WO2010050586A1 - 組み換え蛋白質の発現を増強する方法 - Google Patents
組み換え蛋白質の発現を増強する方法 Download PDFInfo
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- WO2010050586A1 WO2010050586A1 PCT/JP2009/068682 JP2009068682W WO2010050586A1 WO 2010050586 A1 WO2010050586 A1 WO 2010050586A1 JP 2009068682 W JP2009068682 W JP 2009068682W WO 2010050586 A1 WO2010050586 A1 WO 2010050586A1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
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- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18811—Sendai virus
- C12N2760/18841—Use of virus, viral particle or viral elements as a vector
- C12N2760/18843—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to a method for enhancing the expression of a recombinant protein in an RNA virus vector, a method for preparing a recombinant protein useful for a pharmaceutical or the like using the method, and an RNA virus vector for expressing the recombinant protein.
- RNA virus vectors for gene transfer applied to gene therapy and the like.
- the expression level of the loaded gene is directly linked to the function of the vector itself, so that the expression level can be increased. If possible, the effectiveness of the vector can be improved, and its use / application range can be greatly expanded.
- AB5 toxin produced by bacteria is a proteinous exotoxin that is produced by toxin-producing bacteria and secreted outside the cell.
- a subunit (Active subunit) that has activity as a toxin 1 And 5 B subunits (Binding subunits) that have binding activity with cells.
- Cholera toxin (cholera toxin) is one such AB5 toxin, and the non-toxic cholera toxin B subunit (CTB) is used as a molecular adjuvant as an influenza virus vaccine (Isaka M. et al., Microbiol Immunol. 2008). ; 52 (2): 55-63.), Pertussis vaccine (Isaka M.
- the present invention has been made in view of the above circumstances, and the problem to be solved by the present invention is to enhance the expression of recombinant proteins and peptides useful for pharmaceutical compositions in RNA viral vectors, and to provide recombinant proteins and peptides.
- Gene for therapeutic use in gene therapy, gene vaccines, monoclonal antibody production, etc. by providing a method for facilitating the preparation of RNA and introducing this technology into gene transfer vectors derived from RNA viruses It is to provide a more effective gene transfer vector with a high expression level.
- the present inventors have found that when expressing proteins and peptides from RNA viruses, there are not a few examples in which the amount of expression is small and preparation is difficult depending on the properties of the protein or peptide to be expressed. In particular, it was found that the expression efficiency was extremely low when a short peptide or a protein with low solubility was expressed from an RNA virus vector.
- AB toxin B subunit is a protein of more than a dozen KD, and its three-dimensional structural analysis has revealed that it constitutes a pentamer (McCann JA et al., Biochemistry. 1997; 36 ( 30): 9169-78., Pickens JC et al., Chem Biol. 2004; 11 (9): 1205-1215).
- Choleratoxin B (CTB) one of AB5B, has been used as an adjuvant so far, but the change in protein expression by using CTB has not been known.
- the present inventors significantly increased the expression level when expressing this fusion protein using a Sendai virus vector compared to expressing the recombinant protein as it is. I found out.
- corticotropin releasing factor corticotropin releasing factor
- endothelin-1 EGF: corticotropin releasing factor
- NPY neuropeptide Y
- GLP-2 Glucagon-Like Peptide-2
- amyloid ⁇ -42 A ⁇ 42: amyloid ⁇
- AB5B markedly improved the expression level of RNA viral vectors.
- carrier proteins other than AB5B such as PEDI and IL-4
- AB5B showed a significantly higher expression enhancing effect. It should be noted that it functions more effectively than spherical proteins such as IL-4.
- RNA virus vectors have particularly low expression efficiency of proteins with relatively short chain lengths, and it has been difficult to express sufficient amounts of these proteins.
- these proteins as fusion proteins with AB5B based on the present invention, the expression level can be remarkably increased even for proteins having a short chain length.
- the enhancement of the expression level is effective for preparing the recombinant protein itself.
- the use of the expressed fusion protein as it is may be considered, but it may be desirable to remove AB5B, in which case, between the target recombinant protein / peptide, Cleavage by introducing protease cleavage sequences such as enterokinase recognition sequence (DDDDK ⁇ ), Factor Xa recognition sequence (IEGR ⁇ ), thrombin recognition sequence (LVPR ⁇ GS), HRV 3C protease recognition sequence (LEVLFQ ⁇ GP) ⁇ Can be released.
- protease cleavage sequences such as enterokinase recognition sequence (DDDDK ⁇ ), Factor Xa recognition sequence (IEGR ⁇ ), thrombin recognition sequence (LVPR ⁇ GS), HRV 3C protease recognition sequence (LEVLFQ ⁇ GP) ⁇
- DDDDK ⁇ enterokinase recognition sequence
- IEGR ⁇
- CTB Cholera toxin
- AB5B fusion functions effectively to enhance the expression of recombinant proteins in vivo, ex vivo, and in vitro, such as gene therapy, gene vaccine, and monoclonal antibody production using gene-transferred RNA viral vectors.
- Expression enhancement is directly linked to the function of the vector itself, and it is possible to increase the effectiveness of the vector.
- the present invention relates to a method for enhancing the expression of a recombinant protein in an RNA viral vector by fusing the recombinant protein with the AB5B protein, a method for preparing a recombinant protein useful for pharmaceuticals and the like using the method, and AB5B More specifically, it relates to an RNA virus vector encoding a protein fusion protein, and more specifically [1] an RNA virus vector encoding a recombinant protein, wherein the protein is encoded as a fusion protein with AB5 toxin B subunit.
- the present invention makes it possible to increase the expression level of a recombinant protein from an RNA virus vector.
- recombinant proteins such as short peptidic proteins and low-solubility proteins
- these proteins from RNA virus vectors can be obtained by applying the present invention to peptides whose expression level decreases, particularly in RNA virus vectors. It is possible to effectively increase the expression level of.
- RNA virus vector of the present invention as a vector for gene introduction, effective gene therapy, gene vaccine, monoclonal antibody production, etc. can be carried out.
- SeV cDNA (pSeV18 + CTB-mCRF / ⁇ F, pSeV18 + CTB-mET1 / ⁇ F, pSeV18 + CTB-mPYY / ⁇ F, pSeV18 + CTB-mGLP2 / ⁇ F, pSeV18 + mCRF / ⁇ F, pSeV18 + mET1 / ⁇ F + YPVYY It is a figure which shows the structure of (DELTA (DELTA) F, (epsilon) pSeV18 + mGLP2 / (DELTA) F).
- CTB-mCRF, CTB-mET1, CTB-mPYY, CTB-mGLP2, mCRF, mET1, mPYY, and mGLP2 genes contained in the NotI site of pCI were excised and inserted into the NotI site of pSeV18 + NotI / ⁇ F.
- CTB-A ⁇ 15 NotI fragment construction diagram CTB-A ⁇ 15x2, CTB-A ⁇ 15x4, CTB-A ⁇ 15x8 NotI fragment construction diagram.
- FIG. 1 It is a figure which shows the A (beta) antibody titer of the Tg2576 mouse
- the SeV18 + CTB-A ⁇ 15x8 / ⁇ F administration group (right column) clearly showed a decrease in the area ratio in each part of the brain, especially in the hippocampus. It can be seen that is large.
- the present invention is an RNA viral vector encoding a recombinant protein, wherein the protein is encoded as a fusion protein with an AB5 toxin B subunit (AB5B) protein, a method for producing the vector,
- the present invention relates to a composition containing the vector and a method for increasing the expression level of a recombinant protein using the vector.
- the present inventors have found that the expression of a target protein can be significantly increased by using a nucleic acid encoding AB5B in combination with an RNA virus vector. That is, by expressing the target protein as a fusion protein with AB5B from an RNA virus vector, the expression of the protein can be remarkably increased.
- an RNA virus refers to a virus having an RNA genome and having no DNA phase in the life cycle.
- RNA viruses do not have reverse transcriptase (ie, do not include retroviruses). That is, in virus propagation, the viral genome is replicated by RNA-dependent RNA polymerase without DNA.
- RNA viruses include single stranded RNA viruses (including positive and negative stranded RNA viruses) and double stranded RNA viruses.
- a virus having an envelope (enveloped viruses) and a virus having no envelope (non-enveloped viruses) are used, but a vector derived from an envelope virus is preferably used.
- RNA viruses specifically include viruses belonging to the following families.
- Arenaviridae such as Lassa virus Orthomyxoviridae such as influenza virus (including Orthomyxoviridae; including Infuluenza virus A, B, C, and Thogoto-like viruses) Coronaviridae such as SARS virus Togaviridae such as rubella virus Paramyxoviridae such as mumps virus, measles virus, Sendai virus, RS virus Picornaviridae such as Poliovirus, Coxsackie virus, Echovirus Filoviridae, such as Marburg virus and Ebola virus Flaviviridae such as yellow fever virus, dengue virus, hepatitis C virus, hepatitis G virus Bunyaviridae (including Bunyaviridae; including Bunyavirus, Hantavirus, Nairovirus, and Phlebovirus genera) Rhabdoviridae such as rabies virus Reoviridae
- a viral vector is a vector (carrier) for having a genomic nucleic acid derived from the virus and expressing the gene from the genomic nucleic acid by incorporating a transgene into the nucleic acid.
- the virus vector includes infectious virus particles, virus core, complex of virus genome and virus protein, non-infectious particles (non-infectious virus-like particles or non-infectious virus particles), and the like. A complex that has the ability to express a gene carried by introduction into a cell.
- RNA virus a ribonucleoprotein (viral core portion) comprising a viral genome and a viral protein that binds to it can be introduced into the cell to express the transgene in the cell (WO00 / 70055).
- ribonucleoprotein (RNP) is also included in the viral vector in the present invention.
- Introduction into cells can be performed using a transfection reagent or the like as appropriate.
- RNP containing viral genomic RNA and non-infectious viral particles include calcium phosphate (Chen, C. & Okayama, H. (1988) BioTechniques 6: 632-638; Chen, C. and Okayama, H., 1987, Mol.Cell. Biol. 7: 2745), DEAE-dextran (Rosenthal, N. (1987) Methods Enzymol. 152: 704-709)
- Various liposome-based transfection reagents (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)), electroporation (Ausubel, F.
- transfection reagents are DOTMA (Roche), Superfect Transfection Ragent (QIAGEN, Cat No. 301305), DOTAP, DOPE, DOSPER (Roche # 1811169), TransIT-LT1 (Mirus, Product No.
- Envelope viruses are known to take up host cell-derived proteins during virus particle formation, and such proteins are considered to cause antigenicity and cytotoxicity when introduced into cells ( J. Biol. Chem. (1997) 272, 16578-16584). Therefore, there is an advantage in using RNP from which the envelope has been removed (WO00 / 70055).
- RNA virus vector in the present invention is a minus-strand RNA virus vector.
- the minus-strand RNA viral vector is a viral vector derived from a virus that contains minus-strand RNA (strand that encodes a viral protein in an antisense manner) as a genome. Negative strand RNA is also called negative strand RNA.
- a single-stranded minus-strand RNA virus also referred to as a non-segmented minus-strand RNA virus
- a “single-stranded negative strand RNA virus” refers to a virus having a single-stranded negative strand [ie, minus strand] RNA in the genome.
- viruses examples include paramyxovirus (including Paramyxoviridae; Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus), rhabdoviridae; Vesiculovirus, Lyssavirus, Lyssavirus, and Ephemerovirus etc.
- Viruses belonging to this family are included, and taxonomologically belongs to the order of Mononegavirales. (Virus Vol.57 No.1, pp29-36, 2007; Annu. Rev. Genet. 32, 123-162, 1998; Fields virology fourth edition, Philadelphia, Lippincott-Raven, 1305-1340,2001; Microbiol. Immunol. 43, 613-624, 1999; Field Virology, Third edition pp. 1205-1241, 1996).
- examples of the minus-strand RNA virus vector include a paramyxovirus vector.
- Paramyxovirus vectors are viral vectors derived from the Paramyxoviridae virus.
- the Sendai virus of the Paramyxoviridae virus can be mentioned.
- Newcastle disease virus (Newcastle disease virus), mumps virus (Mumps virus), measles virus (Measles virus), RS virus (Respiratory syncytial virus), rinderpest virus, distemper virus (distemper virus) , Simian parainfluenza virus (SV5), human parainfluenza virus types 1,2,3, orthomyxoviridae influenza virus (Influenza virus), rhabdoviridae vesicular stomatitis virus (Vesicular stomatitis virus) ), And rabies virus (Rabies virus).
- Sendai virus SeV
- HPIV-1 human parainfluenza virus-1
- HPIV-3 human parainfluenza virus-3
- PDV canine
- Sendai virus SeV
- human parainfluenza virus-1 HPIV-1
- human parainfluenza virus-3 HPIV-3
- phocine distemper virus PDV
- canine distemper virus CDV
- dolphin molbillivirus DMV
- Peste-des-petits-ruminants virus PDPR
- melesles virus MV
- rinderpest virus RSV
- Hendra virus Hendra
- the vector used in the present invention is, for example, a virus belonging to the Paramyxovirus subfamily (including the Respirovirus genus, Rubravirus genus, and Morbillivirus genus) or a derivative thereof, such as the Respirovirus genus (genus Respirovirus). ) (Also referred to as Paramyxovirus) or a derivative thereof.
- Derivatives include viruses in which viral genes have been modified, chemically modified viruses, and the like so as not to impair the ability to introduce genes by viruses.
- respirovirus viruses to which the present invention can be applied examples include human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 3 (HPIV-3), and bovine parainfluenza virus type 3 (BPIV-3).
- HPIV-1 human parainfluenza virus type 1
- HPIV-3 human parainfluenza virus type 3
- BPIV-3 bovine parainfluenza virus type 3
- Sendai virus also referred to as mouse murine parainfluenza virus type 1
- SPIV-10 simian parainfluenza virus type 10
- RNA virus vectors may be derived from natural strains, wild strains, mutant strains, laboratory passage strains, artificially constructed strains, and the like. Further, it may or may not have propagation ability.
- the term “transmissibility” refers to the ability of a virus vector to produce infectious virus particles by replicating a virus in a host cell.
- the viral vector may be a viral vector having the same structure as a virus isolated from nature, or a viral vector artificially modified by genetic recombination. For example, any gene possessed by the wild-type virus may be mutated or defective. It is also possible to use incomplete viruses such as DI particles (J. Virol. 68: 8413-8417, 1994).
- a virus having a mutation or deletion in at least one gene encoding a viral envelope protein or outer shell protein can be preferably used.
- Such viral vectors can replicate the genome in infected cells, but cannot form infectious viral particles.
- Such a replication-defective virus vector is highly safe because there is no concern of spreading infection around it.
- proteins necessary for genome replication are encoded in genomic RNA
- the genome can be amplified in infected cells.
- a gene is deleted from the viral genome, and the deleted gene product or a protein capable of complementing it is supplied exogenously in virus-producing cells (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74 (14) 6564-6569 (2000)).
- a method for recovering a viral vector as a non-infectious viral particle (VLP) without completely complementing a defective viral protein is also known (WO00 / 70070).
- VLP non-infectious viral particle
- the vector can be produced without complementing the envelope protein.
- a virus vector containing a protein different from the envelope protein inherent in the virus in the envelope can be prepared.
- a virus containing this can be produced by expressing a desired foreign envelope protein in a virus-producing cell during virus production.
- Proteins such as a desired adhesion factor, a ligand, and a receptor which provide the infectious ability to a mammalian cell, are used.
- Specific examples include G protein (VSV-G) of vesicular stomatitis virus (VSV).
- VSV-G protein may be derived from any VSV strain.
- a VSV-G protein derived from a Indiana serotype strain J. Virology 39: 519-528 (1981)
- RNA virus vector of the present invention encodes a recombinant protein as a fusion protein with AB5 toxin B subunit.
- AB5 toxin is a toxin consisting of one A subunit and five B subunits, and is common in many pathogenic bacteria (Merritt E, and Hol W (1995) "AB5 toxins", Curr Opin Struct Biol 5 (2): 165-71; Lencer W, and Saslowsky D (2005) "Raft trafficking of AB5 subunit bacterial toxins", Biochim Biophys Acta 1746 (3): 314-21).
- AB5 toxins examples include Campylobacter jejuni enterotoxin, cholera toxin (Vibrio cholerae), heat-labile enterotoxins (e.g. LT and LT-II) (pertussis toxin), pertussis
- LT and LT-II heat-labile enterotoxins
- pertussis examples include Shiga-like toxin or ⁇ ⁇ verotoxin produced by Bordetella pertussis, Shiga ⁇ toxinin, Shigella dysenteriae, and other enterohemorrhagic bacteria.
- the toxicity of these toxins is borne by the A subunit, and the B subunit forms a pentamer and is thought to be involved in cell adhesion.
- AB5 toxins in the present invention include cholera toxin and E. coli heat-labile enterotoxin, both of which are very similar both structurally and functionally (Hovey BT et al., J Mol Biol., 1999, 285). (3): 1169-78; Ricci S. et al., Infect Immun. 2000, (68 (2):-760-766; Tinker JK et al., Infect Immun. 2005, 73 (6): 3627-3635).
- Specific B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin include accession number ZP_01954889.1, ZP_01976878.1, NP_231099.1, P13811.1, ABV01319.1, P32890, and SEQ ID NO: 68, or those Examples include proteins containing mature proteins (excluding 21 amino acids at the N-terminus; eg, 22 to 124 amino acids). Base sequences encoding these include NZ_AAWE01000267.1, NC_002505.1, M17874.1, EU113246.1, and M17873.1, or sequences encoding their mature proteins (for example, 63 bases of 5 'were excluded) Sequence) and the like.
- Suitable AB5 toxin B subunits in the present invention include those containing an amino acid sequence encoded by these amino acid sequences or base sequences, or having high similarity to these amino acid sequences.
- the AB5 toxin B subunit may have a mutation as well as the natural sequence. Unless there is a significant decrease in the expression of the fusion protein compared to the case of using the natural B subunit, for example, 1 or a small number (for example, several, three, five, ten, less, fifteen) B subunits having an amino acid sequence of up to 20 amino acids) added, deleted, substituted and / or inserted.
- polypeptide in which 1 to several residues (for example, 2, 3, 4, 5, 6, 10, 15 or 20 residues) of amino acids at the N-terminal and / or C-terminal are deleted or added, and 1 to several Polypeptides in which amino acids of residues (for example, 2, 3, 4, 5, 6, 10, 15 or 20 residues) are substituted can also be used.
- Variants that can be used include, for example, fragments of natural proteins, analogs, derivatives, and fusion proteins with other polypeptides (eg, those added with heterologous signal peptides or antibody fragments).
- an activity that includes a sequence in which one or more amino acids of a wild-type amino acid sequence are substituted, deleted, and / or added, and increases the expression of a fused recombinant protein equivalent to the wild-type B subunit.
- a polypeptide having can be used as the AB5 toxin B subunit.
- a variant of AB5 toxin B subunit preferably retains the activity of forming a pentamer.
- a wild-type protein fragment it usually contains a continuous region of 70% or more, preferably 80% or more, more preferably 90% or more of the wild-type polypeptide (in the case of a secreted protein, the mature form).
- Amino acid sequence variants can be prepared, for example, by introducing mutations into DNA encoding a natural polypeptide (Walker and Gaastra, eds. Techniques in Molecular Biology (MacMillan Publishing Company, New York, 1983); Kunkel Proc. Natl. Acad. Sci. USA 82: 488-492, 1985; Kunkel et al., Methods Enzymol. 154: 367-382, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harboratory Press, Plainview, NY), 1989; US Pat. No., 4,873,192).
- Guidance for substitution of amino acids so as not to affect biological activity includes, for example, Dayhoff et al.
- AB5B includes, for example, the R192G mutant of E. coli enterotoxin LT (Lemere et al., Neurobiol. Aging 2002, 23: 991-1000; Seabrook et al., Neurobiol. Aging, 2004, 25: 1141-1151; Seabrook et al ., Vaccine, 2004, 22: 4075-7083).
- the number of amino acids to be modified is not particularly limited, but for example, within 30%, preferably within 25%, more preferably within 20%, more preferably within 15%, more preferably within the total amino acids of a natural mature polypeptide. Within 10%, within 5%, within 3% or within 1%, for example within 15 amino acids, preferably within 10 amino acids, more preferably within 8 amino acids, more preferably within 5 amino acids, more preferably within 3 amino acids. is there.
- substituting an amino acid it can be expected to maintain the activity of the protein by substituting an amino acid having a similar side chain property. Such substitution is referred to as conservative substitution in the present invention.
- Conservative substitutions include, for example, basic amino acids (eg, lysine, arginine, histidine), acidic amino acids (eg, aspartic acid, glutamic acid), uncharged polar amino acids (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non- Each of polar amino acids (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched amino acids (eg threonine, valine, isoleucine), and aromatic amino acids (eg tyrosine, phenylalanine, tryptophan, histidine) Examples include substitution between amino acids in the group.
- basic amino acids eg, lysine, arginine, histidine
- acidic amino acids eg, aspartic acid, glutamic acid
- uncharged polar amino acids
- the modified protein shows high homology with the amino acid sequence of the wild type protein.
- High homology is, for example, an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 96% or more identity.
- Amino acid sequence identity can be determined, for example, using the BLASTP program (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990). For example, on the BLAST web page of NCBI (National Center ch Biothchnology Information), search can be performed using default parameters (Altschul SF et al., Nature Genet.
- the identity value for the entire amino acid sequence of a natural protein is calculated.
- the ratio of the number of matching amino acids in the total number of amino acids of wild type protein cocoon (or mature type in the case of a secreted protein) cocoon is calculated.
- AB5 toxin B subunit is a protein encoded by a nucleic acid that hybridizes under stringent conditions with part or all of the coding region of a gene encoding a wild type protein, and has the same activity (RNA And a protein having an activity to increase expression in a virus.
- the protein preferably forms a pentamer.
- a probe is prepared from either a nucleic acid containing a sequence of the coding region of a wild-type protein gene or a complementary sequence thereof, or a nucleic acid to be hybridized, and whether it hybridizes to the other nucleic acid. Can be identified by detecting.
- the stringent hybridization conditions are, for example, 5x SSC, 7% (W / V) SDS, 100 ⁇ g / ml denatured salmon sperm DNA, 5x Denhardt solution (1x Denhardt solution is 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, And 0.2% Ficoll) at 50 ° C., preferably 60 ° C., more preferably 65 ° C., followed by 2 ⁇ SSC, preferably 1 ⁇ SSC, more preferably 0.5 at the same temperature as the hybridization.
- the condition is that the washing is performed in xSSC, more preferably in 0.1xSSC for 2 hours while shaking.
- the signal peptide (typically the first 21 amino acids) originally possessed by the AB5 toxin B subunit may be left alone or fused to the recombinant protein or removed, eg, from a protein from a eukaryotic cell.
- a signal peptide may be added to the N-terminus or replaced with the original signal peptide (see Examples).
- a signal sequence of a desired secretory protein such as immunoglobulin-kappa-light chain, interleukin- (IL) -2, tissue-plasminogen-activator (tPA) can be used, but is not limited thereto.
- the fusion protein can further include a tag, a linker, and a spacer.
- the target recombinant protein and AB5 toxin B subunit may be directly bonded or may be bonded via a linker (or spacer).
- the sequence of the linker / spacer is not particularly limited, and may be, for example, a sequence of 1 to 15 amino acids, preferably 1 to 8 amino acids, for example 2 to 6 amino acids, for example, about 4 amino acids. ), GP (glycine-proline), GPGP (glycine-proline-glycine-proline), GGS (glycine-glycine-serine), GGGS, GGGGS, repetitions thereof, or any combination thereof. It is not limited to.
- the target recombinant protein and AB5B are usually fused so that AB5B is located on the N-terminal side of the recombinant protein, that is, the target recombinant protein is positioned on the C-terminal side of AB5B.
- the recombinant protein to be fused with AB5 toxin B subunit is not particularly limited, but it can significantly increase the expression level of proteins that have been difficult to express from RNA virus vectors. it can. These include, for example, proteins consisting of short peptide chains.
- the short polypeptide is, for example, 300 amino acids or less, preferably 250, 220, 210, 200, 180, 160, 140, 120, 100, 90, 80, 75, 70, 65, 62, 60, 55, 50, 45. , 40, 35, 30, 25, 20, or any peptide consisting of the following amino acids (excluding the AB5 toxin B subunit portion).
- the recombinant protein to be expressed is, for example, 7 amino acids or more, preferably 8, 10, or 15 amino acids or more (for example, 20 or more, 25 or more, 30 or more, 40 or more, 41 or more). That is, the present invention is an RNA viral vector encoding a recombinant protein, wherein the protein is encoded as a fusion protein with AB5 toxin B subunit, and the recombinant protein is, for example, a peptide having 120 amino acids or less, or a peptide having 50 amino acids or less A vector is provided.
- “recombinant protein” refers to a protein that has been subjected to fusion with the AB5 toxin B subunit.
- the length of the recombinant protein does not include AB5 toxin B subunit.
- the length of the recombinant protein is the length of the portion obtained by removing the AB5 toxin B subunit from the fusion protein.
- proteins and peptides with low water solubility are also suitable as recombinant proteins.
- a protein with low water solubility means that the amount of water or physiological saline required to dissolve 1 mg of protein within 30 minutes is 1 ml or more when shaken vigorously at 20 ⁇ 5 ° C. every 5 minutes for 30 seconds.
- the amount of water or physiological saline required to dissolve 1 mg of protein within 30 minutes is 10 ml or more, more preferably 100 ml or more.
- proteins to be fused with AB5B include neuropeptides and endocrine peptides.
- RNA viral vector of the present invention that expresses a recombinant protein as a fusion protein with AB5B has, for example, a protein expression level that is, for example, compared to the case where the same recombinant protein is expressed from the same RNA viral vector as a single body without being fused with AB5B.
- a protein expression level that is, for example, compared to the case where the same recombinant protein is expressed from the same RNA viral vector as a single body without being fused with AB5B.
- the host cell may be appropriately selected. For example, BHK21 cells (ATCC CCL-10) may be used.
- the target protein includes a desired protein that is difficult to be highly expressed in an RNA virus vector.
- an expression plasmid vector incorporating a nucleic acid encoding the protein is used to express the protein.
- the ratio of the expression level of the protein when the protein is expressed from the same nucleic acid sequence in the RNA virus vector to the expression level of the protein when expressed is, for example, mouse
- the relative ratio is 0.8 or less, preferably 0.75 or less, 0.7 or less, 0.65 or less, 0.6 or less, 0.55 or less, 0.5 or less compared to the ratio measured using the nucleic acid sequence encoding IL-4 (SEQ ID NOs: 81 and 82).
- 0.45 or less, 0.4 or less, 0.35 or less, 0.3 or less, 0.25 or less, or 0.2 or less protein may be mentioned.
- an expression plasmid vector for example, an expression plasmid vector having a CMV promoter such as pcDNA TM 3.1 (Invitrogen) is used, and transfection is performed under appropriate conditions (for example, using a 24-well plate, 1 ⁇ g of plasmid is transferred to Lipofectamine TM 2000 ( Invitrogen)).
- RNA viral vectors are introduced, for example, with MOI 3 using the same host cell. Cells are collected 72 hours after gene transfer, and if it is a secreted protein, the protein concentration in the culture supernatant or the amount of expressed recombinant protein relative to the total protein mass of the collected cells is calculated. This can be measured for mouse IL-4 and the protein of interest, and the values of both can be compared.
- the vector of the present invention may have another foreign gene at a position other than the insertion of the gene encoding the fusion protein of AB5 toxin B subunit.
- it may be a marker gene for monitoring infection of a vector, or it may be a cytokine, hormone, receptor, antibody, fragment thereof, or other gene that regulates the immune system.
- Recombination of the recombinant RNA virus vector may be performed using a known method. Specifically, (a) in the presence of a viral protein that constitutes an RNP containing viral genomic RNA, it encodes the genomic RNA of the RNA virus that encodes the recombinant protein as a fusion protein with the AB5 toxin B subunit or its complementary strand. It can be produced by introducing RNA into a cell or transcribing it in the cell, and (b) collecting the produced virus or RNP containing the genomic RNA.
- the above-mentioned viral proteins constituting RNP typically refer to proteins that form RNP together with viral genomic RNA and constitute nucleocapsid. These are a group of proteins required for genome replication and gene expression.
- N also referred to as nucleocapsid (or nucleoprotein (NP))
- P phospho
- L Large protein
- desired mammalian cells and avian cells can be used.
- desired mammalian cells and avian cells can be used.
- desired mammalian cells and avian cells can be used.
- ATCC CCL-7 monkey kidney-derived LLC-MK 2 cells
- CV-1 cells examples thereof include cultured cells such as ATCC CCL-70
- hamster kidney-derived BHK cells for example, ATCC CCL-10
- human-derived cells and the like.
- a method for producing viral vectors using eggs has already been developed (Nakanishi et al., (1993), "Advanced Protocol III for Neuroscience Research, Molecular Neuronal Physiology", Koseisha, Osaka, pp.153-172. ). Specifically, for example, fertilized eggs are placed in an incubator and cultured at 37-38 ° C. for 9-12 days to grow embryos. A viral vector is inoculated into the allantoic cavity, eggs are cultured for several days (for example, 3 days) to proliferate the viral vector, and the urine fluid containing the virus is collected. Isolation and purification of virus vectors from urine can be performed according to conventional methods (Tatsuto Tashiro, “Virus Experiment Protocol”, supervised by Nagai and Ishihama, Medical View, pp. 68-73, (1995)).
- the viral protein necessary for particle formation may be expressed from the transcribed viral genomic RNA, or may be supplied to trans from other than genomic RNA.
- N, P, and L proteins can be supplied by introducing an expression plasmid or the like that expresses them into cells.
- the transcribed genomic RNA is replicated in the presence of these viral proteins to form infectious viral particles.
- a vector in which a DNA encoding the protein or genome is linked downstream of an appropriate promoter is introduced into a host cell. Examples of the promoter include CMV promoter (Foecking, MK, and Hofstetter H.
- the defective protein and / or other viral proteins that can complement its function are expressed in the virus-producing cells to complement the infectivity of the virus produced. can do.
- it can be pseudotyped with an envelope protein of a minus-strand RNA virus that has a different origin from the genome of the viral vector.
- an envelope protein for example, the G protein (VSV-G) of vesicular stomatitis virus (VSV) (J.
- genes to be deleted from the genome include spike protein genes such as F, HN, H and G, envelope lining protein genes such as M, and any combination thereof. Deletion of spike protein gene is effective for making non-transmissible, for example, minus-strand RNA viral vectors, and deletion of protein protein on the back of envelope such as M protein makes particle formation from infected cells impossible.
- F gene-deleted negative-strand RNA viral vectors Li, H.-O. et al., J.Virol. 74, 6564-6569 (2000)
- M gene-deleted negative-strand RNA viral vectors Inoue , M. et al., J.Virol. 77, 6419-6429 (2003)
- a vector lacking any combination of at least two genes of F, HN (or H), and M is more secure.
- both M and F gene deletion vectors are non-transmissible and lack particle formation while maintaining high levels of infectivity and gene expression.
- viruses can be produced more efficiently by using host cells in which the F gene is integrated into the chromosome (WO00 / 70070).
- expression can be induced using a sequence-specific recombinant enzyme such as Cre / loxP or FLP / FRT and its target sequence so that the F gene can be induced and expressed (WO00 / 70055).
- WO 00/70070 Hasan, M. K.
- an envelope protein gene is incorporated into a vector having a recombinant enzyme target sequence such as Cre / loxP inducible expression plasmid pCALNdlw (Arai, raiT. Et al., J. Virology 72, 1998, p1115-1121).
- a recombinant enzyme target sequence such as Cre / loxP inducible expression plasmid pCALNdlw (Arai, raiT. Et al., J. Virology 72, 1998, p1115-1121).
- Induction of expression is performed, for example, by infecting adenovirus AxCANCre with MOI 3-5 (Saito et al., Nucl. Acids Res. 23: 3816-3821 (1995); Arai, T.et al., J. Virol 72,1115-1121 (1998)).
- any viral gene contained in the vector is modified from a wild-type gene in order to reduce immunogenicity due to a viral protein, or to increase RNA transcription efficiency or replication efficiency.
- a viral gene contained in the vector is modified from a wild-type gene in order to reduce immunogenicity due to a viral protein, or to increase RNA transcription efficiency or replication efficiency.
- Good for example, in a minus-strand RNA viral vector, it is considered that at least one of N, P, and L genes that are replication factors is modified to enhance the function of transcription or replication.
- HN protein which is one of the envelope proteins, has both hemagglutinin activity and neuraminidase activity, which are hemagglutinins.
- the former activity can be weakened, for example, It may be possible to improve the stability of the virus, and for example, by modifying the activity of the latter, it is also possible to regulate the infectivity.
- membrane fusion ability can be regulated by modifying the F protein.
- an antigen-presenting epitope of F protein or HN protein that can be an antigen molecule on the cell surface is analyzed, and a viral vector having a reduced antigen-presenting ability with respect to these proteins can be produced.
- a temperature-sensitive mutation can be introduced into a viral gene for the purpose of suppressing secondary release particle (or VLP: virus-like particle) release (WO2003 / 025570).
- viral protein mutations include the 86th Glu (E86) mutation of the SeV P protein, the substitution of the 511st Leu (L511) of the SeV P protein with other amino acids, or other negative strands.
- examples include substitution of homologous sites of RNA virus P protein. Specific examples include substitution of the 86th amino acid with Lys and substitution of the 511st amino acid with Phe.
- substitution of the 1197th Asn (N1197) and / or the 1795th Lys (K1795) of the SeV L protein with other amino acids, or substitution of homologous sites of other minus-strand RNA virus L proteins Specific examples include substitution of the 1197th amino acid with Ser, substitution of the 1795th amino acid with Glu, and the like.
- Mutations in the P gene and the L gene can remarkably enhance the effects of persistent infectivity, suppression of secondary particle release, or suppression of cytotoxicity.
- G69E, T116A, and A183S can be introduced for the M gene
- A262T, G264, and K461G can be introduced for the HN gene.
- the mutations that can be introduced are not limited to these (WO2003 / 025570). .
- production of a minus-strand RNA virus can be carried out using the following known methods (WO97 / 16539; WO97 / 16538; WO00 / 70055; WO00 / 70070; WO01 / 18223; WO03 / 025570; WO2005 / 071092; WO2006 / 137517; WO2007 / 083644; WO2008 / 007581; Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997, Kato, A. et al., 1997, EMBO J. 16: 578-587 and Yu, D.
- minus-strand RNA viruses including parainfluenza, vesicular stomatitis virus, rabies virus, measles virus, Linder pest virus, Sendai virus and the like can be reconstituted from DNA.
- Examples of the method for producing a plus (+) strand RNA virus include the following examples. 1) Coronavirus Enjuanes L, Sola I, Alonso S, Escors D, Zuniga S. Coronavirus reverse genetics and development of vectors for gene expression. Curr Top Microbiol Immunol. 2005; 287: 161-97. Review. 2) Toga virus Yamanaka R, Zullo SA, Ramsey J, Onodera M, Tanaka R, Blaese M, Xanthopoulos KG. Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus. Cancer Gene Ther. 2001 Oct; 8 (10): 796-802.
- RNA virus propagation methods and recombinant virus production methods refer to Virology Experimental Studies, 2nd revised edition (edited by National Institute of Preventive Health, Alumni Association, Maruzen, 1982).
- the present invention also provides a method for producing a recombinant protein, wherein the protein is expressed from an RNA viral vector encoded as a fusion protein with AB5 toxin B subunit, and the expressed protein is recovered.
- a method for producing a recombinant protein comprising: Specifically, the vector is introduced into a cell, and the fusion protein is expressed from the vector in the cell. By recovering the expressed fusion protein, a recombinant protein can be obtained as a fusion protein. If necessary, only the recombinant protein portion can be separated from the fusion protein and recovered.
- the AB5B moiety can be cleaved.
- the cells are not limited as long as the vector can be introduced, but mammals such as LLC-MK 2 cells (ATCC CCL-7), CV-1 cells (eg ATCC CCL-70), BHK cells (eg ATCC CCL-10), etc.
- proteins may be expressed using chicken eggs (Nakanishi et al., (1993), "Advanced Protocol III for Neuroscience Research, Molecular Neuronal Physiology", Koseisha, Osaka, pp.153-172).
- the present invention also relates to a composition comprising the vector of the present invention or a cell into which the vector has been introduced. Since the composition of the present invention efficiently expresses a recombinant protein, it is useful for a desired purpose in which high expression of the protein is required. That is, the composition of the present invention is a composition for increasing protein expression and a composition for use in obtaining increased protein expression.
- the vector or cell can be combined with a desired pharmacologically acceptable carrier or vehicle as required.
- “Pharmaceutically acceptable carrier or vehicle” includes a desired solution capable of suspending a vector or cells, such as phosphate buffered saline (PBS), sodium chloride solution, Ringer's solution, culture solution, and the like.
- the composition containing the vector may contain a carrier or a medium such as deionized water or a 5% dextrose aqueous solution.
- a carrier or a medium such as deionized water or a 5% dextrose aqueous solution.
- vegetable oils, suspending agents, surfactants, stabilizers, biocides and the like may be contained. Preservatives or other additives can also be added.
- the composition of the present invention can be used as a desired reagent or as a pharmaceutical composition.
- composition of the present invention may be combined with organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof as carriers. it can.
- organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof as carriers. it can.
- the present invention also provides the use of a nucleic acid encoding the AB5 toxin B subunit to enhance protein expression.
- the present invention also provides a reagent for enhancing protein expression, comprising AB5 toxin B subunit or a nucleic acid encoding the subunit.
- the present invention also provides an expression enhancer comprising AB5 toxin B subunit or a nucleic acid encoding the subunit.
- the present invention also provides a method for increasing the expression level of a recombinant protein, comprising the step of expressing the recombinant protein as a fusion protein with AB5 toxin B subunit.
- the AB5 toxin B subunit is, for example, cholera toxin B (CTB).
- CTB cholera toxin B
- the recombinant protein is, for example, a peptide of 120 amino acids or less, or 50 amino acids or less, and the recombinant protein may be a neuropeptide or an endocrine peptide.
- the recombinant protein refers to a protein attached to the fusion with AB5 toxin B subunit.
- the recombinant protein is, for example, a low solubility protein.
- the recombinant protein is expressed, for example, from an RNA viral vector, preferably, for example, a minus-strand RNA viral vector.
- the present invention also provides use of AB5 toxin B subunit or a nucleic acid encoding the same for increasing protein expression.
- the present invention also relates to an AB5 toxin B subunit or a nucleic acid encoding the same for use in increasing protein expression.
- the present invention also relates to a composition for use in increasing protein expression, comprising the subunit or a nucleic acid encoding the subunit.
- the increase in expression can be confirmed by, for example, a step of expressing a recombinant protein not fused with CTB on the same vector and comparing the expression level.
- the present invention also provides a recombinant protein as a fusion protein with an AB5 toxin B subunit in the production of an RNA virus vector having an increased expression level of the recombinant protein, a cell into which the vector has been introduced, or a composition comprising any of them.
- the present invention relates to the use of genomic RNA of RNA virus encoding RNA or its complementary strand (antigenomic RNA), or DNA encoding at least one of them.
- the present invention also relates to a DNA encoding genomic RNA of RNA virus encoding the recombinant protein or its complementary strand (antigenomic RNA) as a fusion protein with AB5 toxin B subunit for increasing the expression level of the recombinant protein.
- a recombinant protein as a fusion protein with an AB5 toxin B subunit in the production of an RNA virus vector having an increased expression level of the recombinant protein, a cell into which the vector has
- the present invention also relates to a composition for obtaining enhanced expression of the recombinant protein, comprising an RNA virus vector encoding the recombinant protein as a fusion protein with AB5 toxin B subunit.
- the present invention also relates to the use of an RNA viral vector encoding a recombinant protein as a fusion protein with AB5 toxin B subunit for increasing the expression of the recombinant protein.
- the present invention also provides an agent (expression enhancer) for use in increasing the expression of the recombinant protein, including an RNA viral vector encoding the recombinant protein as a fusion protein with the AB5 toxin B subunit.
- the present invention also provides the use of an RNA viral vector encoding a recombinant protein as a fusion protein with AB5 toxin B subunit in the manufacture of a drug, reagent and / or medicament with increased expression of the recombinant protein.
- the composition containing the vector of the present invention or the composition containing cells into which the vector has been introduced can be used in vitro, in vivo administration, and in ex vivo administration via cells.
- an RNA virus vector is administered via a cell
- the vector is introduced into an appropriate cultured cell or a cell collected from an inoculated animal.
- infecting cells with an RNA virus outside the body for example, in a test tube or petri dish
- in vitro (or ex vivo) in a desired physiological aqueous solution such as culture medium, physiological saline, blood, plasma, serum, body fluid, etc.
- the MOI multiplicity of infection; the number of infected viruses per sputum cell
- the dose of the composition of the present invention varies depending on the disease, patient's weight, age, sex, symptoms, administration purpose, administration composition form, administration method, transgene, etc., but those skilled in the art will appropriately determine the dose. Is possible.
- the administration route can be appropriately selected, and the inoculation amount may be appropriately adjusted according to the inoculated animal, the inoculation site, the number of inoculations, and the like.
- the administration target of the composition containing the vector of the present invention is preferably a mammal (including human and non-human mammals). Specifically, non-human primates such as humans and monkeys, rodents such as mice and rats, and all other mammals such as rabbits, goats, sheep, pigs, cows, dogs and cats are included.
- the vector of the present invention is also preferably used as a virus-like particle (VLP), and can also be used as a commonly known virus particle.
- VLP virus-like particle
- mCRF fragment was digested with XhoI and EcoRV
- EcoRV-EIS fragment was digested with EcoRV and NotI
- subcloned into pCI vector Promega
- Sendai virus transcription signal added mCRF gene NotI fragment SEQ ID NO: 12 Constructed (pCI-mCRF).
- the F gene deletion-type SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) was digested with NotI, and the mCRF gene NotI fragment (SEQ ID NO: 12) was inserted into the NotI site.
- An atypical SeV cDNA (pSeV18 + mCRF / ⁇ F) was constructed (FIG. 1).
- Example 2 Construction of SeV vector carrying mCRF and CTB fusion gene CTB-A ⁇ 42 NotI fragment (Example 10, SEQ ID NO: 14) was used as a template to perform PCR, and CTB-mCRF fragment (SEQ ID NO: 16) was synthesized.
- the sequences of the primers used are SEQ ID NOs: 3, 4, 5, 17, and 18.
- the CTB-mCRF fragment was digested with XhoI and EcoRV, and the pCI-mCRF prepared above was digested with XhoI and EcoRV to construct a CTB-mCRF gene NotI fragment (SEQ ID NO: 19) to which a Sendai virus transcription signal was added. .
- the FB gene-deleted SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) is digested with NotI, and the CTB-mCRF gene NotI fragment (SEQ ID NO: 19) is inserted into the NotI site.
- the CTB-mCRF gene An onboard F gene deletion type SeV cDNA (pSeV18 + CTB-mCRF / ⁇ F) was constructed (FIG. 1).
- Example 3 Construction of SeV vector carrying mET-1 gene Human A ⁇ 42 gene (SEQ ID NO: 1) to which Ig ⁇ secretion signal was added was used as a template, and Ig ⁇ secretion signal was added to mouse ET1 peptide gene (accession number NP_034234).
- the mET1 fragment (SEQ ID NO: 21) was synthesized by PCR.
- the sequences of the primers used are SEQ ID NOs: 7, 22, 23 and 24.
- PCR was performed using the CTB-A ⁇ 42 NotI fragment (SEQ ID NO: 14) as a template to synthesize an EcoRV-PstI-EIS fragment (SEQ ID NO: 25).
- the sequences of the primers used are SEQ ID NOS: 11 and 26.
- the mET1 fragment was digested with XhoI and EcoRV, the EcoRV-PstI-EIS fragment was digested with EcoRV and NotI, subcloned into the pCI vector, and the mET1 gene NotI fragment (SEQ ID NO: 27) with Sendai virus transcription signal added was constructed. (PCI-mET1).
- the F gene deletion-type SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) is digested with NotI, the mET1 gene NotI fragment (SEQ ID NO: 27) is inserted into the NotI site, and the mET1 gene-loaded F gene is missing.
- An atypical SeV cDNA (pSeV18 + mET1 / ⁇ F) was constructed (FIG. 1).
- Example 4 Construction of a SeV vector carrying a fusion gene of mET-1 and CTB Using the CTB-A ⁇ 42 NotI fragment (SEQ ID NO: 14) as a template, PCR was performed to synthesize a CTB-mET1 fragment (SEQ ID NO: 29) did. The sequences of the primers used are SEQ ID NOs: 17, 22, 23 and 30. Next, the CTB-mET1 fragment was digested with XhoI and EcoRV, and the pCI-mET1 prepared above was digested with XhoI and EcoRV to construct a CTB-mET1 gene NotI fragment (SEQ ID NO: 31) to which a Sendai virus transcription signal was added. .
- the FB gene-deleted SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) is digested with NotI, and the CTB-mET1 gene NotI fragment (SEQ ID NO: 31) is inserted into the NotI site.
- the CTB-mET1 gene An onboard F gene deletion type SeV cDNA (pSeV18 + CTB-mET1 / ⁇ F) was constructed (Fig. 1).
- Example 5 Construction of SeV vector carrying mPYY gene mPYY with Ig ⁇ secretion signal added to mouse PYY peptide gene (accession number NM_145435.1) with A ⁇ 42 gene (SEQ ID NO: 1) added with Ig ⁇ secretion signal as template A fragment (SEQ ID NO: 33) was synthesized by PCR.
- the sequences of the primers used are SEQ ID NOs: 7, 34, 35, 36, and 37.
- the mPYY fragment was then digested with XhoI and EcoRV, the EcoRV-EIS fragment (SEQ ID NO: 9) was digested with EcoRV and NotI, subcloned into the pCI vector, and the Sendai virus transcription signal added NotI fragment (SEQ ID NO: 38). ) was built.
- the F gene-deficient SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) is digested with NotI, and the mPYY gene NotI fragment (SEQ ID NO: 38) prepared above is inserted into its NotI site.
- An onboard F gene deletion type SeV cDNA (pSeV18 + mPYY / ⁇ F) was constructed (FIG. 1).
- Example 6 Construction of a SeV vector carrying a fusion gene of mPYY and CTB Using the CTB-A ⁇ 42 NotI fragment (SEQ ID NO: 14) as a template, PCR was performed to synthesize a CTB-mPYY fragment (SEQ ID NO: 40).
- the sequences of the primers used are SEQ ID NOs: 17, 34, 35, 36, and 41.
- the CTB-mPYY fragment was digested with XhoI and EcoRV
- the EcoRV-EIS fragment (SEQ ID NO: 9) was digested with EcoRV and NotI, subcloned into a pCI vector, and the Sendai virus transcription signal added CTB-mPYY gene NotI fragment (SEQ ID NO: 42) was constructed.
- the FB gene-deleted SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) is digested with NotI, and the CTB-mPYY gene NotI fragment (SEQ ID NO: 42) is inserted into the NotI site.
- the CTB-mPYY gene An onboard F gene deletion type SeVSe cDNA (pSeV18 + CTB-mPYY / ⁇ F) was constructed (FIG. 1).
- Example 7 Construction of SeV vector carrying mGLP-2 gene A ⁇ 42 gene (SEQ ID NO: 1) to which Ig ⁇ secretion signal was added was used as a template, and Ig ⁇ secretion signal was assigned to mouse GLP-2 peptide gene (accession number NM_175681.2).
- the mGLP-2 fragment (SEQ ID NO: 44) to which was added was synthesized by PCR.
- the sequences of the primers used are SEQ ID NOs: 7, 45, 46, 47 and 48.
- the mGLP-2 fragment was digested with XhoI and EcoRV, the EcoRV-PstI-EIS fragment (SEQ ID NO: 25) was digested with EcoRV and NotI, subcloned into the pCI vector, and the mGLP-2 gene with a Sendai virus transcription signal added.
- a NotI fragment (SEQ ID NO: 49) was constructed.
- F gene deletion type SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) is digested with NotI, mGLP-2 gene NotI fragment (SEQ ID NO: 49) is inserted into its NotI site, and mGLP-2 gene Onboard F gene deletion type SeV cDNA (pSeV18 + mGLP-2 / ⁇ F) was constructed (FIG. 1).
- Example 8 Construction of SeV vector carrying GLP-2 and CTB fusion gene CTB-A ⁇ 42 NotI fragment (SEQ ID NO: 14) was used as a template for PCR and CTB-mGLP-2 fragment (SEQ ID NO: 51) was synthesized.
- the sequences of the primers used are SEQ ID NOs: 17, 45, 46, 47 and 52.
- the CTB-mGLP-2 fragment was digested with XhoI and EcoRV, the EcoRV-PstI-EIS fragment (SEQ ID NO: 25) was digested with EcoRV and NotI, subcloned into the pCI vector, and added to the Sendai virus transcription signal CTB- The mGLP-2 gene NotI fragment (SEQ ID NO: 53) was constructed.
- the F gene-deficient SeV vector (WO00 / 70070) cDNA (pSeV18 + NotI / ⁇ F) was digested with NotI, and the CTB-mGLP-2 gene NotI fragment (SEQ ID NO: 53) was inserted into the NotI site.
- An F gene deletion-type SeV cDNA (pSeV18 + CTB-mGLP-2 / ⁇ F) loaded with mGLP-2 gene was constructed (FIG. 1).
- Example 9 Construction of SeV vector carrying A ⁇ 42 gene (1) Construction of Not I fragment of A ⁇ 42 gene Based on the human amyloid ⁇ peptide sequence (1-42) (SEQ ID NO: 55), the A ⁇ 42 gene was covered by a plurality of primers and bound by PCR.
- the base sequence of A ⁇ 42 is optimized in consideration of human codon usage, and has a structure in which an Ig ⁇ secretion signal is linked to the N-terminal side and a Sendai virus transcription signal is added to the C-terminal side. ( Figure 2, SEQ ID NO: 56)
- the construction method is shown in FIG.
- the six long primers F1 (SEQ ID NO: 58), F2 (SEQ ID NO: 59), R1 (SEQ ID NO: 60), R2 (SEQ ID NO: 61), R3 (SEQ ID NO: 62) covering the entire length of the Ig ⁇ signal and A ⁇ 42 region.
- R4 (SEQ ID NO: 63) are mixed at the same time, PCR is carried out without using a template, and then the two primers F1-1 (SEQ ID NO: 64) and R4 introduced with the restriction enzyme EcoRI recognition sequence using the PCR product as a template.
- -1 (SEQ ID NO: 65) was further subjected to PCR.
- the obtained PCR product was cleaved with the restriction enzyme EcoRI, subcloned into the EcoRI site of the pCI expression plasmid (Promega), the base sequence was confirmed, and a clone with the correct sequence was selected.
- PCR with primer NotI-A ⁇ -F SEQ ID NO: 66
- NotI recognition sequence SEQ ID NO: 66
- SendI virus transcription signal primer NotI-polyA-R
- Example 10 Construction of a SeV vector carrying a fusion gene of A ⁇ 42 and CTB (CTB-A ⁇ 42) (1) Construction of a NotI fragment of CTB-A ⁇ 42 gene
- the NotI fragment of CTB-A ⁇ 42 is a human amyloid ⁇ sequence (1- 42) a cholera toxin B subunit sequence (SEQ ID NO: 68) containing a secretion signal on the N-terminal side of the sequence of Fig. 42) is connected by a GPGP amino acid linker, and a Sendai virus transcription signal is added to the C-terminal side (Fig. 4, SEQ ID NO: 69).
- the base sequences of CTB and A ⁇ were changed without changing the amino acid sequence according to human codon usage.
- the full length was synthesized by PCR using a plurality of long primers covering the full length. Specifically, eight types of long primers covering the entire length of the CTB-A ⁇ region [CTB-A ⁇ F-1 (SEQ ID NO: 71), F-2 (SEQ ID NO: 72), F-3 (SEQ ID NO: 73), F -4 (SEQ ID NO: 74), R-1 (SEQ ID NO: 75), R-2 (SEQ ID NO: 76), R-3 (SEQ ID NO: 77), R-4 (SEQ ID NO: 78)]
- CTB-A ⁇ F-1 SEQ ID NO: 71
- F-2 SEQ ID NO: 72
- F-3 SEQ ID NO: 73
- F -4 SEQ ID NO: 74
- R-1 SEQ ID NO: 75
- R-2 SEQ ID NO: 76
- R-3 SEQ ID NO: 77
- R-4 SEQ ID
- the C-terminal fragment containing the Sendai virus transcription signal is composed of two primers CTB-A ⁇ F1-2 (SEQ ID NO: 79) and CTB-A ⁇ R5-2 (SEQ ID NO: 80) using the pCI plasmid (Promega) as a template. PCR was performed to obtain a full length CTB-A ⁇ PCR fragment. The PCR fragment was subcloned into pGEM-T Easy plasmid (Promega) by TA cloning, the nucleotide sequence was confirmed, and the plasmid was amplified. The desired CTB-A ⁇ 42 NotI fragment (SEQ ID NO: 69) was constructed by digesting the plasmid with the restriction enzyme NotI.
- Example 11 Construction of SeV vector carrying fusion gene of A ⁇ 42 and IL-4 (1) Construction of NotI fragment of fusion gene of A ⁇ 42 and IL-4 The fusion of A ⁇ 42 gene and mouse IL-4 is partly over The method was performed by wrapping and binding by PCR.
- the A ⁇ 42 gene uses a plasmid containing the A ⁇ 42 EcoRI fragment (Example 9: FIG. 3), and the mouse IL-4 gene (SEQ ID NO: 81) extracts mRNA from the spleen of the mouse (BALB / cA) Reverse transcription using 4 specific primers, amplification by PCR, and subcloning into a cloning plasmid were obtained in the form of cDNA.
- This plasmid incorporating mouse IL-4 cDNA was used for the construction. Specifically, using mouse IL-4 plasmid as a template, PCR was performed with two primers NotI-IL4-F (SEQ ID NO: 83) and primer IL4-R (SEQ ID NO: 84), while A ⁇ 42 plasmid was used as a template. PCR was performed with primer A ⁇ 42-F (SEQ ID NO: 85) and primer NotI-A ⁇ 42-R (SEQ ID NO: 86) to obtain a PCR fragment of IL-4 and A ⁇ 42.
- Primers IL4-R and A ⁇ 42-F are designed to partially overlap, so mix PCR fragments of IL-4 and A ⁇ 42 as a template, and use Primer NotI-IL4-F and Primer NotI-A ⁇ 42-R. Two genes were combined as one fusion gene by PCR. This PCR fragment was subcloned into a cloning plasmid, and after confirming the nucleotide sequence, it was cleaved with restriction enzyme NotI to construct the target fusion gene NotI fragment (SEQ ID NO: 87) of A ⁇ 42 and IL-4.
- Example 12 Reconstitution and amplification of Sendai virus vector Reconstitution and amplification of virus were reported by Li et al. (Li, H.-O. et al., J. Virology 74. 6564-6569 (2000), WO00 / 70070) and its improved method (WO2005 / 071092). Since the vector used was the F gene deletion type, F protein helper cells that express F protein using the Cre / loxP expression induction system were used. This system uses the plasmid pCALNdLw (Arai, T. et al., J. Virol. 72: 1115-1121 (1988)) designed to induce and express gene products with Cre DNA recombinase.
- a recombinant adenovirus (AxCANCre) expressing Cre DNA recombinase as a plasmid transformant was prepared by the method of Saito et al. (Saito, I. et al., Nucl. Acid. Res. 23, 3816-3821 (1995), Arai, T. et. al., J. Virol. 72, 1115-1121 (1998)) to express the inserted gene.
- Vectors (SeV18 + CTB-mCRF / ⁇ F, SeV18 + CTB-mET1 / ⁇ F, SeV18 + CTB-mPYY / ⁇ F, SeV18 + CTB-mGLP2 / ⁇ F, SeV18 + mCRF / ⁇ F, SeV18 + mET1 / ⁇ F, SeV18 + mPYY / ⁇ F, SeV18 + mGLP2 / ⁇ F, SeV18 + A ⁇ 42 / ⁇ F, SeV18 + CTB-A ⁇ 42 / ⁇ F, SeV18 + mIL4-A ⁇ 42 / ⁇ F
- SeV vector carrying A ⁇ 42 and PEDI fusion gene (1) Construction of SeV vector cDNA carrying A ⁇ 42-PEDI fusion gene Primer PEDI-1F (SEQ ID NO: 89), PEDI-2R (SEQ ID NO: 90) , PEDI-3F (SEQ ID NO: 91), PEDI-4R (SEQ ID NO: 92), PEDI-5F (SEQ ID NO: 93), PEDI-6R (SEQ ID NO: 94), PEDI-7F (SEQ ID NO: 95), PEDI-8R ( The PEDI gene was amplified by PCR using a total of 10 species (SEQ ID NO: 96), PEDI-9F (SEQ ID NO: 97) and PEDI-10R (SEQ ID NO: 98).
- Example 14 Effect of CTB fusion on mCRF expression: Comparison of expression ability between SeV vector expressing mCRF alone and SeV vector expressing CTB-mCRF fusion protein (1) SeV18 + mCRF / ⁇ F, SeV18 + CTB-mCRF / ⁇ F in BHK21 cells And the expression levels of mCRF and CTB-mCRF peptide were evaluated.
- BHK21 cells were seeded at 1 ⁇ 10 6 in a collagen-coated 6-well plate and infected with each SeV vector diluted with serum-free medium (VPSFM) so as to have MOI 10. After 1 hour, GMEM containing 10% FBS was added. After 24 hours, the medium was replaced with serum-free medium (VPSFM). After 48 hours, the culture supernatant and cells were collected to prepare a cell disruption solution. Non-infected cells were used as a control.
- Example 15 Effect of CTB fusion on mET-1 expression: Comparison of expression ability between SeV vector expressing mET-1 alone and SeV vector expressing CTB-mET1 fusion protein (1) SeV18 + mET-1 / ⁇ F, SeV18 in BHK21 cells + CTB-mET-1 / ⁇ F was infected, and the expression levels of mET-1 and CTB-mET1 peptides were evaluated. BHK21 cells were seeded at 1 ⁇ 10 6 in a collagen-coated 6-well plate and infected with each SeV vector diluted with serum-free medium (VPSFM) so as to have MOI 10. After 1 hour, GMEM containing 10% FBS was added. After 24 hours, the medium was replaced with serum-free medium (VPSFM). After 48 hours, the culture supernatant and cells were collected to prepare a cell disruption solution. Non-infected cells were used as a control.
- Example 16 Effect of CTB fusion on mPYY expression: Comparison of expression ability between SeV vector expressing mPYY alone and SeV vector expressing CTB-mPYY fusion protein (1) SeV18 + mPYY / ⁇ F, SeV18 + CTB-mPYY / ⁇ F in BHK21 cells And the expression levels of mPYY and CTB-mPYY peptides were evaluated.
- BHK21 cells were seeded at 1 ⁇ 10 6 in a collagen-coated 6-well plate and infected with each SeV vector diluted with serum-free medium (VPSFM) so as to have MOI 10. After 1 hour, GMEM containing 10% FBS was added. After 24 hours, the medium was replaced with serum-free medium (VPSFM). After 48 hours, the culture supernatant and cells were collected to prepare a cell disruption solution. Non-infected cells were used as a control.
- Example 17 Effect of CTB fusion on mGLP-2 expression: Comparison of expression ability between mGLP-2 single expression SeV vector and CTB-mGLP-2 fusion protein expression SeV vector (1) SeV18 + mGLP-2 / ⁇ F in BHK21 cells SeV18 + CTB-mGLP-2 / ⁇ F was infected, and the expression levels of mGLP-2 and CTB-mGLP-2 peptides were evaluated. BHK21 cells were seeded at 1 ⁇ 10 6 in a collagen-coated 6-well plate and infected with each SeV vector diluted with serum-free medium (VPSFM) so as to have MOI 10. After 1 hour, GMEM containing 10% FBS was added. After 24 hours, the medium was replaced with serum-free medium (VPSFM). After 48 hours, the culture supernatant and cells were collected to prepare a cell disruption solution. Non-infected cells were used as a control.
- VPSFM serum-free medium
- Example 18 Comparison of the effects of CTB fusion, PEDI fusion, and IL-4 fusion on A ⁇ 42 expression: CTB-A ⁇ 42 fusion protein expression SeV vector, PEDI-A ⁇ 42 fusion protein expression SeV vector and IL-4-A ⁇ 42 fusion protein expression SeV Comparison of expression by vector (1) Infect BHK21 cells with SeV18 + A ⁇ 42 / ⁇ F, SeV18 + IL-4-A ⁇ 42 / ⁇ F, SeV18 + PEDI-A ⁇ 42 / ⁇ F, SeV18 + CTB-A ⁇ 42 / ⁇ F Evaluation was performed.
- BHK21 cells were seeded in a 6-well plate coated with collagen at 1 ⁇ 10 6 cells / well, and infected with each SeV vector diluted with serum-free medium (VPSFM) to a MOI of 10. After 1 hour, GMEM containing 10% FBS was added. After 24 hours, the medium was replaced with serum-free medium (VPSFM). After 48 hours, the culture supernatant and cells were collected to prepare a cell disruption solution.
- VPSFM serum-free medium
- Example 19 Effect of CTB fusion on A ⁇ 42 expression: Comparison of expression ability between SeV vector expressing A ⁇ 42 alone and SeV vector expressing CTB-A ⁇ 42 fusion protein BHK-21 cells seeded the day before confluence (3 ⁇ 10 5 cells / well seeded, 12-well plate) was infected with SeV vector loaded with A ⁇ 42 alone and SeV vector loaded with CTB-A ⁇ 42 at MOI 10 and cultured at 37 ° C, 5% CO 2 in 1 ml / well VP-SFM medium. Kiyoshi and cell lysate were collected. The culture supernatant was concentrated 10 times with acetone precipitation, and a sample was prepared with 1x SDS Loading Buffer.
- Cell lysate was prepared using 150 ⁇ l / well of 1 ⁇ SDS Loading Buffer. After the prepared culture supernatant and cell lysate were treated at 98 ° C for 10 min, SDS-PAGE (using 15% Wako gel) / Western blotting (using 6E10 antibody) with A ⁇ 42 peptide at 1-0.5-0.25-0.125ng / lane as control ) And protein quantification.
- SDS-PAGE using 15% Wako gel
- Western blotting using 6E10 antibody
- the expression level of A ⁇ by the SeV vector loaded with A ⁇ 42 alone was only 4.4 ng / well in the cell lysate and 7.2 ⁇ 10 ⁇ 3 ng / well in the supernatant.
- the expression level of A ⁇ by the SeV vector loaded with A ⁇ 42-CTB fusion gene was 2500 ng / well in the cell lysate, 200 ng / well in the supernatant, 568 times in the lysate, and 27778 times in the supernatant. It was.
- Example 21 Construction of SeV vector carrying A ⁇ 15 and CTB fusion gene (CTB-A ⁇ 15), and A ⁇ 15 tandem type and CTB fusion gene (CTB-A ⁇ 15x2, CTB-A ⁇ 15x4, or CTB-A ⁇ 15x8) (1 ) Construction of CTB-A ⁇ 15 gene Not1 fragment The CTB-A ⁇ 15 gene NotI fragment was constructed based on the CTB-A ⁇ 42 gene. ( Figure 10) Using the plasmid containing the CTB-A ⁇ 42 gene NotI fragment as a template, perform inverse PCR with two primers A ⁇ 15-EcoR-R (SEQ ID NO: 107) and A ⁇ 15-EcoR-F (SEQ ID NO: 108) with EcoRV restriction enzyme site added.
- the PCR product obtained was cleaved with the restriction enzyme EcoRV.
- the plasmid containing the CTB-A ⁇ 15 fragment was obtained by self-ligation.
- the plasmid was cleaved with the restriction enzyme NotI to obtain a NotI fragment (SEQ ID NO: 109) of the target CTB-A ⁇ 15 gene.
- (2) Construction of CTB-A ⁇ 15 tandem type (CTB-A ⁇ 15x2, CTB-A ⁇ 15x4, or CTB-A ⁇ 15x8) gene NotI fragment The CTB-A ⁇ 15 tandem gene NotI fragment was constructed using two genes. (Fig.
- the method introduces a restriction enzyme site except for the A ⁇ 42 part of the plasmid containing CTB-A ⁇ 42, A ⁇ 15 tandem fragment to which restriction enzyme sites were added by PCR was incorporated into that portion.
- a plasmid containing a NotI fragment of CTB-A ⁇ 42 (Example 10: SEQ ID NO: 69) as a template
- two primers CTB-SmaI-R (SEQ ID NO: 111) and CTB added with a restriction enzyme SmaI site
- Inverse PCR was performed with -SmaI-F (SEQ ID NO: 112), and the obtained PCR product was cleaved with SmaI and self-ligated to obtain a plasmid from which A ⁇ 42 was removed.
- a ⁇ 15 tandem fragment was incorporated into the SmaI site of the plasmid.
- the A ⁇ 15 tandem fragment was prepared based on a plasmid containing an A ⁇ 15 8 tandem NotI fragment (SEQ ID NO: 113).
- PCR using the plasmid as a template and PCR with two primers A ⁇ 15-SmaI-F (SEQ ID NO: 115) and A ⁇ 15-EcoRV-R (SEQ ID NO: 116) with restriction enzyme sites added.
- a product is obtained. After they are TA cloned and the nucleotide sequence is confirmed, they are excised with two restriction enzymes SmaI and EcoRI to obtain blunt-ended A ⁇ 15 tandem fragments.
- the fragment was inserted into the SmaI site of the plasmid from which A ⁇ 42 was removed, the plasmid was amplified, and excised with the restriction enzyme NotI to obtain the target CTB-A ⁇ 15x2 NotI fragment (SEQ ID NO: 117), CTB-A ⁇ 15x4 NotI fragment (SEQ ID NO: 119) And a CTB-A ⁇ 15x8 NotI fragment (SEQ ID NO: 121) was obtained.
- Example 22 Comparison of A ⁇ peptide expression ability (1) Western blotting The infection and expression of the constructed vector were evaluated by Western blot. The cell lysate and culture supernatant infected with the SeV vector were mixed with an equal volume of SDS-PAGE sample buffer and heat-denatured at 98 ° C. for 5 minutes. SDS-PAGE was performed on a 15% acrylamide gel, and transferred to a PVDF membrane by a semi-driving method.
- Block with 5% milk / TBS-T react with anti-A ⁇ antibody (6E10), then react with HRP-labeled anti-mouse IgG as a secondary antibody and CCD camera using chemiluminescent substrate SuperSignal West Femto Detection was performed by As a result, expression of CTB-A ⁇ 42, CTB-A ⁇ 15, CTB-A ⁇ 15x2, CTB-A ⁇ 15x4, and CTB-A ⁇ 15x8 in BHK cells and secretion into the medium were confirmed.
- CTB-A ⁇ 15x8 was greater than the expression level of CTB-A ⁇ 42, indicating that CTB-A ⁇ 15x8 was also secreted into the medium very much relative to the secretion of CTB-A ⁇ 42 (FIG. 12).
- GM1-ELISA The binding of CTB to GM1 was evaluated using a plate on which ganglioside GM1 was immobilized.
- Ganglioside GM1 (5 ⁇ g / mL) was immobilized on each well of a 96-well plate (Nunc, MaxiSorp plate), blocked with 20% BlockingOne (Nacalai Tesque), and then the culture supernatant of cells infected with SeV vector ( 20-fold to 2 million-fold dilution) was added, reacted with HRP-labeled 6E10 antibody, and detected using a TMB chromogenic substrate. The amount of binding was evaluated by measuring absorbance (OD450) with a plate reader.
- CTB-A ⁇ 42, CTB-A ⁇ 15, CTB-A ⁇ 15x2, CTB-A ⁇ 15x4 and CTB-A ⁇ 15x8 secreted into the medium bind to GM1
- CTB-A ⁇ 15 is CTB -It was shown that the binding ability to GM1 decreased as the number of A ⁇ 15 repeats increased (Fig. 13).
- Example 23 Evaluation of anti-A ⁇ antibody-inducing ability in normal mice using various constructed SeV vectors (1) Normal mice (comparison between intramuscularly administered CTB-A ⁇ 42 and CTB-A ⁇ 15x8) SeB vectors loaded with CTB-A ⁇ 42 gene, CTB-A ⁇ 15x8 gene, and GFP gene were intramuscularly administered to C57BL / 6N mice with 5 ⁇ 10 7 CIU / head titers (right hind limb), and the antibody titer was evaluated. 14 days after the treatment, blood was collected from the mice and the amount of anti-A ⁇ antibody in the plasma was measured.
- a ⁇ 1-42 peptide (5 ⁇ g / mL) was immobilized on each well of a 96-well plate (Nunc, MaxiSorp plate), blocked with 20% BlockingOne (Nacalai Tesque), and then mouse plasma was added (300 to 300,000 times) Diluted), reacted with peroxidase-labeled anti-mouse IgG antibody, and detected using TMB chromogenic substrate.
- the A ⁇ antibody titer was evaluated by measuring absorbance (OD450) with a plate reader.
- Anti-A ⁇ antibody (6E10) was used as a standard antibody.
- the antibody titer of the CTB-A ⁇ 15x8 gene administration group was 12.23 times that of the CTB-A ⁇ 42 gene administration group.
- mice intramuscular, intradermal, nasal administration
- the SeV vector loaded with was administered intramuscularly (right hind limb) with a 5 ⁇ 10 7 CIU / head titer, and the antibody titer was evaluated. 14 days after the treatment, blood was collected from the mice and the amount of anti-A ⁇ antibody in the plasma was measured. As a result, the A ⁇ antibody titer increased in all administration groups except the Control group.
- the intradermal administration group had a lower antibody titer than the other administration groups, and the intranasal administration group had a higher antibody titer than the intramuscular administration group of the same titer (FIG. 15).
- Example 24 Evaluation of boosting effect in the induction of anti-A ⁇ antibody in normal mice with various constructed SeV vectors
- Normal mouse muscle injection
- Boost with purified CTB-A ⁇ 42 protein C57BL / 6N mice with CTB-A ⁇ 42
- the SeV vector loaded with the gene was administered intramuscularly (right hind limb) with 5 ⁇ 10 7 CIU / head titers, and CTB-A ⁇ 42 protein produced in E. coli after 14 and 28 days was 20 ⁇ g / PBS / head and 100 ⁇ g / PBS /, respectively.
- the antibody titer was evaluated by intramuscular administration (right hind limb) with head, 100 ⁇ g / IFA (incomplete Freund's adjuvant) / head. Every 14 days of treatment, blood was collected from the mice and the amount of anti-A ⁇ antibody in the plasma was measured. As a result, in the group immunized with CTB-A ⁇ 42 gene and additionally immunized with CTB-A ⁇ 42 protein, a marked increase in anti-A ⁇ antibody was obtained (FIG. 17).
- the A ⁇ antibody titer after the second boost was 32 ⁇ g / ml in the 20 ⁇ g boost group, 107 ⁇ g / ml in the 100 ⁇ g boost group, and 25.9 ⁇ g / ml in the 100 ⁇ g + IFA boost group.
- mice SeV vector boosted C57BL / 6N mice were injected intranasally with 5x10 6 CIU / head and 5x10 7 CIU / head titers of SeV vector carrying CTB-A ⁇ 15x8 gene. One day later, the same SeV vector was administered intranasally with the same titer, and the antibody titer was evaluated. After 14 and 28 days of the treatment, blood was collected from the mice and the amount of anti-A ⁇ antibody in the plasma was measured. As a result, a significant increase in A ⁇ antibody titer was obtained at a rate of 3/3 in the CTB-A ⁇ 15x8 gene boost group (FIG. 20).
- Example 25 Efficacy evaluation in APP model mice using various constructed SeV vectors: intramuscular injection (1) Anti-A ⁇ antibody titer In APP transgenic mice (Tg2576) (13 months old), a model mouse for Alzheimer's disease SeV18 + CTB-A ⁇ 18x5 / ⁇ F (also referred to as CTB-A ⁇ 15x8), or SeV18 + CTB-A ⁇ 42 / ⁇ F (also referred to as CTB-A ⁇ 42), a SeV vector carrying a GFP gene as a control group (SeV18 + GFP / ⁇ F; (Also referred to as “GFP”) was administered intramuscularly (right hind limb) at 5 ⁇ 10 7 CIU / head.
- GFP GFP
- CTB-A ⁇ 42 protein produced in E. coli was intramuscularly administered (right hind limb) to half of the CTB-A ⁇ 42 gene administration group.
- the A ⁇ antibody titer in plasma was measured 14, 28, 42, and 56 days after administration of SeV vector.
- a marked increase in A ⁇ antibody titer was observed in the CTB-A ⁇ 15x8 gene administration group.
- the CTB-A ⁇ 42 gene administration group there were half of the individuals in which the A ⁇ antibody titer hardly increased, and the increase in the A ⁇ antibody titer was lower than that in the CTB-A ⁇ 15x8 gene administration group.
- the CTB-A ⁇ 42 protein boost group there was no increase in the A ⁇ antibody titer due to the boost (FIG. 21).
- Brain A ⁇ content ELISA Brain tissue was collected from the APP mice 56 days after the start of SeV vector administration, and the amount of A ⁇ in the left hemisphere of the brain tissue was measured by ELISA. Brain tissue was ultrasonically homogenized in TBS, centrifuged at 35,000g for 1 hour, supernatant was collected as A ⁇ soluble fraction, precipitate was ultrasonically homogenized in 10% formic acid, neutralized with 1M Tris, A ⁇ Collected as insoluble fraction. The amount of A ⁇ in the brain was measured using A ⁇ 42 ELISA kit and A ⁇ 40 ELISA kit manufactured by Wako Pure Chemical Industries.
- the amount of A ⁇ in the insoluble fraction was reduced to about 80% in the CTB-A ⁇ 15x8 gene administration group compared to the GFP gene administration group.
- the CTB-A ⁇ 42 gene administration group there was no decrease in the amount of A ⁇ .
- the CTB-A ⁇ 42 protein boost group there was a slight decrease in the amount of A ⁇ .
- the amount of A ⁇ in the soluble fraction was reduced to about 50% in the CTB-A ⁇ 15x8 gene administration group compared to the GFP gene administration group.
- the CTB-A ⁇ 42 gene administration group there was no decrease in the amount of A ⁇ .
- the amount of A ⁇ in the CTB-A ⁇ 42 protein boost group was reduced to 60-70% (FIG. 22).
- microglia activation was observed around senile plaques in both groups of animals, but in parallel to the tendency that senile plaques tended to decrease in animals in the vector administration group, the area ratio occupied by microglia also decreased. There was a trend.
- the present invention makes it possible to increase the expression level of a recombinant protein from an RNA virus vector.
- RNA virus vector of the present invention as a vector for gene introduction, effective gene therapy, gene vaccine, monoclonal antibody production and the like can be carried out.
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Abstract
Description
遺伝子治療等に応用されている遺伝子導入用RNAウイルスベクターにおいても同様である。遺伝子導入用RNAウイルスベクターを利用した、遺伝子治療・遺伝子ワクチン・モノクローナル抗体作製等への応用の場合に、搭載遺伝子の発現量はベクターの機能そのものに直結するため、その発現量を増強することが可能になれば、ベクターの有効性を向上し、その利用・適用範囲を大きく広げることができる。
また、AB5Bの融合は、遺伝子導入RNAウイルスベクターを利用した、遺伝子治療・遺伝子ワクチン・モノクローナル抗体作製など、in vivo, ex vivoおよびin vitroでのリコンビナント蛋白質の発現増強に有効に機能する。発現増強はベクターの機能そのものに直結し、ベクターの有効性を高めることが可能である。
〔1〕組み換え蛋白質をコードするRNAウイルスベクターであって、該蛋白質が、AB5トキシンBサブユニットとの融合蛋白質としてコードされていることを特徴とするベクター、
〔2〕AB5トキシンBサブユニットがコレラトキシンB(CTB)である、〔1〕に記載のベクター、
〔3〕該組み換え蛋白質が120アミノ酸以下のペプチドである、〔1〕または〔2〕に記載のベクター、
〔4〕該組み換え蛋白質が50アミノ酸以下のペプチドである、〔3〕に記載のベクター、
〔5〕該組み換え蛋白質が神経ペプチドまたは内分泌性ペプチドである、〔1〕から〔4〕のいずれかに記載のベクター、
〔6〕該組み換え蛋白質が低溶解性蛋白質である、〔1〕から〔5〕のいずれかに記載のベクター、
〔7〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔1〕から〔6〕のいずれかに記載のベクター、
〔8〕〔1〕から〔7〕のいずれかに記載のベクターおよび薬学的に許容される担体を含む医薬組成物、
〔9〕〔1〕から〔7〕のいずれかに記載のベクターが導入された細胞、
〔10〕〔1〕から〔7〕のいずれかに記載のベクターを発現させる工程を含む、組み換え蛋白質の製造方法、
〔11〕組み換え蛋白質の発現量が上昇したRNAウイルスベクターの製造方法であって、該蛋白質をAB5トキシンBサブユニットと融合させた融合蛋白質をコードするRNAウイルスベクターを製造する工程を含む方法、
〔12〕AB5トキシンBサブユニットがコレラトキシンB(CTB)である、〔11〕に記載の方法、
〔13〕該組み換え蛋白質が120アミノ酸以下のペプチドである、〔11〕または〔12〕に記載の方法、
〔14〕該組み換え蛋白質が50アミノ酸以下のペプチドである、〔13〕に記載の方法、
〔15〕該組み換え蛋白質が神経ペプチドまたは内分泌性ペプチドである、〔11〕から〔14〕のいずれかに記載の方法、
〔16〕該組み換え蛋白質が低溶解性蛋白質である、〔11〕から〔15〕のいずれかに記載の方法、
〔17〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔11〕から〔16〕のいずれかに記載の方法、
〔18〕RNAウイルスベクターからの組み換え蛋白質の発現量を上昇させる方法であって、該組み換え蛋白質をAB5トキシンBサブユニットとの融合蛋白質としてRNAウイルスベクターから発現させる工程を含む方法、
〔19〕AB5トキシンBサブユニットがコレラトキシンB(CTB)である、〔18〕に記載の方法、
〔20〕該組み換え蛋白質が120アミノ酸以下のペプチドである、〔18〕または〔19〕に記載の方法、
〔21〕該組み換え蛋白質が50アミノ酸以下のペプチドである、〔20〕に記載の方法、
〔22〕該組み換え蛋白質が神経ペプチドまたは内分泌性ペプチドである、〔18〕から〔21〕のいずれかに記載の方法、
〔23〕該組み換え蛋白質が低溶解性蛋白質である、〔18〕から〔22〕のいずれかに記載の方法、
〔24〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔18〕から〔23〕のいずれかに記載の方法、
〔25〕〔1〕から〔7〕のいずれかに記載のベクターからなるウイルス様粒子、等に関する。
なお上記の各項において、同一の項を引用する各項に記載の発明の2つまたはそれ以上を任意に組み合わせた発明は、それらに引用される上位の項に記載の発明において、既に意図されている。また、本明細書に記載した任意の発明要素、技術的要素、およびそれらの任意の組み合わせは、本明細書において意図されている。また、それらの発明において、本明細書に記載の任意の要素またはその任意の組み合わせを除外した発明も、本明細書に意図されている。また本明細書は、明細書中に例えば好ましいとしてある特定の態様を記載した場合、それを開示するのみならず、その態様を含むより上位の本明細書に開示された発明から、その態様を除外した発明も開示するものである。
ラッサウィルスなどのアレナウイルス科(Arenaviridae)
インフルエンザウイルスなどのオルソミクソウイルス科(Orthomyxoviridae;Infuluenza virus A, B, C, および Thogoto-like viruses 等を含む)
SARSウイルスなどのコロナウイルス科(Coronaviridae)
風疹ウイルスなどのトガウイルス科(Togaviridae)
ムンプスウイルス、麻疹ウイルス、センダイウイルス、RSウイルスなどのパラミクソウイルス科(Paramyxoviridae)
ポリオウイルス、コクサッキーウイルス、エコーウイルスなどのピコルナウイルス科(Picornaviridae)
マールブルグウイルス、エボラウイルスなどのフィロウイルス科(Filoviridae)
黄熱病ウイルス、デング熱ウイルス、C型肝炎ウイルス、G型肝炎ウイルスなどのフラビウイルス科(Flaviviridae)
ブンヤウイルス科(Bunyaviridae;Bunyavirus, Hantavirus, Nairovirus, および Phlebovirus属等を含む)
狂犬病ウイルスなどのラブドウイルス科(Rhabdoviridae)
レオウイルス科(Reoviridae)
すなわち本発明は、組み換え蛋白質をコードするRNAウイルスベクターであって、該蛋白質がAB5トキシンBサブユニットとの融合蛋白質としてコードされており、該組み換え蛋白質が例えば120アミノ酸以下、または50アミノ酸以下のペプチドであるベクターを提供する。ここで「組み換え蛋白質」とは、AB5トキシンBサブユニットとの融合に付された蛋白質を言う。すなわち組み換え蛋白質の長さには、AB5トキシンBサブユニットは含まない。例えば組み換え蛋白質の長さは、融合蛋白質からAB5トキシンBサブユニットを除いた部分の長さである。
1)コロナウイルス
Enjuanes L, Sola I, Alonso S, Escors D, Zuniga S.
Coronavirus reverse genetics and development of vectors for gene expression.
Curr Top Microbiol Immunol. 2005;287:161-97. Review.
2)トガウイルス
Yamanaka R, Zullo SA, Ramsey J, Onodera M, Tanaka R, Blaese M, Xanthopoulos KG.
Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus.
Cancer Gene Ther. 2001 Oct;8(10):796-802.
Datwyler DA, Eppenberger HM, Koller D, Bailey JE, Magyar JP.
Efficient gene delivery into adult cardiomyocytes by recombinant Sindbis virus.
J Mol Med. 1999 Dec;77(12):859-64.
3)ピコルナウイルス
Lee SG, Kim DY, Hyun BH, Bae YS.
Novel design architecture for genetic stability of recombinant poliovirus: the manipulation of G/C contents and their distribution patterns increases the genetic stability of inserts in a poliovirus-based RPS-Vax vector system.
J Virol. 2002 Feb;76(4):1649-62.
Mueller S, Wimmer E.
Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames.
J Virol. 1998 Jan;72(1):20-31.
4)フラビウイルス
Yun SI, Kim SY, Rice CM, Lee YM.
Development and application of a reverse genetics system for Japanese encephalitis virus.
J Virol. 2003 Jun;77(11):6450-65.
Arroyo J, Guirakhoo F, Fenner S, Zhang ZX, Monath TP, Chambers TJ.
Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE).
J Virol. 2001 Jan;75(2):934-42.
5)レオウイルス
Roner MR, Joklik WK.
Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome.
Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):8036-41. Epub 2001 Jun 26.
その他のRNAウイルスの増殖方法および組み換えウイルスの製造方法については、ウイルス学実験学 各論、改訂二版(国立予防衛生研究所学友会編、丸善、1982)を参照のこと。
本発明のベクターはウイルス様粒子(VLP)としても好ましく用いられ、通常知られるウイルス粒子のように用いることもできる。
Igκ分泌シグナルを付加したヒトAβ42遺伝子(配列番号1)をtemplateにし、マウスCRFペプチド遺伝子(accession number NM_205769)にIgκ分泌シグナルを付加したmCRFフラグメント(配列番号3)をPCRにより合成した。用いたプライマーの配列は、配列番号4~8である。次にCTB-Aβ42 NotIフラグメント(実施例10、配列番号14)をtemplateにして、PCRを行い、EcoRV-EISフラグメント(配列番号9)を合成した。用いたプライマーの配列は配列番号10および11である。次にmCRFフラグメントをXhoIとEcoRVで消化し、EcoRV-EISフラグメントをEcoRVとNotIで消化し、pCIベクター(Promega)にサブクローニングし、センダイウイルスの転写シグナル付加したmCRF遺伝子NotIフラグメント(配列番号12)を構築した(pCI-mCRF)。
CTB-Aβ42 NotIフラグメント(実施例10、配列番号14)をtemplateにして、PCRを行い、CTB-mCRFフラグメント(配列番号16)を合成した。用いたプライマーの配列は、配列番号3、4、5、17、18である。次にCTB-mCRFフラグメントをXhoIとEcoRVで消化し、上記で作製したpCI-mCRFをXhoIとEcoRVで消化し、センダイウイルスの転写シグナル付加したCTB-mCRF遺伝子NotIフラグメント(配列番号19)を構築した。
Igκ分泌シグナルを付加したヒトAβ42遺伝子(配列番号1)をtemplateにし、マウスET1ペプチド遺伝子(accession number NP_034234)にIgκ分泌シグナルを付加したmET1フラグメント(配列番号21)をPCRにより合成した。用いたプライマーの配列は、配列番号7、22、23、24である。次にCTB-Aβ42 NotIフラグメント(配列番号14)をtemplateにして、PCRを行い、EcoRV-PstI-EISフラグメント(配列番号25)を合成した。用いたプライマーの配列は、配列番号11、および26である。次にmET1フラグメントをXhoIとEcoRVで消化し、EcoRV-PstI-EISフラグメントをEcoRVとNotIで消化し、pCIベクターにサブクローニングし、センダイウイルスの転写シグナル付加したmET1遺伝子NotIフラグメント(配列番号27)を構築した(pCI-mET1)。
CTB-Aβ42 NotIフラグメント(配列番号14)をtemplateにして、PCRを行い、CTB-mET1フラグメント(配列番号29)を合成した。用いたプライマーの配列は、配列番号17、22、23、30である。次にCTB-mET1フラグメントをXhoIとEcoRVで消化し、上記で作製したpCI-mET1をXhoIとEcoRVで消化し、センダイウイルスの転写シグナル付加したCTB-mET1遺伝子NotIフラグメント(配列番号31)を構築した。
Igκ分泌シグナルを付加したAβ42遺伝子(配列番号1)をtemplateにし、マウスPYYペプチド遺伝子(accession number NM_145435.1)にIgκ分泌シグナルを付加したmPYYフラグメント(配列番号33)をPCRにより合成した。用いたプライマーの配列は、配列番号7、34、35、36、37である。次にmPYYフラグメントをXhoIとEcoRVで消化し、EcoRV-EISフラグメント(配列番号9)をEcoRVとNotIで消化し、pCIベクターにサブクローニングし、センダイウイルスの転写シグナル付加したmPYY遺伝子NotIフラグメント(配列番号38)を構築した。
CTB-Aβ42 NotIフラグメント(配列番号14)をtemplateにして、PCRを行い、CTB-mPYYフラグメント(配列番号40)を合成した。用いたプライマーの配列は、配列番号17、34、35、36、41である。次にCTB-mPYYフラグメントをXhoIとEcoRVで消化し、EcoRV-EISフラグメント(配列番号9)をEcoRVとNotIで消化し、pCIベクターにサブクローニングし、センダイウイルスの転写シグナル付加したCTB-mPYY遺伝子NotIフラグメント(配列番号42)を構築した。
Igκ分泌シグナルを付加したAβ42遺伝子(配列番号1)をtemplateにし、マウスGLP-2ペプチド遺伝子(accession number NM_175681.2)にIgκ分泌シグナルを付加したmGLP-2フラグメント(配列番号44)をPCRにより合成した。用いたプライマーの配列は、配列番号7、45、46、47、48である。次にmGLP-2フラグメントをXhoIとEcoRVで消化し、EcoRV-PstI-EISフラグメント(配列番号25)をEcoRVとNotIで消化し、pCIベクターにサブクローニングし、センダイウイルスの転写シグナル付加したmGLP-2遺伝子NotIフラグメント(配列番号49)を構築した。
CTB-Aβ42 NotIフラグメント(配列番号14)をtemplateにして、PCRを行い、CTB-mGLP-2フラグメント(配列番号51)を合成した。用いたプライマーの配列は、配列番号17、45、46、47、52である。次にCTB-mGLP-2フラグメントをXhoIとEcoRVで消化し、EcoRV-PstI-EISフラグメント(配列番号25)をEcoRVとNotIで消化し、pCIベクターにサブクローニングし、センダイウイルスの転写シグナル付加したCTB-mGLP-2遺伝子NotIフラグメント(配列番号53)を構築した。
(1) Aβ42遺伝子のNot Iフラグメントの構築
Aβ42遺伝子はヒトアミロイドβペプチド配列(1-42)(配列番号55)を元に、全長を複数のプライマーでカバーしてPCRで結合した。Aβ42の塩基配列はヒトcodon usageを考慮して最適化し、N端側にはIgκの分泌シグナルを結合させ、C端側にはセンダイウイルスの転写シグナルを付加した構造をしている。(図2, 配列番号56)
構築方法は図3に示した。まずIgκシグナルとAβ42領域の全長をカバーする6種類の長いプライマーF1 (配列番号58), F2 (配列番号59), R1 (配列番号60), R2 (配列番号61), R3 (配列番号62), R4 (配列番号63) を同時に混ぜて、テンプレート入れずにPCRを行い、次にそのPCR産物をテンプレートとして制限酵素EcoRIの認識配列を導入した2つのプライマーF1-1 (配列番号64) とR4-1 (配列番号65) で更にPCRを行った。そして得られたPCR産物を制限酵素EcoRIで切断し、pCI発現プラスミド(Promega社)のEcoRIサイトへサブクローニングし、塩基配列の確認を行い正しい配列のクローンを選択した。
選択したプラスミドをテンプレートにし、NotI認識配列を付加したプライマーNotI-Aβ-F(配列番号66)とセンダイウイルス転写シグナルとNotI認識配列を付加したプライマーNotI-polyA-R(配列番号67)とでPCRを行ない、得られたPCR産物を制限酵素NotIで消化し、目的のAβ42 NotIフラグメント(図2, 配列番号56)を構築した。
(2)Aβ42遺伝子搭載センダイウイルスcDNAの構築
F遺伝子欠失型SeVベクター(WO00/70070)のcDNA(pSeV18+NotI/ΔF)をNotIで消化し、そのNotI部位にAβ42 NotIフラグメントを挿入し、Aβ42遺伝子搭載F遺伝子欠失型SeV cDNA(pSeV18+Aβ42/ΔF)を構築した。
(1)CTB-Aβ42遺伝子のNotIフラグメントの構築
CTB-Aβ42のNotIフラグメントは、ヒトアミロイドβ配列(1-42)の配列のN末端側に分泌シグナルを含むコレラトキシンBサブユニット配列(配列番号68)をGPGPアミノ酸リンカーでつなぎ、C末端側にセンダイウイルスの転写シグナルを付加した構造をしている(図4, 配列番号69)。更に発現効率を増すためにCTBとAβの塩基配列はヒトのcodon usageに従ってアミノ酸配列を変えずに塩基配列を変更した。
この遺伝子の構築には、全長をカバーする長い複数のプライマーを用いてPCRにより全長を合成した。具体的には、CTB-Aβ領域の全長をカバーする長いプライマーを8種類 [CTB-AβF-1 (配列番号71), F-2 (配列番号72), F-3 (配列番号73), F-4 (配列番号74), R-1 (配列番号75), R-2 (配列番号76), R-3 (配列番号77), R-4 (配列番号78)] 合成し、それら8種類を混ぜてPCRを行うことによりN端からAβ42までに対応するフラグメントを得た。センダイウイルス転写シグナルを含むC端側のフラグメントは、pCIプラスミド (Promega社) をテンプレートにして2種のプライマー CTB-Aβ F1-2 (配列番号79) CTB-Aβ R5-2 (配列番号80) でPCRを行いCTB-Aβ全長のPCRフラグメントを得た。そのPCRフラグメントはTAクローニングによりpGEM-T Easyプラスミド (Promega社) にサブクローニングして、塩基配列を確認し、プラスミドを増幅した。そのプラスミドを制限酵素NotIで消化することにより、目的のCTB-Aβ42 NotIフラグメント (配列番号69) を構築した。
F遺伝子欠失型SeVベクター(WO00/70070)のcDNA(pSeV18+NotI/ΔF)をNotIで消化し、そのNotI部位にCTB-Aβ42 NotIフラグメントを挿入し、CTB-Aβ42遺伝子搭載F遺伝子欠失型SeV cDNA(pSeV18+CTB-Aβ42/ΔF)を構築した。
(1)Aβ42とIL-4の融合遺伝子のNotIフラグメントの構築
Aβ42遺伝子とマウスIL-4の融合は一部をオーバーラップさせてPCRで結合させる方法で行った。
Aβ42遺伝子はAβ42 EcoRIフラグメント(実施例9:図3)が入ったプラスミドを利用し、マウスIL-4遺伝子(配列番号81)は、マウス(BALB/cA)の脾臓からmRNAを抽出し、IL-4特異的プライマーを用いて逆転写し、PCRで増幅し、クローニングプラスミドにサブクローニングすることによりcDNAの形で取得した。このマウスIL-4 cDNAが組み込まれたプラスミドを構築に用いた。
具体的には、マウスIL-4プラスミドをテンプレートして、2つのプライマー NotI-IL4-F(配列番号83)とプライマー IL4-R(配列番号84)でPCRを行い、一方でAβ42プラスミドをテンプレートとし、プライマーAβ42-F(配列番号85)とプライマー NotI-Aβ42-R(配列番号86)でPCRを行い、IL-4とAβ42のPCRフラグメントを得た。プライマーIL4-RとAβ42-Fは一部がオーバーラップするようデザインされているため、IL-4とAβ42のPCRフラグメントを混ぜてテンプレートとし、プライマー NotI-IL4-Fとプライマー NotI-Aβ42-RでPCRすることで、2つの遺伝子を一つの融合遺伝子として結合させた。このPCRフラグメントをクローニングプラスミドにサブクローニングし、塩基配列を確認後、制限酵素NotIで切断することにより、目的のAβ42とIL-4の融合遺伝子NotIフラグメント(配列番号87)を構築した。
F遺伝子欠失型SeVベクター(WO00/70070)のcDNA(pSeV18+NotI/ΔF)をNotIで消化し、そのNotI部位に上記で作製したmIL4-Aβ42 NotIフラグメントを挿入し、Aβ42遺伝子搭載F遺伝子欠失型SeV cDNA(pSeV18+mIL4-Aβ42/ΔF)を構築した。
ウィルスの再構成および増幅はLiらの報告(Li, H.-O. et al., J. Virology 74. 6564-6569 (2000), WO00/70070)およびその改良法(WO2005/071092)に従って行った。使用したベクターはF遺伝子欠失型であるので、Cre/loxP発現誘導システムによりF蛋白質を発現するF蛋白のヘルパー細胞を利用した。当該システムはCre DNA リコンビナーゼにより遺伝子産物を誘導発現するように設計されたプラスミドpCALNdLw(Arai, T. et al., J. Virol. 72: 1115-1121 (1988))を利用したものであり、同プラスミドのトランスフォーマントにCre DNAリコンビナーゼを発現する組み換えアデノウィルス(AxCANCre)をSaitoらの方法(Saito, I. et al., Nucl. Acid. Res. 23, 3816-3821 (1995), Arai, T. et al., J. Virol. 72, 1115-1121 (1998))で感染させて挿入遺伝子を発現させる。
これらの方法によって、CTB-mCRF, CTB-mET1, CTB-mPYY, CTB-mGLP2, mCRF, mET1, mPYY, mGLP2, Aβ42, CTB-Aβ42, mIL4-Aβ42の各遺伝子を搭載したF遺伝子欠失型SeVベクター(それぞれSeV18+CTB-mCRF/ΔF, SeV18+CTB-mET1/ΔF, SeV18+CTB-mPYY/ΔF, SeV18+CTB-mGLP2/ΔF, SeV18+mCRF/ΔF, SeV18+mET1/ΔF, SeV18+mPYY/ΔF, SeV18+mGLP2/ΔF, SeV18+Aβ42/ΔF, SeV18+CTB-Aβ42/ΔF, SeV18+mIL4-Aβ42/ΔF)を調製した。
(1)Aβ42-PEDI融合遺伝子搭載SeVベクターcDNAの構築
プライマー PEDI-1F (配列番号89)、PEDI-2R (配列番号90)、PEDI-3F (配列番号91)、PEDI-4R (配列番号92)、PEDI-5F (配列番号93)、PEDI-6R (配列番号94)、PEDI-7F (配列番号95)、PEDI-8R (配列番号96)、PEDI-9F (配列番号97)とPEDI-10R (配列番号98) 計10種を用いたPCRでPEDI遺伝子を増幅した。PEDI遺伝子とAβ42を融合してSeVベクターへ搭載するため、Aβ42搭載SeVベクターをTemplateとして、プライマー SeVF6 (配列番号99)とS-PEDI-C (配列番号100)を用いたPCRで得られたFragment 1、同Templateでプライマー PEDI-Ab-N (配列番号101)と SEVR280 (配列番号102)を用いたPCRで得られたFragment 3、PEDI遺伝子をTemplateとして、プライマー PEDI-N (配列番号103)と PEDI-C (配列番号104)を用いたPCRで得られたFragment 2をTemplateとして、オーバーラップPCRでPEDI-Aβ42 NotIフラグメント(配列番号105)が得られ、pSeV18+/ΔFのNotIサイトへ挿入し、PEDI-Aβ42融合遺伝子搭載F欠失型SeVベクターのcDNAが得られた。
PEDI-Aβ42遺伝子を搭載するSeVベクター(SeV18+PEDI-Aβ42/ΔF)の再構成は、実施例12と同様に、Liらの方法(Li, H.-O. et al., J. Virology 74. 6564-6569 (2000), WO00/70070)およびその改良法(WO2005/071092)に従って行った。
(1) BHK21細胞にSeV18+mCRF/ΔF、SeV18+CTB-mCRF/ΔFを感染させ、mCRF およびCTB-mCRFペプチド発現量の評価を行った。BHK21細胞をコラーゲンコートされた6穴プレートに1x106個になるよう播き、MOI 10 になるように無血清培地(VPSFM)で希釈された各SeVベクターを感染させた。1時間後に10% FBS含有GMEMを加え、24時間後に無血清培地(VPSFM)に置換し、48時間後に培養上清と細胞を回収し、細胞破砕液を調製した。コントロールとして非感染細胞を用いた。
CRFの測定は矢内原研究所のCRF ELISAキット用いた。発現量の評価は、プレートリーダーでの吸光度測定(O.D.450)により行った。
結果を図5に示す。mCRFはほとんど発現していないのに対し、CTB-mCRFは顕著に発現が増大していることが判明した。
(1) BHK21細胞にSeV18+mET-1/ΔF、SeV18+CTB-mET-1/ΔFを感染させ、mET-1およびCTB-mET1ペプチド発現量の評価を行った。BHK21細胞をコラーゲンコートされた6穴プレートに1x106個になるよう播き、MOI 10 になるように無血清培地(VPSFM)で希釈された各SeVベクターを感染させた。1時間後に10% FBS含有GMEMを加え、24時間後に無血清培地(VPSFM)に置換し、48時間後に培養上清と細胞を回収し、細胞破砕液を調製した。コントロールとして非感染細胞を用いた。
ET1の測定は直接サンプルを96穴プレートに固相化し、1% BSA/TBS-T bufferでブロックした後、抗マウスET1抗体とペルオキシダーゼ標識抗ウサギIgG抗体で検出した。発現量の評価は、プレートリーダーでの吸光度測定(O.D.450)により行った。
結果を図6に示す。mET1はほとんど発現していないのに対し、CTB-mET1は顕著に発現が増大していた。
(1) BHK21細胞にSeV18+mPYY/ΔF、SeV18+CTB-mPYY/ΔFを感染させ、mPYY およびCTB-mPYYペプチド発現量の評価を行った。BHK21細胞をコラーゲンコートされた6穴プレートに1x106個になるよう播き、MOI 10 になるように無血清培地(VPSFM)で希釈された各SeVベクターを感染させた。1時間後に10% FBS含有GMEMを加え、24時間後に無血清培地(VPSFM)に置換し、48時間後に培養上清と細胞を回収し、細胞破砕液を調製した。コントロールとして非感染細胞を用いた。
mPYYの測定は矢内原研究所のラットPYY ELISAキット用いた。発現量の評価は、プレートリーダーでの吸光度測定(O.D.450)により行った。
結果を図7に示す。mPYYはほとんど発現していないのに対し、CTB-mPYYは顕著に発現が増大していた。
(1) BHK21細胞にSeV18+ mGLP-2/ΔF、SeV18+CTB-mGLP-2/ΔFを感染させ、mGLP-2 およびCTB-mGLP-2ペプチド発現量の評価を行った。BHK21細胞をコラーゲンコートされた6穴プレートに1x106個になるよう播き、MOI 10 になるように無血清培地(VPSFM)で希釈された各SeVベクターを感染させた。1時間後に10% FBS含有GMEMを加え、24時間後に無血清培地(VPSFM)に置換し、48時間後に培養上清と細胞を回収し、細胞破砕液を調製した。コントロールとして非感染細胞を用いた。
mGLP-2の測定は矢内原研究所のGLP-2 ELISAキット用いた。発現量の評価は、プレートリーダーでの吸光度測定(O.D.450)により行った。
結果を図8に示す。mGLP-2はほとんど発現していないのに対し、CTB-mGLP-2は顕著に発現が増大していた。
(1) BHK21細胞にSeV18+Aβ42/ΔF、SeV18+IL-4-Aβ42/ΔF、SeV18+PEDI-Aβ42/ΔF、SeV18+CTB-Aβ42/ΔFを感染させ、Aβ42抗原量の評価を行った。BHK21細胞をコラーゲンコートされた6穴プレートに1x106個/wellになるよう播き、MOI 10になるように無血清培地(VPSFM)で希釈された各SeVベクターを感染させた。1時間後に10% FBS含有GMEMを加え、24時間後に無血清培地(VPSFM)に置換し、48時間後に培養上清と細胞を回収し、細胞破砕液を調製した。
Aβ42の測定は和光純薬工業のヒトAβ42 ELISAキット用いた。発現量の評価は、プレートリーダーでの吸光度測定(O.D.450)により行った。
結果を図9に示す。Aβ42はほとんど発現していないのに対し、IL-4-Aβ42、PEDI-Aβ42、CTB-Aβ42は発現が増大している。細胞内における発現量はそれぞれkkAβ42の1395倍、171.5倍、12608倍であった。
コンフルエントになった前日播種したBHK-21細胞(3x105 cells/well播種, 12-well plate)へAβ42単独搭載SeVベクターとCTB-Aβ42搭載SeVベクターをMOI 10 で感染させ、1 ml/well VP-SFM培地で37℃, 5% CO2培養し、4日目の培養上清と細胞lysateを回収した。培養上清アセトン沈殿で10倍濃縮し、1x SDS Loading Bufferでサンプルを調製した。細胞Lysateは150μl/wellの1x SDS Loading Bufferを用いて調製した。調製した培養上清と細胞Lysateを98℃, 10min処理した後、Aβ42ペプチドを1-0.5-0.25-0.125ng/laneでコントールとしてSDS-PAGE(15% Wakoゲル使用)/Western blotting(6E10抗体使用)を行い、蛋白定量をした。Aβ42単独搭載SeVベクターによるAβの発現量は、細胞Lysateでは4.4ng/well、上清では7.2x10-3 ng/wellしかなかった。一方、Aβ42-CTB融合遺伝子搭載SeVベクターによるAβ発現量は、細胞Lysateでは2500ng/well、上清では200ng/wellであり、Lysateでは568倍、上清では27778倍と大幅改善されたことがわかった。
(1)CTB-Aβ15遺伝子のNot1フラグメントの構築
CTB-Aβ15遺伝子のNotIフラグメントは、CTB-Aβ42遺伝子をもとに構築した。(図10)
CTB-Aβ42遺伝子NotIフラグメントが入ったプラスミドをテンプレートとし、EcoRV制限酵素サイトを付加した2種類のプライマーAβ15-EcoR-R (配列番号107) およびAβ15-EcoR-F (配列番号108) でinverse PCRを行い、得られたPCR産物を制限酵素EcoRVで切断した。そしてセルフライゲーションすることにより、CTB-Aβ15フラグメントが入ったプラスミドを得た。そのプラスミドを制限酵素NotIで切断することにより、目的のCTB-Aβ15遺伝子のNotIフラグメント (配列番号109) を得た。
(2)CTB-Aβ15タンデムタイプ(CTB-Aβ15x2, CTB-Aβ15x4, あるいはCTB-Aβ15x8)遺伝子のNotIフラグメントの構築
CTB-Aβ15タンデム遺伝子のNotIフラグメントは、2つの遺伝子を利用して構築した。(図11)
方法はCTB-Aβ42の入ったプラスミドのAβ42部分を除いて制限酵素サイトを導入し、
その部分にPCRで制限酵素サイトを付加したAβ15タンデムのフラグメントを組み込んだ。
具体的にはCTB-Aβ42のNotIフラグメント(実施例10:配列番号69)が入ったプラスミドをテンプレートとし、制限酵素SmaIサイトを付加した2種のプライマーCTB-SmaI-R(配列番号111)およびCTB-SmaI-F(配列番号112)でinverse PCRを行い、得られたPCR産物をSmaIで切断し、セルフライゲーションすることにより、Aβ42を除去したプラスミドを得た。そしてそのプラスミドのSmaIサイトにAβ15タンデムフラグメントを組み込んだ。
Aβ15タンデムフラグメントの作成方法は、Aβ15の8タンデムNotIフラグメント(配列番号113)が入ったプラスミドを基に作成した。そのプラスミドをテンプレートとし、制限酵素サイトを付加した2種のプライマーAβ15-SmaI-F(配列番号115)およびAβ15-EcoRV-R (配列番号116) でPCRを行うと、Aβ15のリピート数が異なるPCR産物が得られる。それらをTAクローニングして塩基配列確認後に、2種の制限酵素SmaI, EcoRIで切り出すと平滑末端のAβ15タンデムフラグメントが得られる。そのフラグメントをAβ42を除去したプラスミドのSmaIサイトへ組み込んでプラスミドを増幅し、制限酵素NotIで切り出すことにより、目的のCTB-Aβ15x2 NotIフラグメント(配列番号117), CTB-Aβ15x4 NotIフラグメント(配列番号119)およびCTB-Aβ15x8 NotIフラグメント(配列番号121)を得た。
F遺伝子欠失型SeVベクター(WO00/70070)のcDNA(pSeV18+NotI/ΔF)をNotIで消化し、そのNotI部位にCTB-Aβ15, CTB-Aβ15x2, CTB-Aβ15x4, CTB-Aβ15x8フラグメントを挿入し、CTB-Aβ15, CTB-Aβ15x2, CTB-Aβ15x4, CTB-Aβ15x8遺伝子搭載F遺伝子欠失型SeV cDNA(pSeV18+CTB-Aβ15/ΔF, pSeV18+CTB-Aβ15x2/ΔF, pSeV18+CTB-Aβ15x4/ΔF, pSeV18+CTB-Aβ15x8/ΔF)を構築した。
(1)Western blotting
構築したベクターの感染と発現をウエスタンブロット法により評価した。
SeVベクターを感染させた細胞の破砕液および培養上清は等量のSDS-PAGE用サンプルbufferと混和し、98℃で5分間熱変成した。15%のアクリルアミドゲルでSDS-PAGEを行い、セミドライブロッティング法によりPVDF膜へ転写を行った。5%ミルク/TBS-Tでブロッキングを行い、抗Aβ抗体(6E10)と反応させた後、2次抗体としてHRP標識された抗マウスIgGと反応させ、化学発光基質SuperSignal West Femtoを用いてCCDカメラにより検出を行った。
その結果、CTB-Aβ42、CTB-Aβ15、CTB-Aβ15x2、CTB-Aβ15x4、CTB-Aβ15x8のBHK細胞内における発現と培地中への分泌が確認された。CTB-Aβ15x8の発現量はCTB-Aβ42の発現量より多く、CTB-Aβ15x8の培地中へ分泌も、CTB-Aβ42の培地中へ分泌に対して非常に多いことが示された(図12)。
ガングリオシドGM1を固相化したプレートを用いてCTBのGM1に対する結合を評価した。
ガングリオシドGM1 (5μg/mL)を96ウェルプレート (Nunc,MaxiSorp plate)の各ウェルに固相化させ、20% BlockingOne(ナカライテスク)でブロッキングした後、SeVベクターを感染させた細胞の培養上清(20倍~2百万倍希釈)を加え、HRP標識された6E10抗体と反応させ、TMB発色基質を用いて検出した。結合量の評価はプレートリーダーでの吸光度測定(O.D.450)により行った。
その結果、培地中に分泌されたCTB-Aβ42、CTB-Aβ15、CTB-Aβ15x2、CTB-Aβ15x4、CTB-Aβ15x8はGM1に結合し、CTB-Aβ15x8はCTB-Aβ42の10倍、CTB-Aβ15はCTB-Aβ15x8の100倍結合しており、Aβ15の繰り返しが増えるほどGM1に対する結合能が低下していることが示された(図13)。
(1)正常マウス(筋肉内投与CTB-Aβ42とCTB-Aβ15x8の比較)
C57BL/6Nマウス にCTB-Aβ42遺伝子、CTB-Aβ15x8遺伝子、GFP遺伝子を搭載したSeVベクターを各5x107 CIU/headのタイターで筋肉内投与(右後肢)し、抗体価の評価を行った。上記処置14日後、マウスから血液を採取し、血漿中の抗Aβ抗体量を測定した。Aβ1-42ペプチド(5μg/mL)を96ウェルプレート (Nunc,MaxiSorp plate)の各ウェルに固相化させ、20% BlockingOne(ナカライテスク)でブロッキングした後、マウス血漿を加え(300~30万倍希釈)、ペルオキシダーゼ標識抗マウスIgG抗体を反応させ、TMB発色基質を用いて検出した。Aβ抗体価の評価は、プレートリーダーでの吸光度測定(O.D.450)により行った。標準抗体として抗Aβ抗体(6E10)を用いた。
その結果、CTB-Aβ42遺伝子投与群(n=6)およびCTB-Aβ15x8遺伝子投与群(n=6)においてAβ抗体価が上昇し、ControlであるGFP遺伝子投与群(n=6)ではAβ抗体価の上昇が見られなかった(図14)。CTB-Aβ15x8遺伝子投与群の抗体価はCTB-Aβ42遺伝子投与群の12.23倍であった。
C57BL/6Nマウス にCTB-Aβ15x8遺伝子を搭載したSeVベクターを5x106 CIU/headと5x107 CIU/headのタイターで鼻腔内、皮内、筋肉内(右後肢)に投与し、Control群としてGFP遺伝子を搭載したSeVベクターを5x107 CIU/headのタイターで筋肉内投与(右後肢)し、抗体価の評価を行った。上記処置14日後、マウスから血液を採取し、血漿中の抗Aβ抗体量を測定した。
その結果、Control群を除く全ての投与群においてAβ抗体価が上昇した。皮内投与群では他の投与群に比べて抗体価が低く、鼻腔内投与群では同タイターの筋肉内投与群に比べ高い抗体価が得られた(図15)。
C57BL/6Nマウス にCTB-Aβ15、CTB-Aβ15x2、CTB-Aβ15x4、CTB-Aβ15x8、Control群としてGFP遺伝子を搭載したSeVベクターを5x107 CIU/headのタイターで鼻腔内に投与し、抗体価の評価を行った。
その結果、いずれも投与群においても、Control群に比べて有意に抗体価が上昇した。CTB-Aβ15x8投与群に比べ、CTB-Aβ15、CTB-Aβ15x4投与群において特に高いAβ抗体価が得られた(図16)。
(1) 正常マウス(筋注):精製CTB-Aβ42蛋白によるブースト
C57BL/6Nマウス にCTB-Aβ42遺伝子を搭載したSeVベクターを各5x107 CIU/headのタイターで筋肉内投与(右後肢)し、14、28日後に大腸菌で生産したCTB-Aβ42タンパクをそれぞれ20μg/PBS/head、100μg/PBS/head、100μg/IFA(不完全フロイントアジュバント)/headで筋肉内投与(右後肢)し、抗体価の評価を行った。上記処置14日ごとに、マウスから血液を採取し、血漿中の抗Aβ抗体量を測定した。
その結果、CTB-Aβ42遺伝子で免役し、CTB-Aβ42タンパクを追加免役した群では顕著な抗Aβ抗体上昇が得られた(図17)。2回ブースト後のAβ抗体価は20μgブースト群で32μg/ml、100μgブースト群で107μg/ml、100μg+IFAブースト群で25.9μg/mlであった。
C57BL/6Nマウス にCTB-Aβ42遺伝子およびCTB-Aβ15x8遺伝子を搭載したSeVベクターを5x107 CIU/headのタイターで筋肉内投与(右後肢)し、56日後に同SeVベクターを同タイターで筋肉内投与(右後肢)し、抗体価の評価を行った。
上記処置14、28日後、マウスから血液を採取し、血漿中の抗Aβ抗体量を測定した。
その結果、CTB-Aβ15x8遺伝子ブースト群において顕著なAβ抗体価の上昇が得られた(図18)。一方、CTB-Aβ42遺伝子ブースト群では、上記のCTB-Aβ15x8遺伝子ブースト群と比べると、Aβ抗体価の上昇は弱かった。
C57BL/6Nマウス にCTB-Aβ15x8遺伝子またはCTB-Aβ42遺伝子を搭載したSeVベクターを5x106 CIU/head および5x107 CIU/headのタイターで筋肉内投与(右後肢)し、56日後に同SeVベクターを同タイターで筋肉内投与(右後肢)し、抗体価の評価を行った。
上記処置14、28日後、マウスから血液を採取し、血漿中の抗Aβ抗体量を測定した。
その結果、両ベクターともブーストの明確な効果が得られたが、CTB-Aβ15x8遺伝子ブースト群において得に顕著なAβ抗体価の上昇が得られた(図19)。
C57BL/6Nマウス にCTB-Aβ15x8遺伝子を搭載したSeVベクターを5x106 CIU/head および 5x107 CIU/headのタイターで鼻腔内投与し、56日後に同SeVベクターを同タイターで鼻腔内投与し、抗体価の評価を行った。
上記処置14、28日後、マウスから血液を採取し、血漿中の抗Aβ抗体量を測定した。
その結果、CTB-Aβ15x8遺伝子ブースト群において3/3の割合で顕著なAβ抗体価の上昇が得られた(図20)。
(1) 抗Aβ抗体価
アルツハイマー病のモデルマウスであるAPPトランスジェニックマウス(Tg2576)(13ヶ月齢)にSeV18+CTB-Aβ18x5/ΔF(CTB-Aβ15x8とも記す)、又はSeV18+CTB-Aβ42/ΔF(CTB-Aβ42とも記す)、対照群としてGFP遺伝子を搭載したSeVベクター(SeV18+GFP/ΔF;以下、「GFP」とも記す)を5x107 CIU/headで筋肉内投与(右後肢)した。14、28日後、CTB-Aβ42遺伝子投与群の半分に大腸菌で生産したCTB-Aβ42タンパクを筋肉内投与(右後肢)した。SeVベクター投与から14、28、42、56日後に血漿中のAβ抗体価を測定した。
その結果、CTB-Aβ15x8遺伝子投与群において顕著なAβ抗体価の上昇が観察された。CTB-Aβ42遺伝子投与群においてはAβ抗体価がほとんど上昇しない個体が半分存在し、Aβ抗体価の上昇もCTB-Aβ15x8遺伝子投与群に対して低かった。CTB-Aβ42タンパクブースト群においてはブーストによるAβ抗体価の上昇は見られなかった(図21)。
上記APPマウスより、SeVベクター投与開始56日後に脳組織を採材し、脳組織左半球中のAβ量をELISAにより測定した。脳組織をTBS中で超音波ホモジナイズし、35,000gで1時間遠心後、上清をAβ可溶性画分として採取、沈殿を10%ギ酸中で超音波ホモジナイズし、1M Trisを用いて中和、Aβ不溶性画分として採取した。和光純薬工業のAβ42 ELISAキットおよびAβ40 ELISAキットを用いて脳内Aβの量を測定した。その結果、不溶性画分中のAβ量はCTB-Aβ15x8遺伝子投与群においてGFP遺伝子投与群に比べ80%程度に低下していた。CTB-Aβ42遺伝子投与群ではAβ量の低下は見られなかった。CTB-Aβ42タンパクブースト群においては若干のAβ量低下が見られた。可溶性画分中のAβ量はCTB-Aβ15x8遺伝子投与群においてGFP遺伝子投与群に比べ50%程度に低下していた。CTB-Aβ42遺伝子投与群ではAβ量の低下は見られなかった。CTB-Aβ42タンパクブースト群におけるAβ量は60~70%に低下していた(図22)。
センダイウイルスベクターを筋注投与したマウスから、投与後8週間(15ヶ月齢)の時点で各群を解剖し、右脳半球を病理組織検査用に10%中性緩衝ホルマリン液に浸漬固定し、パラフィン包埋後、脳正中裂より約2mmの部位の脳組織縦断切片を得た。上記切片組織中のAβ蛋白や老人斑を検出するために、70%ギ酸で処理し、5% H2O2で内因性ペルオキシダーゼ活性を失活させた。抗Aβ抗体(6E10抗体、1000倍希釈)と反応させた後、ペルオキシダーゼ標識二次抗体を加え、DAB発色を行った。また、顕微鏡に連結させた3CCDカメラ用いて撮影し、各例20-30枚の画像ファイルを合成し(図23)、嗅球、大脳新皮質および海馬の各領域におけるAβ蓄積部分の占める面積を画像解析ソフトNIH imageを用いて全例同一条件にて測定した。そして、各測定部位に占めるAβ蓄積部分の面積率を計算した。また、そのときの計測に使用された老人斑の個数も比較した。その結果、図24に示すように、とくに海馬における老人斑面積率が減少傾向を示した。
治療群およびコントロール群から、上記(3)と同様にして投与後8週間経過時(15ヶ月齢)のパラフィン切片のHE染色標本および抗Iba-1抗体(ミクログリア)染色標本を用いて中枢神経系における炎症性細胞浸潤およびミクログリア活性化の有無を確認した。その結果、コントロール群、治療群ともに、脳のいずれの部位においても、炎症細胞浸潤は全く観察されず、本発明のベクターが中枢神経系の炎症を起こさないことが実証された。
また、ミクログリアの活性化は両群の動物の老人斑周囲に観察されたが、ベクター投与群の動物では老人斑が減少する傾向にあったことと平行して、ミクログリアの占める面積率も減少する傾向にあった。
Claims (25)
- 組み換え蛋白質をコードするRNAウイルスベクターであって、該蛋白質が、AB5トキシンBサブユニットとの融合蛋白質としてコードされていることを特徴とするベクター。
- AB5トキシンBサブユニットがコレラトキシンB(CTB)である、請求項1に記載のベクター。
- 該組み換え蛋白質が120アミノ酸以下のペプチドである、請求項1または2に記載のベクター。
- 該組み換え蛋白質が50アミノ酸以下のペプチドである、請求項3に記載のベクター。
- 該組み換え蛋白質が神経ペプチドまたは内分泌性ペプチドである、請求項1から4のいずれかに記載のベクター。
- 該組み換え蛋白質が低溶解性蛋白質である、請求項1から5のいずれかに記載のベクター。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項1から6のいずれかに記載のベクター。
- 請求項1から7のいずれかに記載のベクターおよび薬学的に許容される担体を含む医薬組成物。
- 請求項1から7のいずれかに記載のベクターが導入された細胞。
- 請求項1から7のいずれかに記載のベクターを発現させる工程を含む、組み換え蛋白質の製造方法。
- 組み換え蛋白質の発現量が上昇したRNAウイルスベクターの製造方法であって、該蛋白質をAB5トキシンBサブユニットと融合させた融合蛋白質をコードするRNAウイルスベクターを製造する工程を含む方法。
- AB5トキシンBサブユニットがコレラトキシンB(CTB)である、請求項11に記載の方法。
- 該組み換え蛋白質が120アミノ酸以下のペプチドである、請求項11または12に記載の方法。
- 該組み換え蛋白質が50アミノ酸以下のペプチドである、請求項13に記載の方法。
- 該組み換え蛋白質が神経ペプチドまたは内分泌性ペプチドである、請求項11から14のいずれかに記載の方法。
- 該組み換え蛋白質が低溶解性蛋白質である、請求項11から15のいずれかに記載の方法。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項11から16のいずれかに記載の方法。
- RNAウイルスベクターからの組み換え蛋白質の発現量を上昇させる方法であって、該組み換え蛋白質をAB5トキシンBサブユニットとの融合蛋白質としてRNAウイルスベクターから発現させる工程を含む方法。
- AB5トキシンBサブユニットがコレラトキシンB(CTB)である、請求項18に記載の方法。
- 該組み換え蛋白質が120アミノ酸以下のペプチドである、請求項18または19に記載の方法。
- 該組み換え蛋白質が50アミノ酸以下のペプチドである、請求項20に記載の方法。
- 該組み換え蛋白質が神経ペプチドまたは内分泌性ペプチドである、請求項18から21のいずれかに記載の方法。
- 該組み換え蛋白質が低溶解性蛋白質である、請求項18から22のいずれかに記載の方法。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項18から23のいずれかに記載の方法。
- 請求項1から7のいずれかに記載のベクターからなるウイルス様粒子。
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US20120088819A1 (en) | 2012-04-12 |
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EP2363472A1 (en) | 2011-09-07 |
EP2363472A4 (en) | 2012-08-01 |
JPWO2010050586A1 (ja) | 2012-03-29 |
CN102272302A (zh) | 2011-12-07 |
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