovine growth hormone polyA sequence. Nucleotide sequence, Nucleotide sequence SEQ ID NO: 36: Nucleotide sequence of human IDS SEQ ID NO: 37: Nucleotide sequence of human CMV enhancer Nucleotide sequence No. 38: Nucleotide sequence of chicken β-actin promoter SEQ ID NO: 39: Nucleotide 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: Nucleotide sequence of primer 3, Nucleotide sequence of primer 42: Nucleotide sequence of primer 4, Nucleotide sequence of probe 1 SEQ ID NO: 44: Nucleotide sequence of Primer 5, Nucleotide sequence Sequence No. 45: Nucleotide sequence of Primer 6, Nucleotide sequence No. 46: Nucleotide sequence of probe 2, Nucleotide sequence No. 47: Contains human CMV enhancer and chicken β-actin promoter. Nucleotide sequence, Nucleotide sequence SEQ ID NO: 48: Nucleotide sequence of primer 7, Nucleotide sequence No. 49: Nucleotide sequence of primer 8, Nucleotide sequence No. 50: Human CMV enhancer, chicken β-actin promoter, chicken β-actin / MVM chimeric intron, Nucleotide sequence containing the bovine growth hormone poly A sequence, Nucleotide sequence SEQ ID NO: 51: Nucleotide sequence containing the human IDS gene, Nucleotide sequence

Claims (46)

Nucleic acid molecule containing at least the following base sequence;
(A) Nucleotide sequence encoding the Rep protein of adeno-associated virus or its functional equivalent,
(B) Nucleotide sequence encoding cap protein of adeno-associated virus or its functional equivalent,
(C) Nucleotide sequence containing the reverse terminal repeat of the first adeno-associated virus (first ITR) or its functional equivalent,
(D) Nucleotide sequence containing the reverse terminal repeat of the second adeno-associated virus (second ITR) or its functional equivalent,
(E) A base sequence for inserting a base sequence encoding a foreign protein and / and a base sequence encoding a foreign protein located between the first ITR and the second ITR,
(F) Nucleotide sequence containing the E2A region of adenovirus or its functional equivalent,
(G) A base sequence containing the E4 region of adenovirus or its functional equivalent, and (h) a base sequence containing the VA1 RNA region of adenovirus or its functional equivalent. A base sequence containing a first gene expression control site that controls the expression of the Rep protein,
The first aspect of claim 1, further comprising a base sequence containing a second gene expression control site that controls the expression of the Cap protein, and a base sequence containing a third gene expression control site that controls the expression of the foreign protein. Nucleic acid molecule. The nucleic acid molecule of claim 1 or 2, further comprising a base sequence containing a drug resistance gene. Downstream of the base sequence containing the first gene expression control site,
Nucleotide sequence encoding the Rep protein,
Further downstream, it contains a base sequence containing the second gene expression control site, and further downstream contains a base sequence encoding the Cap protein.
The nucleic acid molecule of claim 2 or 3. Downstream of the base sequence containing the first ITR,
Nucleotide sequence containing the third gene expression control site,
A base sequence for inserting a base sequence encoding the foreign protein and / and a base sequence encoding the foreign protein further downstream,
Further downstream, it contains a base sequence containing the second ITR.
The nucleic acid molecule according to any one of claims 2 to 4. Downstream of the nucleotide sequence of the E2A region of the adenovirus or its functional equivalent,
Nucleotide sequence of the E4 region of the adenovirus or its functional equivalent,
Further downstream, it contains the base sequence of the adenovirus VA1 RNA region or its functional equivalent.
The nucleic acid molecule according to any one of claims 2 to 5. Downstream of the base sequence encoding the Cap protein,
Nucleotide sequence containing the first reverse terminal repeat (ITR),
Nucleotide sequence containing the third gene expression control site further downstream,
A base sequence for inserting a base sequence encoding the foreign protein and / and a base sequence encoding the foreign protein further downstream,
Further downstream, it contains a base sequence containing the second reverse terminal repeat (ITR).
The nucleic acid molecule of claim 4. Downstream of the base sequence containing the second reverse terminal repeat (ITR),
Nucleotide sequence of the E2A region of the adenovirus or its functional equivalent,
Further downstream, the base sequence of the E4 region of the adenovirus or its functional equivalent,
Further downstream, it contains the base sequence of the adenovirus VA1 RNA region or its functional equivalent.
The nucleic acid molecule of claim 7. The nucleic acid molecule according to any one of claims 1 to 8, further comprising a base sequence containing a drug resistance gene. The nucleic acid molecule of claim 9, wherein the base sequence containing the drug resistance gene is contained downstream of the base sequence encoding the Cap protein and upstream of the base sequence containing the first ITR. The nucleic acid molecule according to any one of claims 1 to 10, wherein a base sequence having an enhancer function for increasing the expression level of the Rep protein and the Cap protein is contained downstream of the base sequence encoding the Cap protein. Any of claims 1-11, 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. Nucleic acid molecule. The nucleic acid molecule of claim 12, wherein the Rep protein comprises Rep68 and / and Rep78. Any of claims 1 to 13, 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. Nucleic acid molecule. The nucleic acid molecule of claim 14, wherein the Cap protein comprises VP1. The nucleic acid molecule according to any one of claims 2 to 15, wherein the first gene expression control site is controlled by the E2A region of the adenovirus or a functional equivalent thereof. The nucleic acid molecule of claim 16, wherein the first gene expression control site is the P5 promoter of an adeno-associated virus. The nucleic acid molecule according to any one of claims 2 to 17, wherein the second gene expression control site is controlled by VA1 RNA of adenovirus or a functional equivalent thereof. The nucleic acid molecule of claim 18, wherein the second gene expression control site is the P40 promoter of an adeno-associated virus. Any of claims 1-19, wherein the adeno-associated virus serotype of the ITR is selected from the group consisting of 1,2,3,4,5,6,7,8,9,10 and 11. Nucleic acid molecule. The nucleic acid molecule of any of claims 1-19, wherein the adeno-associated virus serotype of the ITR is 2. The nucleic acid molecule according to any one of claims 1 to 21, wherein the base sequence for encoding the foreign protein includes a multicloning site. The serotypes of the adenovirus 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 to 30, 37, 40, 41, AdHu2, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdHu49, AdHu50, AdC6, It is selected from the group consisting of serotype, dog Ad2 type, sheep Ad, and porcine Ad3 type.
The nucleic acid molecule according to any one of claims 1 to 22. 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, claims 1-23. Any nucleic acid molecule. Any of claims 1 to 24, wherein the functional equivalent of the E4 region of the adenovirus is derived from any of the groups selected from the group consisting of herpes simplex virus, vaccinia virus, cytomegalovirus, and pseudorabies. Nucleic acid molecule. 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, claims 1-25. Nucleic acid molecule of any. The nucleic acid molecule of any of claims 2 to 26, wherein the E2A region of the adenovirus is controlled by E1B of the adenovirus or a functional equivalent thereof. The nucleic acid molecule of any of claims 2-27, wherein the E4 region of the adenovirus is regulated by E1B of the adenovirus or a functional equivalent thereof. The nucleic acid molecule according to any one of claims 9 to 28, wherein the drug resistance gene imparts resistance to a drug corresponding to the drug resistance gene to prokaryotic cells. The third gene expression control site is represented by a promoter derived from cytomegalovirus, an SV40 early promoter, a human elongation factor-1alpha (EF-1α) promoter, a human ubiquitin C promoter, a β-actin promoter, and SEQ ID NO: 47. It is selected from the group consisting of a promoter containing a base sequence and a promoter containing the base sequence represented by SEQ ID NO: 47 downstream of the promoter containing the base sequence represented by SEQ ID NO: 47.
The nucleic acid molecule according to any one of claims 2 to 29. The nucleic acid molecule according to any one of claims 11 to 30, wherein the base sequence having the enhancer function is the P5 promoter of an adeno-associated virus or a functional equivalent thereof. The nucleic acid molecule according to any one of claims 1 to 31, wherein the foreign protein is of human origin. The nucleic acid molecule according to any one of claims 1 to 31, 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. The foreign proteins are growth hormone, lysosome enzyme, somatomedin, insulin, glucagon, lysosome enzyme, cytokine, phosphokine, blood coagulation factor, antibody, fusion protein of antibody and other proteins, granulocyte macrophage colony stimulator (GM- CSF), granulocyte colony stimulator (G-CSF), macrophage colony stimulator (M-CSF), erythropoetin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicular stimulator (FSH), gonads Stimulant hormone releasing hormone (GnRH), gonadotropin, DNasel, thyroid stimulating hormone (TSH), nerve growth factor (NGF), hairy neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3 , Neurotrophin 4/5, Neurotrophin 6, Neurotrophin 6, Neurogulin 1, Activin, Basic fibroblast growth factor (bFGF), Fibroblast growth factor 2 (FGF2), Epithelial cell growth factor (EGF), Vascular endothelium Has the activity of degrading growth factors (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), beta amyloid. It is selected from the group consisting of enzymes, etanercept, pegbisomant, metrereptin, avatacept, ashotase, and GLP-1 receptor agonists.
The nucleic acid molecule according to any one of claims 1 to 32. The foreign protein is a lysosomal enzyme, and the lysosomal enzyme is α-L-izronidase, isulonic acid-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activated protein, β-hexosaminidase A, β. -Hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartyl glucosaminidase, α-N- Acetylgalactosaminidase, acidic sphingomyelinase, α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetylCoAα-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, It is selected from the group consisting of acidic ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidylpeptidase-1, hyaluronidase-1, CLN1 and CLN2.
The nucleic acid molecule according to any one of claims 1 to 32. The foreign proteins are anti-IL-6 antibody, anti-beta amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-HER2 antibody, anti-PCSK9 antibody, anti-TNF-α antibody. , And the nucleic acid molecule of any of claims 1-33, which is selected from the group consisting of antibodies having an affinity for proteins present on the surface of vascular endothelial cells. The proteins present on the surface of the vascular endothelial cells are transferase receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transport carrier 1, A group consisting of organic anion transporters, monocarboxylic acid transporters, low-density lipoprotein receptor-related proteins 1, low-density lipoprotein receptor-related proteins 8, and membrane-bound precursors of heparin-binding epithelial growth factor-like growth factors. 36. The nucleic acid molecule of claim 36, which is selected from. The foreign protein is a fusion protein of an antibody and another protein.
The antibody is selected from the group consisting of mouse antibodies, humanized antibodies, human-mouse chimeric antibodies, and human antibodies, and the other proteins are growth hormones, lysosome enzymes, cytokines, lymphocaines, blood coagulation. Factors, antibodies, fusion proteins of antibodies to other proteins, granulocyte macrophage colony stimulator (GM-CSF), granulocyte colony stimulator (G-CSF), macrophage colony stimulator (M-CSF), erythropoetin, darbepoetin , Tissue plasminogen activator (t-PA), thrombomodulin, follicular stimulating hormone, DNasel, thyroid stimulating hormone (TSH), nerve growth factor (NGF), hairy neurotrophic factor (CNTF), glial cell line neuronutrition Factors (GDNF), Neurotrophin 3, Neurotrophin 4/5, Neurotrophin 6, Neurotrophin 6, Neurogulin 1, Activin, Basic Fibroblast Growth Factor (bFGF), Fibroblast Growth Factor 2 (FGF2), Epithelium Cell growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor) , And an enzyme that has the activity of degrading beta-amyloid.
The nucleic acid molecule according to any one of claims 1-31. The foreign protein is a fusion protein of an antibody and another protein.
The antibody is selected from the group consisting of mouse antibody, humanized antibody, human-mouse chimeric antibody, and human antibody, and the other protein is a lysosomal enzyme, and the lysosomal enzyme is α-L-. Izlonidase, isulonic acid-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activating 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, acidic sphingomyelinase, α-galactosidase A, β-glucuronidase, Heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl CoAα-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acidic seramidase, amilo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1 , Tripeptidylpeptidase-1, hyaluronidase-1, CLN1 and CLN2.
The nucleic acid molecule according to any one of claims 1-31. The nucleic acid molecule of claim 38 or 39, wherein the other protein is of human origin. The antibody has an affinity for proteins present on the surface of vascular endothelial cells.
The nucleic acid molecule according to any one of claims 38 to 40. The proteins present on the surface of the vascular endothelial cells are transferase receptor, insulin receptor, leptin receptor, insulin-like growth factor I receptor, insulin-like growth factor II receptor, lipoprotein receptor, glucose transport carrier 1, A group consisting of organic anion transporters, monocarboxylic acid transporters, low-density lipoprotein receptor-related proteins 1, low-density lipoprotein receptor-related proteins 8, and membrane-bound precursors of heparin-binding epithelial growth factor-like growth factors. The nucleic acid molecule of claim 41, which is selected from. The nucleic acid molecule according to any one of claims 1-42, which is a cyclic plasmid. A method for producing recombinant AAV particles, which comprises the step of introducing the nucleic acid molecule according to any one of claims 1 to 43 into a host cell. The production method according to claim 44, wherein the genome of the host cell contains a base sequence containing an E1A region and an E1B region of adenovirus. The production method according to claim 45, wherein the host cell is a HEK293 cell.

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