WO2005108581A1 - リンパ球細胞へ遺伝子導入するための組換えウイルスベクター - Google Patents
リンパ球細胞へ遺伝子導入するための組換えウイルスベクター Download PDFInfo
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- WO2005108581A1 WO2005108581A1 PCT/JP2005/008250 JP2005008250W WO2005108581A1 WO 2005108581 A1 WO2005108581 A1 WO 2005108581A1 JP 2005008250 W JP2005008250 W JP 2005008250W WO 2005108581 A1 WO2005108581 A1 WO 2005108581A1
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Definitions
- the present invention relates to a recombinant viral vector for introducing a desired gene into lymphocyte cells, particularly a recombinant human herpesvirus vector prepared using BAC (E. coli artificial chromosome), and such a vector. It relates to a pharmaceutical composition comprising a viral vector. Furthermore, the present invention relates to a vector containing a human herpesvirus genomic gene and a BAC vector sequence, and a cell containing such a vector. Further, the present invention relates to a method for producing a recombinant human herpesvirus. The present invention also relates to a nucleic acid cassette comprising a fragment capable of homologous recombination with a herpesvirus genome and a BAC vector sequence.
- lymphocyte cells For the treatment of various diseases targeting lymphocyte cells, such as human immunodeficiency virus (HIV) infection, establishment of a technique for gene therapy on lymphocyte cells is desired.
- HIV human immunodeficiency virus
- a sufficient vector system for introducing a desired gene into lymphocyte cells has been developed.
- Herpesvirus is a general term for viruses belonging to the family Herpesviridae.
- Human herpesvirus 6 or 7 (HHV-6 or HHV-7) is a double-stranded DNA virus belonging to the herpesviridae 13 herpesvirus subfamily, both of which have a sudden rash (Exanthom subitum ) Cause virus.
- Non-patent document 1 and Non-patent document 2 There are two strains of HHV-6: HHV-6A and HHV-6B. It is a viral infection characterized by sudden high fever and rashes before and after antipyretic that are often affected in infancy, and the prognosis is generally good.
- Non-patent Document 3 Since HHV-7 tends to be infected later than HHV-6 (Non-patent Document 3), a sudden rash caused by HHV-7 is clinically often experienced as a second sudden rash. Sero-epidemiological studies on HHV-6 and HHV-7 have found that most children are antibody-positive by the age of 2 to 3 years, with subclinical infection reported as 20 to 40%. Yes.
- HHV-7 was developed in 1990 by Frenkel et al., Who cultured healthy human peripheral blood CD4 + T lymphocytes. It is a herpes virus that was newly discovered due to the cytopathic effect that occurs (Non-Patent Document 4). It is a virus isolated from human peripheral blood mononuclear cells, and both HHV-6 and -7 are CD4 + T lymphotropic viruses. The cell-side receptor for HHV-7 infection is CD4, which can only grow on human T lymphocytes. Therefore, it is a virus that can be used for gene modification of human T lymphocytes.
- HHV-7 The genome of HHV-7 is a double-stranded DNA, which is about 145 kbp.
- the entire nucleotide sequence has been determined by Nicholas et al., And it is known that there are at least 101 genes on the genome. (Non-patent document 5)
- HHV-7 viruses are supposed to be harmless to healthy people V
- genes containing antigenic determinants of various viruses for example, Mumbus
- Expression in HHV-7 is considered to be useful as a vaccine.
- a recombinant virus when used as a vaccine, it is not preferable from the viewpoint of quality control and quality assurance that the genotype changes with the subculture. Therefore, when a recombinant virus is used as a vaccine, it is necessary to stably supply a virus derived from a single recombinant genotype. Therefore, it is desired to develop a method for producing a HHV-7 recombinant virus having a single genotype.
- HHV-6A U1102 strain
- HHV-7 MSO strain
- the HHV-7 strain in which CD4 is the cell-side receptor, showed good growth in SupTl cells, but failed to establish infection in SupTlZHIV cells.
- the HHV-6A strain infects HIV-infected SupTl (Sup-TlZHIV) cells and recognizes clear CPE (Non-patent Document 6).
- Patent Document 1 Yamanishi K et al. "Identification of human herpesvirus 6 asa casual agent for exanthem subitum.” Lancet 1988; i: 1065-1010 7
- Non-patent literature 2 Tanaka K et al "Human herupesvirus 7: Another casual ag ent for roseola (exanthem subitum) J pediatr. 1994; 125: 1 ⁇ ; — 6 and —7 in children of different ages and detectio n of those two viruses in throat swabs by polymerase chain react ion "Jounal of Medical Virology. 1996; 48: 88-94
- Non-Patent Document 4 Frankel N et al., "Isolation of a new herpesvirus from hum an CD4 + T cells.” ProNAS USA 87: 749- 752, ProNAS USA 87: 7 49-752, 1990
- Non-patent document 5 John N. et al., Journal of Virology, Sep. 1996, pages 5975-5989
- Non-patent document 6 Masao Yamada et al. HIV persistent infection Sup— Trial of HHV-6 and 7 double infection on T1 cells, Abstract No. 122, 7th Annual Meeting of AIDS Society, Abstracts, 1993, Tokyo
- An object of the present invention is to provide a recombinant viral vector for introducing a desired gene into lymphocyte cells, particularly a recombinant human herpesvirus vector prepared using BAC (E. coli artificial chromosome), and such It is to provide a pharmaceutical composition comprising a viral vector.
- Another object of the present invention is to provide a vector containing a human herpesvirus genomic gene and a BAC vector sequence, and a cell containing such a vector.
- an object of the present invention is to provide a method for producing a recombinant human herpesvirus.
- Another object of the present invention is to provide a nucleic acid cassette comprising a fragment capable of homologous recombination with a herpesvirus genome and a BAC vector sequence.
- a recombinant HHV-7 and a method for producing the same for example, using BAC (E. coli artificial chromosome), a single virus strain, recombinant HHV-7 or A method of making HHV-6 is provided.
- BAC E. coli artificial chromosome
- the ideal HIV vaccine is to provide complete and long-term protection against all types of HIV.
- some of the advantages and disadvantages of traditional inactive HIV vaccines are as follows.
- Recombinant vaccines are produced using conventional methods, but it is difficult to maintain immunogenicity (because of low immunogenicity), so high antigen load, adjuvant and frequent inoculation are required. And its safety is the biggest concern.
- subunit vaccines containing either native or recombinant subunits are limited by the selection of unsafe force subunits and low immunogenicity.
- HIV has the power to attack immune cells that are normally activated by vaccines, in addition to being integrated with cells.
- Gpl20 binds to CD4 when it infects HIV cells.
- CD4 is a receptor on the surface of HIV-infected immune cells (T cells) and can be said to be the gateway to cells.
- HHV-7 can suppress the proliferation of HIV by being on the surface of CD4 + cells and HHV-6 in the cell.
- the present invention provides a pharmaceutical composition for preventing HIV infection and a pharmaceutical composition for treating HIV infection.
- the present inventors have completed the present invention by developing a method for producing a recombinant herpesvirus using a BAC vector sequence.
- the present invention provides the following.
- a recombinant herpesvirus for gene transfer into lymphocytes 1.
- the recombinant herpesvirus according to item 1 comprising a BAC vector sequence.
- the BAC vector sequence comprises a recombinant protein dependent recombinant sequence.
- the recombinant herpesvirus according to item 3 wherein the herpesvirus genome is derived from HHV-6A U1102 strain or HHV-6B HST strain.
- BAC vector sequence is a vector having the sequence set forth in SEQ ID NO: 401.
- a pharmaceutical composition comprising the virus according to item 1.
- a vector comprising a human herpesvirus essential gene and a BAC vector sequence.
- non-essential region is selected as a group force that also has the following region power of HHV-7: Region within ORF of gene HI, region within ORF of gene DR1, region within ORF of gene DR2, region within ORF of gene H2, region within ORF of gene DR6, region within ORF of gene DR7, gene H3 ORF region, gene H4 ORF region, gene U2 ORF region, gene U3 ORF region, gene U4 ORF region, gene U5Z7 ORF region, gene U8 region Region in ORF, region in ORF of gene U10, region in ORF of gene U12, region in ORF of gene U13, region in ORF of gene U15, region in ORF of gene U16, ORF of gene U17Ex Region within the ORF of gene U17, region within the ORF of gene U17a, region within the ORF of gene U18, region within the ORF of gene U19, region within the ORF of gene U20, within the ORF of gene U21 Region
- a cell comprising the vector according to item 21.
- the cell according to item 35 which is a mammalian cell.
- the mammalian cell according to item 38 which is a human-derived cell.
- a pharmaceutical composition comprising the virus according to item 40.
- a method for producing a recombinant herpesvirus comprising the following steps:
- BAC vector sequence comprises at least two recombinant protein dependent recombinant sequences.
- non-essential area is selected from the following group powers that are also HHV-7 area powers:
- non-essential region is a region adjacent to the ORF of gene U24 of HHV-7 or a region adjacent to the ORF of gene U24a of HHV-7.
- herpesvirus genome is derived from a wild type strain.
- herpesvirus genome is derived from HHV-6A U1102 strain or HHV-6B HS T strain.
- BAC vector sequence is a vector having the sequence set forth in SEQ ID NO: 401.
- a pharmaceutical composition comprising the virus according to item 61.
- a method for introducing a mutation into the vector according to item 19, comprising the following steps: introducing the vector into a bacterial host cell;
- plasmid vector comprising a fragment that is partly of the herpesvirus genome, wherein the fragment has at least one mutation
- a method for introducing a mutation into the vector according to item 19, comprising the following steps: introducing the vector into a bacterial host cell;
- a first plasmid vector comprising a first fragment that is a part of the herpesvirus genome, wherein the first fragment is at least one Having a mutation
- a second plasmid vector comprising a second fragment of the herpesvirus genome, wherein the second fragment comprises at least one Having a mutation, and wherein the second fragment is different from the first fragment;
- a nucleic acid cassette comprising: a nucleic acid force set, wherein each end of said BAC sequence is linked to a first fragment and a second fragment, respectively.
- nucleic acid cassette of item 66 wherein the first fragment and the second fragment are at least lkb.
- nucleic acid cassette of item 66 wherein the first fragment and the second fragment are at least 80% identical to the sequence of the herpesvirus genome.
- the nucleic acid cassette of item 66, wherein the first fragment and the second fragment are at least 85% identical to the sequence of the herpesvirus genome.
- nucleic acid cassette according to item 66 wherein the first and second fragments are each independently selected as a group force having the following region force of the herpesvirus HHV-7 genome.
- nucleic acid cassette according to item 66 wherein the first and second fragments are each independently selected as a group force having the following region force of the herpesvirus HHV-7 genome.
- nucleic acid cassette according to item 66 wherein the first and second fragments are each independently selected as a group force having the following region force of the herpesvirus HHV-7 genome.
- nucleic acid cassette according to item 66 wherein the first and second fragments are each independently selected as a group force that also has the following region force of the herpesvirus HHV-7 genome. Nucleic acid cassettes that are at least 90% identical to
- nucleic acid cassette according to item 66, wherein the first and second fragments are each independently selected as a group force having the following region force of the herpesvirus HHV-6 genome. Nucleic acid cassette that is at least 90% identical to
- the first and second fragments are each independently a region adjacent to the ORF of HHV-6 gene U5 or the ORF of gene U8 of HHV-6 The described nucleic acid cassette.
- nucleic acid cassette of item 66 wherein the BAC vector sequence comprises a selectable marker.
- selectable marker is a drug selectable marker.
- nucleic acid cassette of item 88 wherein the selectable marker is a gene encoding green fluorescent protein.
- nucleic acid cassette according to item 66, wherein the herpesvirus genome is derived from HHV-7 KHR strain.
- nucleic acid cassette according to item 66, wherein the herpesvirus genome is derived from HHV-6A U1102 strain or HHV-6B HS T strain.
- nucleic acid cassette of item 66, wherein the BAC vector sequence has the nucleic acid sequence of SEQ ID NO: 401.
- a pharmaceutical composition for the prevention, treatment or prognosis of HIV comprising the recombinant herpesvirus according to item 4.
- a pharmaceutical composition for the prevention of HIV comprising the recombinant herpesvirus according to item 4.
- a pharmaceutical composition for prevention, treatment or prognosis of HIV comprising the recombinant herpesvirus according to item 6.
- a pharmaceutical composition for the prevention of HIV comprising the recombinant herpesvirus according to item 6.
- a recombinant herpesvirus and a method for producing the same are provided.
- a method for producing a recombinant herpesvirus using BAC (E. coli artificial chromosome) and a recombinant herpesvirus produced by the method is described.
- BAC E. coli artificial chromosome
- a pharmaceutical composition comprising a recombinant herpesvirus.
- a vector containing a herpesvirus genome gene and a BAC vector sequence a cell containing such a vector, a fragment capable of homologous recombination with the herpesvirus genome, and BAC A nucleic acid cassette containing the vector sequence is provided.
- HHV-7 is known not to cause any harm to healthy individuals, among various proteins of viruses, proteins known to act as vaccines are recombinant HHV- By being incorporated into 7 and expressed, it becomes possible to produce a virus vaccine.
- compositions for the prevention, treatment, and Z or prognosis of HIV infection comprising the recombinant HHV-7 and HHV-6 of the present invention.
- FIG. 1 is a diagram schematically showing insertion of a BAC vector into the HHV-7 genome.
- FIG. 2 is a result showing the expression of GFP introduced into a host cell by the recombinant HHV-7 of the present invention.
- SEQ ID NO: 1 genome sequence of JI strain
- SEQ ID NO: 50 U65 5> 3 'direction 94111 to 95103 amino acid sequence of amino acids 1-331
- SEQ ID NO: 52 U68 5> 3 'direction 97024-97368 amino acid sequence of amino acids 1-115 SEQ ID NO: 53, U71 5> 3' direction 100392-100613 amino acids 1-74 nucleic acid sequence encoding
- SEQ ID NO: 112 nucleic acid sequence encoding U60-U663 ' ⁇ 5' direction 90878-92005 and 95122-95985 amino acids 1-664
- SEQ ID NO: L30, DR4, 5,-> 3 'direction, 2746-3048 amino acid sequence of amino acid 1-101 SEQ ID NO: L31, DR6, 5,->3' direction, 4725-5036, amino acid 1- Amino acid sequence of 104 SEQ ID NO: L32, DR7, 5, —> 3 ′ direction, 5629-6720, amino acid sequence of amino acids 1-364 SEQ ID NO: L33, DR8, 5, —> 3 ′ direction, 7237-7568
- SEQ ID NO: 161 U91, 5, ⁇ 3, direction, 136485-136829, amino acid sequence of amino acids 1-115 SEQ ID NO: 162, U95, 5, ⁇ 3, orientation, 142941-146306, amino acid sequence of amino acids 1-1122 SEQ ID NO: 163, DR1, 5, ⁇ 3, orientation, 151734-152027, amino acid sequence of amino acids 1-98 SEQ ID NO: 164, DR4, 5, ⁇ 3, direction, 153979-154281, amino acid sequence of amino acids 1-101 SEQ ID NO: 165, DR6, 5, ⁇ 3, direction, 155958-156269, amino acid sequence of amino acids 1-104 SEQ ID NO: 166, DR7, 5, ⁇ 3, direction, 156862-157953, amino acid sequence of amino acids 1-364 SEQ ID NO: 167, DR8, 5, ⁇ 3, direction, 158470-158802, amino acid sequence of amino acids 1-111 SEQ ID NO: 168, DR2, Nucleic acid sequence encoding 5 ' ⁇ 3' direction, 79 ⁇
- SEQ ID NO: 172 nucleic acid sequence encoding U50, 5 ' ⁇ 3' direction, 80812 ⁇ 82479, amino acids 1-556
- SEQ ID NO: 175 U59, 'direction, 96239 ⁇ 97291, amino acid sequence of amino acids 1-351
- SEQ ID NO: 176 U63,' direction, 98632 ⁇ 99282
- SEQ ID NO: 177 U63, orientation, 98632-99282, amino acid sequence of amino acids 1-217
- SEQ ID NO: 178 U65, orientation, 100545-101552 nucleic acid sequence encoding amino acids 1-336
- SEQ ID NO: 179 U65, orientation, 100545-101552, amino acid sequence of amino acids 1-336
- SEQ ID NO: 180 nucleic acid sequence encoding U68, orientation, 103519-103863, amino acids 1-115
- SEQ ID NO: 183 U71, 5, ⁇ 3, orientation, 106965-107198, amino acid sequence of amino acids 1-78
- SEQ ID NO: 184 Nucleic acid sequence encoding U74, 5 ′ ⁇ 3 ′ orientation, 110636-112624, amino acids 1-663
- SEQ ID NO: 190 Genomic sequence of U1102 strain (complementary strand)
- SEQ ID NO: 256 nucleic acid sequence encoding U16 exons 1-2, 3, ⁇ 5, direction, 26259-27034 and 27187-27349, amino acids 1-313
- SEQ ID NO: 315 U63, orientation, 99756 ⁇ 100412, amino acid sequence of amino acids 1-219
- SEQ ID NO: 316 U65 amino acid sequence of amino acids 1-219
- orientation, 101675 ⁇ 102682 nucleic acid sequence encoding amino acids 1-336
- HN2 3 '>5' direction, 149749 ⁇ 149913, amino acid sequence of amino acids 1-55 SEQ ID NO: 333.
- U97, 3, -5 'direction, 149352-149651 amino acid sequence of amino acids 1-100 SEQ ID NO: 334.
- U94, 3, -5 'direction, 142683-144155 amino acid sequence of amino acids 1-491 SEQ ID NO: 335.
- U90 5 'direction, 137810-138085 amino acid sequence of amino acids 1--92 SEQ ID NO: 337.
- U55 direction, 88628 ⁇ 90106 Amino acid sequence of amino acids 1-493 SEQ ID NO: 351.
- U47 direction, 76617 ⁇ 78833 Amino acid sequence of amino acids 1-739 SEQ ID NO: 355.
- SEQ ID NO: 389 Nucleic acid sequence encoding U9, 3 ′ ⁇ 5 ′ direction, 18022 ⁇ 18336, amino acids 1-105 SEQ ID NO: 390. U9, 3, ⁇ 5, direction, 18022-18336, amino acid sequence of amino acids 1-105 SEQ ID NO: 391.Nucleic acid sequence encoding U22, 3 ' ⁇ 5' direction, 34690 ⁇ 35298, amino acids 1-203
- SEQ ID NO: 394 Amino acid sequence of U27, 3, ⁇ 5, orientation, 38758-39933, amino acids 1-392 SEQ ID NO: 395. Nucleic acid sequence encoding U34, 3 ′ ⁇ 5 ′ orientation, 54046-54876, amino acids 1-277 Column
- SEQ ID NO: 396 Amino acid sequence of U34, 3, ⁇ 5, orientation, 54046-54876, amino acids 1-277 SEQ ID NO: 397. Nucleic acid sequence encoding U39, 3 ′ ⁇ 5 ′ orientation, 60542 ⁇ 63034, amino acids 1-831 Column
- SEQ ID NO: 400 U61, 3 ' ⁇ 5' direction, 99355-99867, amino acid sequence of amino acids 1-171 SEQ ID NO: 401, BAC vector sequence
- the term "herpesvirus” includes all of HHV-6A, HHV-6B, and HHV-7, and unless otherwise stated. Including both wild type and recombinant type of virus.
- the term “: HHV-6” encompasses HHV-6 81111-68, and unless otherwise noted, the wild type, recombinant Includes any type.
- essential gene of a herpes virus refers to a gene essential for the herpes virus to propagate.
- a “non-essential gene” of a herpes virus is a gene that is not essential for the growth of the herpes virus. ! Uh.
- Non-essential genes for human herpesvirus 7 include, but are not limited to, for example, the following: gene HI, gene DR1, gene DR2, gene H2, gene DR6, gene DR7, gene H3 , Gene H4, Gene U2, Gene U3, Gene U4, Gene U5Z7, Gene U8, Gene U10, Gene U12, Gene U13, Gene U15, Gene U16, Gene U17Ex, Gene U17, Gene U17a, Gene U18, Gene U19, Gene U20, Gene U21, Gene U23, Gene U24, Gene U24 a, Gene U25, Gene U26, Gene U28, Gene U32, Gene U33, Gene U3 4, Gene U35, Gene U36, Gene U37, Gene U40, Gene U42, Gene U4 4, Gene U45, Gene U46, Gene U47, Gene U49, Gene U50, Gene U5 1, gene U52, gene U55A, gene U55B, gene U58, gene U59, gene U62, gene U63, gene U64, gene U65,
- the region in the ORF of the non-essential gene and the region adjacent to Z or ORF can be used as a target for BAC vector insertion.
- a preferred target is, for example, a region within or adjacent to the ORF of gene U24, or a region within or adjacent to the ORF of gene U24a, and preferably a region adjacent to the ORF of gene U24 or the gene.
- the region adjacent to the ORF of U24a but not limited to.
- wild strain of a herpesvirus refers to a herpesvirus strain isolated from nature that has not been artificially modified!
- wild strains include, but are not limited to, JI strains.
- the nucleic acid sequence of this JI strain is set forth in SEQ ID NO: 1.
- the orientation of the reading frame of each ORF of this JI strain, the position on the genome, and the number of amino acid residues of the encoded polypeptide are as follows.
- non-essential genes for human herpesvirus 6 include, but are not limited to, the following: gene LT1, gene DR1, gene DR2, gene DR3, gene DR4, gene DR5, gene DR6, gene DRHN1, gene DR7, gene DRHN2, gene DR8, gene IJ1, gene Ul, gene U2, gene U3, gene U4, gene U5, gene U6, gene U7, gene U8, gene U9, gene U10, gene U12EX , Gene U12, gene U13, gene U15, gene U16, gene U17, gene U18, gene U19, gene U20, gene U21, gene U22, gene U23, gene U24, gene U25, gene U26, gene U28, gene U32 , Gene U33, gene U34, gene U35, gene U36, gene U37, gene U40, heredity U42 gene U44 gene U45 gene U46 gene U47 gene U49, gene U50 gene U51 gene U52 gene U55 gene U58 gene U59, gene U
- a region in the ORF of the non-essential gene and a region adjacent to Z or ORF can be used as a target for BAC vector insertion.
- Preferred targets are, for example, the region within or adjacent to the ORF of gene U5, or the region within or adjacent to the ORF of gene U8, and preferably the region adjacent to the ORF of gene U5 or the region of gene U8.
- the force that is the region adjacent to the ORF is not limited to these.
- wild strain of HHV-6A refers to an HHV-6A strain that has not been artificially modified and has been isolated from nature.
- wild strains include U1102 strain 1S, but is not limited to this.
- the nucleic acid sequence of this U1102 strain is set forth in SEQ ID NO: 128.
- the reading frame direction of each ORF of this U1102 strain, the position on the genome, and the number of amino acid residues of the encoded polypeptide are as follows.
- the “mutant strain” refers to a herpesvirus strain in which a wild-type virus strain is mutagenized by mutagenesis, multiple subcultures, or the like. When mutagenizing herpesvirus strains, this mutagenesis can be random or site-specific mutagenesis.
- genes listed below are not limited to these: genes LT 1, gene DR1, gene DR2, Gene DR3, gene DR4, gene DR5, gene DR 6, gene DRHN1, gene DR7, gene DRHN2, gene DR8, gene LJ1, gene Ul, gene U2, gene U3, gene U4, gene U5, gene U6, gene U7 , Gene U8, gene U9, gene U10, gene U12EX, gene U12, gene U13, gene U15, gene U16, gene U17, gene U18, gene U19, gene U20, gene U21, gene U22, gene U23, gene U24, gene U25, gene U26 , Gene U28, Gene U32, Gene U33, Gene U34, Gene U35, Gene U36
- a region in the ORF of the non-essential gene and a region adjacent to Z or ORF can be used as a target for BAC vector insertion.
- Preferred targets are, for example, the region within or adjacent to the ORF of gene U5, or the region within or adjacent to the ORF of gene U8, and preferably the region adjacent to the ORF of gene U5 or the region of gene U8.
- the force that is the region adjacent to the ORF is not limited to these.
- the wild strain of HHV-6B refers to a herpesvirus strain that has not been artificially modified and has been isolated from nature.
- HHV-6B wild strains include, but are not limited to, the HST strain.
- the nucleic acid sequence of this HST strain is set forth in SEQ ID NO: 272.
- the orientation of the reading frame of each ORF of this HST strain, the position on the genome, and the number of amino acid residues of the encoded polypeptide are as follows.
- a wild strain of a herpesvirus refers to a herpesvirus strain isolated from nature that has not been artificially modified!
- mutant strain refers to a herpesvirus strain in which a wild-type virus strain is mutagenized by mutagenesis, multiple subcultures, or the like. When mutagenizing herpesvirus strains, this mutagenesis may be site-specific, even for random mutagenesis. Even heterogeneous mutagenesis.
- protein protein
- polypeptide oligopeptide
- peptide a polymer of amino acids of any length.
- nucleic acid refers to a nucleotide polymer of any length. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (e.g., degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605—2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91— 9 8 (1994)).
- gene refers to a factor that defines a genetic trait. Usually arranged on a chromosome in a certain order. A gene that defines the primary structure of a protein is called a structural gene, and a region that affects its expression is called a regulatory element. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and Z or “protein” “polypeptide”, “oligopeptide” and “peptide”. In this specification, the “open reading frame” or “ORF” of a gene is one of three frameworks when the base sequence of a gene is divided into 3 bases each, and has a start codon.
- the herpesvirus genome has its entire base sequence determined, at least 101 genes have been identified, and each of the genes is known to have an open reading frame (ORF).
- the “region in the ORF” of a gene in the herpesvirus genome refers to a region in which a base that forms an ORF is present in a gene in the herpesvirus genome.
- the "region adjacent to the ORF" of the gene in the herpesvirus genome means the presence of a base in the vicinity of the ORF in the gene in the herpesvirus genome! A region that exists and does not fall within the ORF of the gene or another gene.
- homology of a gene refers to the degree of identity of two or more gene sequences with respect to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity.
- the ability of two genes to have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions.
- the DNA sequence power between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, If they are 95%, 96%, 97%, 98% or 99% identical, the genes have homology.
- expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo and takes another form.
- it refers to force transcription and translation of genes, polynucleotides, and the like to form a polypeptide, but transcription and production of mRNA may also be an embodiment of expression. More preferably, such polypeptide forms may be post-translationally processed.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC—IUB Biochemica 1 Nomenclature Commission. Nucleotides can also be referred to by the generally accepted single letter code.
- a “fragment” refers to a polypeptide or polynucleotide having a sequence length from 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length n).
- the length of the fragment can be changed as appropriate according to its purpose.
- the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 in the case of a polypeptide.
- 15, 2 Examples include 0, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) may also be appropriate as lower limits.
- nucleotides 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 600, 700, 800 900, 1000 and higher nucleotides, and lengths expressed in integers not specifically listed here (eg, 11 etc.) may also be appropriate as lower limits.
- polypeptide encoded by the gene in the BAC vector has substantially the same action as the naturally occurring polypeptide, one or more (for example, one or several) amino acids in the amino acid sequence are substituted.
- the sugar chain may be substituted, added and Z or deleted, which may be added and Z or deleted.
- sugar chain refers to a compound obtained by connecting one or more unit sugars (monosaccharide and Z or a derivative thereof). When two or more unit sugars are connected, each unit sugar is bonded by dehydration condensation using a glycosidic bond.
- unit sugars monosaccharide and Z or a derivative thereof.
- Such sugar chains include, for example, polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylethyldarcosamine, N-acetylethylgalatatosamine, sialic acid and sialic acids contained in the living body.
- sugar chain can be used interchangeably with “polysaccharide”, “carbohydrate”, and “carbohydrate”.
- the “sugar chain” in the present specification includes both sugar chains and sugar chain-containing substances.
- One amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, a protein equivalent in enzyme activity).
- the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient.
- the hydrophilicity index is also considered in the production of variants. As described in US Pat. No.
- hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+ 3.0 ⁇ 1); glutamic acid (+ 3.0 ⁇ 1) ); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline (0.5 ⁇ 1); Alanine (0.5); histidine (0.5); cystine (1.0); methionine (1.3); valine (-1.5); leucine (-1.8); isoleucine (1 1.8); tyrosine (-2.3); ferulanine (12.5); and tryptophan (13.4).
- an amino acid can be replaced with another that has a similar hydrophilicity index and can still provide a biological equivalent.
- the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
- conservative substitution means that the amino acid substitution is similar to the hydrophilicity index or Z and hydrophobicity index of the amino acid to be replaced with the original amino acid as described above. This refers to substitution. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and palin, leucine, and isoleucine However, it is not limited to these.
- variant refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide has been changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Alleles are genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant that has an allelic relationship with a gene.
- a “species homologue or homolog” is a homology (preferably 60% or more homology, more preferably 80% or more 85% or more, 90% or more, 95% or more homology).
- ortholog also called orthologous gene
- a hemoglobin gene family with multiple gene structures For example, the human and mouse ⁇ -hemoglobin genes are orthologs.
- the human ⁇ -hemoglobin gene and the / 3 hemoglobin gene are paralogs (genes generated by gene duplication). Since orthologs are useful for the estimation of molecular phylogenetic trees, the orthologs of the present invention may also be useful in the present invention.
- Constant (modified) variants applies to both amino acid and nucleic acid sequences.
- Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essential if the nucleic acid does not encode an amino acid sequence. Refers to the same sequence.
- the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
- This variation in nuclear acid is a “silent modification (mutation)” which is one species of conservatively modified mutations.
- Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid.
- each codon in a nucleic acid except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan, produces a functionally identical molecule. It is understood that it can be modified.
- each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
- such modifications can be made to avoid substitution of cysteine, an amino acid that significantly affects the conformation of the polypeptide.
- amino acid additions, deletions, or modifications are also made to produce a BAC vector containing a gene encoding a functionally equivalent polypeptide. It can be carried out.
- Amino acid substitution refers to substitution of one or more of the original peptide, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids.
- the addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain.
- An amino acid deletion is one or more from the original peptide, for example 1-10, preferably 1-5, more preferably Deletion of 1 to 3 amino acids.
- Amino acid modifications include but are not limited to forces including amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like.
- the amino acid to be substituted or added may be a natural amino acid or an unnatural amino acid, or an amino acid analog. Natural amino acids are preferred.
- a nucleic acid form of a polypeptide refers to a nucleic acid molecule capable of expressing the protein form of the polypeptide. As long as the expressed polypeptide has substantially the same activity as the native polypeptide, a part of the nucleic acid sequence is deleted or substituted with other bases as described above. Alternatively, a part of other nucleic acid sequence may be inserted. Alternatively, other nucleic acids may be bound to the 5 ′ end and the Z or 3 ′ end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions to a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.
- Such a nucleic acid can be obtained by a well-known PCR method or chemically synthesized.
- a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.
- substitution, addition or deletion of a polypeptide or polynucleotide refers to an amino acid or its substitute, or nucleotide, respectively, relative to the original polypeptide or polynucleotide. Or its substitute power is replaced, added or removed.
- substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques.
- the number of substitutions, additions or deletions should be one or more, and any number will be sufficient as long as the desired function is maintained in the variant having the substitution, addition or deletion. be able to. For example, such a number can be 1 or several and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length .
- Macromolecular structures are described in terms of various levels of organization. obtain. For a general discussion of this configuration, see, for example, Alberts et al., Molecular Biology of the Cell (3rd edition, 1994), and Cantor and Schimmel, Biophysi cal Chemistry Part I: The Conformation of Biological Macromolecules (1980). See General molecular biology techniques that can be used in the present invention include Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A A person skilled in the art can easily carry out the operation with reference to Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, etc.
- vector refers to a vector that can transfer a target polynucleotide sequence into a target cell.
- examples of such vectors include prokaryotic cells, yeast, animal cells, plant cells, insect cells, forces capable of autonomous replication in host cells such as individual animals and individual plants, or integration into chromosomes. And those containing a promoter at a position suitable for transcription of the polynucleotide of the present invention are exemplified.
- BAC vector is a plasmid created based on the F plasmid of Escherichia coli, and it can stably hold and grow a large DNA fragment of about 3 OOkb or more stably in bacteria such as Escherichia coli. It is a vector that can be made to occur.
- the BAC vector contains at least a region essential for the replication of the BAC vector.
- the region essential for replication includes, for example, oriS, which is the replication origin of F plasmid, or a variant thereof.
- the “BAC vector sequence” refers to a sequence containing a sequence essential for the function as a BAC vector.
- the BAC vector sequence may further comprise a “recombinant protein dependent recombination sequence” and a Z or “selectable marker”.
- homologous recombination includes both “recombinant protein dependent recombination” and “recombinant protein independent recombination”. “Recombinant protein-dependent recombination” means in the presence of recombinant protein. Refers to homologous recombination that occurs in the absence of a recombinant protein.
- Recombinant protein-independent recombination refers to homologous recombination that occurs regardless of the presence or absence of a recombinant protein.
- recombinant protein-dependent recombination sequence refers to a sequence that causes recombination protein-dependent recombination
- recombinant protein-independent recombination sequence refers to a recombination protein-independent recombination sequence. This refers to a sequence that causes recombination.
- Recombinant protein-dependent recombination sequences cause recombination in the presence of the recombinant protein, but not in the absence of the recombinant protein.
- the recombinant protein preferably acts specifically on recombinant protein dependent recombinant sequences and does not act on sequences other than recombinant protein dependent recombinant sequences.
- Representative recombinant protein-dependent recombination sequences and recombinant protein pairs include, but are not limited to, the following: 1 ⁇ (1 ⁇ cus of crossover oi PiJ from Batateliophage P1 ⁇ [Combination of J and Cre (cyclization recombination) protein, Flp protein and FRT site, ⁇ 031 and attB, attP thread combination (Thorpe, Helena M .; Wilson, Stuart E.
- selection marker refers to a gene that functions as an index for selecting a host cell containing a BAC vector.
- Selectable markers include, but are not limited to, fluorescent markers, luminescent markers, and drug selectable markers.
- Fluorescent markers include, but are not limited to, genes encoding fluorescent proteins such as green fluorescent protein (GFP).
- Luminescent marker includes, but is not limited to, a gene encoding a photoprotein such as luciferase.
- a tamper that selects the following group forces as well Include, but are not limited to, genes encoding proteins: dihydrofolate reductase gene, glutamine synthetase gene, aspartate transaminase, metakine thionein (MT), adenosine deaminase (ADA), adenosine Minase (AMP D1, 2), xanthine-guanine monophosphoribosyltransferase, UMP synthase, P glycoprotein, asparagine synthetase, and or-tin decarboxylase.
- genes encoding proteins include, but are not limited to, genes encoding proteins: dihydrofolate reductase gene, glutamine synthetase gene, aspartate transaminase, metakine thionein (MT), adenosine deaminase (ADA), adenosine Minase (AMP D1, 2), xant
- DHFR dihydrofolate reductase gene
- MTX glutamine synthetase
- Msx glutamine synthetase
- AST aspartate transaminase
- PHA N-phosphonacetyl- L-aspartate
- ADA adenosine deaminase
- AMPD1 adenosine deaminase
- azaserine coformacin
- an “expression vector” refers to a nucleic acid sequence in which various regulatory elements are linked in a manner that allows them to operate in a host cell, in addition to a structural gene and a promoter that regulates its expression.
- the regulatory element may preferably include a terminator, a selectable marker such as a drug resistance gene (eg, kanamycin resistance gene, hygromycin resistance gene, etc.) and an enhancer.
- a drug resistance gene eg, kanamycin resistance gene, hygromycin resistance gene, etc.
- the type of expression vector of an organism eg, a plant
- the type of regulatory elements used can vary depending on the host cell.
- the plant expression vector used in the present invention may further have a T-DNA region. The T-DNA region is particularly Increase the efficiency of gene transfer when transforming the plant with kuterium.
- recombinant vector refers to a vector capable of transferring a target polynucleotide sequence into a target cell.
- Such vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or can be integrated into chromosomes. Examples include those containing a promoter at a position suitable for polynucleotide transcription.
- Terminal 1 is a sequence that is located downstream of a protein-coding region of a gene, and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is known that the terminator influences the expression level of a gene by being involved in mRNA stability. Examples of the terminator include, but are not limited to, a CaMV35S terminator, a nopaline synthase gene terminator (Tnos), and a tobacco PRla gene terminator.
- promoter refers to a region upstream of the ORF of DNA that determines the initiation site of gene transcription and directly regulates its frequency, and to which RNA polymerase is bound.
- the putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.
- expression of the promoter of the present invention to be "constitutive” means that all the yarns and weaves of an organism are in the stage of development / viability of the organism. However, it is a property that is expressed in an almost constant amount. Specifically, when Northern plot analysis is performed under the same conditions as in the examples of the present specification, for example, it is the same at any time point (for example, 2 points or more (for example, 5th day and 15th day)). Alternatively, when almost the same level of expression is observed in any of the corresponding sites, the expression is constitutive by the definition of the present invention. Constitutive promoter Is considered to play a role in maintaining the homeostasis of organisms in a normal growth environment. These properties can be determined by extracting any partial RNA of the organism and analyzing the expression level by Northern plot analysis or quantifying the expressed protein by Western plot.
- the enzyme sequence is preferably an enzyme sequence region containing an upstream sequence in the SV40 promoter.
- a plurality of sensors can be used, but one may or may not be used.
- operably linked refers to transcriptional translational regulatory sequences (eg, promoters, enhancers, etc.) or translational regulators that have the expression (operation) of the desired sequence. It is placed under the control of the array. In order for a promoter to be operably linked to a gene, the force with which the promoter is usually placed immediately upstream of the gene does not necessarily have to be placed adjacent.
- transformation As used in the present invention, the terms “transformation”, “transduction” and “transfusion” are used interchangeably unless otherwise stated, and mean the introduction of a nucleic acid into a host cell. Any transformation method can be used as long as it is a method for introducing DNA into a host cell. For example, various well-known methods such as an electoral position method, a method using a particle gun (gene gun), and a calcium phosphate method can be used. Technology.
- Transformant refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, a transformed tissue, a transformed host, etc., depending on the subject, and encompasses all of these forms in this specification, but is specific in a particular context. Can refer to form.
- Prokaryotic cells include prokaryotic cells belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacteria, Syudomonas, etc., for example, Escherichia coll XL 1-Blue, Escherichia coll XL2—Blue, Esch erichia coli DH1, Escherichia coli MC1000, Escherichia coli KY327 6, Eschericnia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.
- animal cells include umbilical cord blood mononuclear cells, peripheral blood mononuclear cells, and Sup-Tl cells.
- animal is used in the broadest sense in the art and includes vertebrates and invertebrates. Examples of animals include, but are not limited to, mammals, birds, reptiles, amphibians, fishes, worms and worms.
- tissue of an organism refers to a population of cells having a certain similar action in the population.
- a tissue can be a part of an organ.
- organ organ
- cells there are many cells having the same function, but there are also cases where slightly different functions are mixed, so in this specification, as long as the tissue shares certain characteristics, You can have a mix of cells.
- an "organ” refers to a structure having one independent form and combining one or more tissues to perform a specific function.
- the plant include, but are not limited to, callus, root, stem, stem, leaf, flower, seed, germ, embryo, and fruit.
- animals include stomach, liver, intestine, spleen, lung, trachea, nose, heart, artery, vein, lymph node (lymphatic system), thymus, ovary, eye, ear, tongue, and skin. It is not limited.
- transgenic means that a specific gene is incorporated into an organism or an organism (for example, a plant or an animal (mouse) that incorporates such a gene. Etc.)).
- the transgenic organism is a microinjection method (microinjection method), viral vector method, ES cell method (embryonic stem cell method), sperm vector method, chromosomal fragment It can be produced using a transgeneic animal production technique utilizing the introduction method (transzomic method), the episome method, or the like. Techniques for producing such transgenic animals are well known in the art.
- screening refers to a large number of candidates for a substance having a specific property of interest, or a host cell or virus, etc., in a specific operation and Z or evaluation method. It means selecting power. In the present invention, it is understood that viruses obtained by screening having a desired activity are also included within the scope of the present invention.
- chip or “microchip” is used interchangeably and refers to a micro integrated circuit that has various functions and is a part of a system.
- the chip include, but are not limited to, a DNA chip, a protein chip, and a cell chip.
- the term "array” refers to a pattern or pattern in which a composition (eg, DNA, protein, cell) containing one or more (eg, 1000 or more) target substances is arranged and arranged.
- a substrate for example, a chip
- An array that is patterned on a small substrate eg, 10 ⁇ 10 mm
- the microarray and the array are used interchangeably. Therefore, even a pattern that is larger than the above-mentioned substrate is sometimes called a microarray.
- an array is composed of a desired set of cells that are themselves immobilized on a solid surface or membrane.
- the array preferably comprises at least 10 2 cells containing the same or different viruses, more preferably at least 10 3 , and even more preferably at least 10 4 , and even more preferably at least 10 5 cells. These cells are preferably placed on a surface of 125 ⁇ 80 mm, more preferably 10 ⁇ 10 mm. Formats of microtiter plates such as a 96-well microtiter plate and a 384-well microtiter plate are contemplated, with sizes that are about the size of a slide glass. Contains target substance to be fixed One or more types of compositions may be used. The number of such types can be any number from 1 to the number of spots. For example, a composition containing about 10, about 100, about 500, and about 1000 target substances can be immobilized.
- any number of target substances can be disposed on a solid surface or membrane such as a substrate, but usually 10 8 per substrate.
- target substances for example, biomolecules such as cells
- up to 10 biomolecules in other embodiments up to 10 7 biomolecules, up to 10 6 biomolecules, up to 10 5 biomolecules, up to 10 4 biomolecules, up to 10 3 biomolecules
- up to 10 2 biomolecules can be arranged, but a composition containing a target substance exceeding 10 8 biomolecules may be arranged. In these cases, the size of the substrate is preferably smaller.
- the spot size of a composition (eg, a cell) containing the target substance can be as small as the size of a single biomolecule (this can be on the order of l-2 nm).
- the minimum substrate area is determined in some cases by the number of biomolecules on the substrate.
- spots of biomolecules may be placed on the array.
- spot refers to a certain set of compositions containing a target substance.
- spotting refers to producing a spot of a composition containing a certain target substance on a certain substrate or plate. Spotting can be done by any method, for example, it can be accomplished by pipetting or the like, or it can be done by automated equipment, such methods are well known in the art.
- the term "address” refers to a unique location on a substrate and may be distinguishable from other unique locations.
- the address is suitable for associating with a spot with that address, and the entity at every address can take an arbitrary shape so that the entity forces at other addresses can also be identified (eg, optical). obtain.
- the shape defining the address can be, for example, a force that can be circular, elliptical, square, rectangular, or an irregular shape. Therefore, “address” indicates an abstract concept, and “spot” can be used to indicate a specific concept, but when it is not necessary to distinguish between the two, And “spot” can be used interchangeably.
- the size defining each address is, among other things, the size of the board, the size on a particular board. It depends on the number of dresses, the amount of the composition containing the target substance and the Z or available reagents, the size of the microparticles and the degree of resolution required for any method in which the array is used.
- the size can be, for example, a force that can range from 1 to 2 nm force several centimeters, and can be any size consistent with the application of the array.
- the spatial layout and shape that defines the address is designed to suit the particular application in which the microarray is used. Addresses can be densely arranged, can be widely distributed, or can be subgrouped into a desired pattern appropriate for a particular type of analyte.
- support refers to a substance capable of carrying cells, bacteria, viruses, polynucleotides or polypeptides.
- the support material can be either covalently bonded or non-covalently bonded, and can be derivatized to have such a property that has the property of binding to cells etc. used in the present invention. Solid materials.
- the material for use as a support can be any material that can form a solid surface.
- Natural and synthetic polymers eg, polystyrene, cellulose, chitosan, dextran, and nylon
- the support includes a moiety that performs hydrophobic bonding.
- the support may be formed of a plurality of layers of different materials.
- inorganic materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
- Organic materials such as silicone resin, polyphenylene oxide, and polysulfone can be used.
- a membrane used for blotting such as a nitrocellulose membrane or a PVDF membrane, can also be used as the support.
- the herpesvirus of the present invention can also be used as a component of a pharmaceutical composition for treatment, prevention, and Z or treatment of infectious diseases.
- the "effective amount" of a drug refers to an amount that allows the drug to exert its intended drug effect.
- the minimum concentration may be referred to as the minimum effective amount.
- Such minimum effective amounts are well known in the art, and usually the minimum effective amount of a drug can be determined by those skilled in the art or determined by those skilled in the art. In order to determine such an effective amount, in addition to actual administration, an animal model or the like can be used. The present invention is also useful in determining such effective amounts.
- pharmaceutically acceptable carrier refers to a substance that is used in the production of agricultural chemicals such as pharmaceuticals or veterinary drugs, and that does not adversely affect active ingredients.
- Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifying agents, suspending agents, Solvents, fillers, bulking agents, buffers, delivery vehicles, excipients, and Z or agricultural or pharmaceutical adjuvants.
- the type and amount of the drug used in the treatment method of the present invention is determined based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, seriousness). It can be easily determined by those skilled in the art in view of the patient's age, weight, sex, medical history, the shape or type of the site of the subject to be administered, and the like.
- the frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, and treatment course. This can be easily determined by those skilled in the art.
- the frequency of monitoring the disease state includes, for example, monitoring once every few months every day (for example, once a week, once a month). It is preferable to perform monitoring once a week, once a month, while monitoring the progress.
- the “instruction” describes the treatment method of the present invention and the like for a person who performs administration such as a doctor or a patient.
- This instruction is a word that instructs to administer the medicine of the present invention, for example, immediately after radiation therapy or immediately before (for example, within 24 hours). Is described.
- This instruction is prepared according to the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.). It will be clearly stated that it has been approved. Instructions are so-called package inserts, usually provided on paper, but not limited to them, for example, electronic media (e.g., homepages provided via the Internet, e-mail). Such a form may also be provided.
- more than one drug may be used in the treatment of the present invention.
- drugs of similar nature or origin may be used, or drugs of different nature or origin may be used.
- Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.
- Micromicroarray Fabrication a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, MJ (1997). Fundamentals of Microfabrication, CRC1 5 Press; Rai—Choud hury, P. (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography, etc., which are incorporated herein by reference for relevant portions.
- the present invention provides a recombinant herpesvirus.
- the herpes virus contains a BAC vector sequence in its genomic sequence.
- the BAC sequence used preferably contains the F plasmid-derived replication origin, but it may be a sequence other than the F plasmid-derived replication origin. Any origin of replication can be used as long as it can be maintained and propagated in the cell.
- the BAC vectors of the invention can be maintained and Z or amplified in bacterial host cells, preferably E. coli cells.
- part of this BAC vector is inserted into a non-essential region of the herpesvirus genome, allowing manipulation as a BAC containing the herpesvirus genome.
- the BAC containing this herpesvirus genome can produce and propagate recombinant herpesviruses when introduced into mammalian cells.
- a recombinant herpesvirus host cell any mammalian cell capable of growing a wild herpesvirus strain can be used.
- the host cell is of human origin and is not limited to, for example, umbilical cord blood mononuclear cells, peripheral blood mononuclear cells, and SupTl cells.
- a BAC vector containing the human herpesvirus genome using a human herpesvirus eg, HHV-6A, HHV-6B, or HHV-7 genome and a BAC vector
- homologous recombination is required. It is possible to use various well-known methods such as the method used.
- Examples of the method using homologous recombination include a method using a nucleic acid having a linear BAC vector sequence in which sequences homologous to the human herpesvirus genome are linked.
- a method for producing a BAC vector containing a human herpesvirus genome using a nucleic acid having a linear BAC vector sequence in which sequences homologous to the human herpesvirus genome are linked is typically (1).
- the nucleic acid is introduced into an appropriate host together with the human herpesvirus genome, and (2) the host cell is cultured and homologous sequence linked to the linear BAC vector sequence.
- the method includes culturing the host cell and extracting circular viral DNA.
- the host cells used in the above method include umbilical cord blood mononuclear cells, peripheral blood mononuclear cells, and SupTl cells, but are not limited to these. Please tell us!
- the method for introducing the BAC vector into the mammalian host is not limited, but includes, but not limited to, the calcium phosphate method, the electopore position method, and the lipofuxion method.
- Amaxa's! / Is the Elect Mouth Positioning method of Bio-Rad which is a method that can efficiently introduce a large amount of genes into T cells.
- the conditions of Amaxa's electoral position are, for example, that cells are washed twice with cell washing buffer (RPMI-1640 medium or Opri-MEM medium) and then washed with electoral position buffer (RPMI-1640). (10 mM glucose, 0.
- a restriction enzyme fragment of a nucleic acid without using homologous recombination is used.
- Various known methods can also be used.
- the non-essential region for introducing the BAC vector sequence is selected from the group consisting of the following regions:
- this nonessential region is the ORF region of gene U24, gene U24a, gene 25, and gene 26, or a region adjacent to these ORFs. This is because gene U24, gene U24a, gene 25, and gene 26 are non-essential genes that are continuous on the HHV-7 genome, and therefore are easy to design nucleic acids for homologous recombination.
- the BAC vector sequence used in the present invention preferably comprises a recombinant protein dependent recombinant sequence and a Z or selectable marker.
- the selectable marker sequence is a drug selectable marker and a gene encoding Z or green fluorescent protein. This is because the presence of a desired gene can be easily confirmed.
- a vector used for producing the virus and a method for producing the virus are also provided.
- pharmaceutical compositions comprising the above viruses and pharmaceutical compositions in the form of vaccines.
- the recombinant human herpesvirus of the present specification can introduce a desired antigen protein. Therefore, for example, when introducing an antigen known to act as a vaccine, the vector of the present invention can be used as a vaccine vector.
- a method for introducing a mutation into a vector for producing the vaccine of the present invention comprises the following steps: introducing a vector into a bacterial host cell; introducing a plasmid vector comprising a fragment of the human herpesvirus genome into the bacterial host cell, wherein Wherein the fragment has at least one mutation; culturing the bacterial host cell; isolating a vector having a BAC vector sequence from the cultured bacterial host cell.
- homologous recombination occurs between a vector for producing the vaccine of the present invention and a plasmid vector containing a fragment consisting of a part of the human herpesvirus genome in a bacterial host cell.
- the vector for producing the vaccine of the present invention has a mutation on a partial fragment of the human herpesvirus genome.
- a plasmid vector containing a fragment consisting of a part of the human herpesvirus genome is transformed into a bacterium. It can be introduced into a host cell.
- a method for introducing mutation into this fragment a method of introducing mutation using PCR is well known, for example, it has no proofreading function, and one of four nucleotides of thermostable polymerase is low! / It is possible to introduce mutations at random by using in. It is also possible to introduce a desired mutation at a desired position by performing PCR using a primer having a mutated base sequence.
- the vector for producing the vaccine of the invention has a mutation on a fragment consisting of a part of the human herpesvirus genome.
- Various well-known methods such as the alkaline method and commercially available kits can also be used to prepare BAC vector sequences for bacterial host cell forces.
- a further method for introducing a mutation into a vector for producing the vaccine of the present invention comprises the following steps: introducing the vector into a bacterial host cell; introducing a first plasmid vector comprising a first fragment comprising a portion of the human herpesvirus genome into the bacterial host cell. Wherein the first fragment has at least one mutation; a second plasmid vector comprising a second fragment that is a part of the human herpesvirus genome is provided in the bacterial host cell. Wherein the second fragment has at least one mutation, and the second fragment is different from the first fragment; culturing the bacterial host cell A step; isolating a vector having a BAC vector sequence from the cultured bacterial host cell.
- a nucleic acid cassette that can be used to produce the vaccine of the present invention.
- This nucleic acid cassette is preferably a first fragment capable of homologous recombination with the human herpesvirus genome in the host cell, a BAC vector sequence, and a homologous recombination with the human herpesvirus genome in the host cell.
- a nucleic acid cassette comprising a possible second fragment, wherein each end of the BAC sequence is linked to a first fragment and a second fragment, respectively.
- the first fragment and the second fragment are preferably at least lkb, at least 1.5 kb, at least 2 kb.
- This first fragment and second fragment are preferably at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical to the sequence of the human herpesvirus genome.
- the first and second fragments are derived from different regions of the human herpesvirus genome.
- the first and second fragments may each independently be derived from a region adjacent to the ORF of gene U24 or a region adjacent to the ORF of gene U24a.
- the BAC vector sequence includes a recombinant protein dependent recombination sequence and a Z or selectable marker.
- This selection marker may be a drug selection marker or a gene encoding a fluorescent protein such as a green fluorescent protein.
- this BAC vector sequence has the nucleic acid sequence set forth in SEQ ID NO: 401.
- the first and second fragments are each independently a group force consisting of the following region forces of the HHV-7 genome: Group power that also has regional power At least 80%, 85%, 90%, and 95% identical to the selected region: the region within the ORF of gene HI, the region within the ORF of gene DR1, and the region within the ORF of gene DR2 A region in the ORF of gene H2, a region in the ORF of gene DR6, a region in the ORF of gene DR7, a region in the ORF of gene H3, a region in the ORF of gene H4, a region in the ORF of gene U2, Region within ORF of gene U3, Region within ORF of gene U4, Region within ORF of gene U5Z7, Region within ORF of gene U8, Region within ORF of gene U10, Region within ORF of gene U12, Gene U13 ORF region of gene, region of ORF of gene U15, region of ORF of gene U16, region of OR
- the first and second fragments are derived from different regions of the human herpesvirus genome.
- the first and second fragments may each independently be derived from a region adjacent to the ORF of gene U24 or a region adjacent to the ORF of gene U24a.
- the BAC vector sequence includes a recombinant protein dependent recombination sequence and a Z or selectable marker.
- This selection marker may be a drug selection marker or a gene encoding a fluorescent protein such as a green fluorescent protein.
- this BAC vector sequence has the nucleic acid sequence set forth in SEQ ID NO: 401.
- Such mutagenesis can be performed using, for example, the following method for HHV-7:
- the HHV-7-U21-27- BAC plasmid (plasmid containing the HHV-7 genome and BAC vector sequence), and (b) the nucleic acid encoding the mutant foreign gene
- the desired foreign A nucleic acid such as a shuttle vector or PCR product having a partial sequence of the herpesvirus genome linked to both ends of the gene and the foreign gene is introduced.
- HHV-7-U21-27-BAC plasmid into which a foreign gene has been introduced can be easily selected and amplified in E. coli.
- Recombinant herpesvirus can be obtained by producing virus from HHV-7-U21-27- BAC with foreign genes (Markus Wagner ⁇ TRENDS in Microbilogy, Vol. 10, No. 10). 7, July 2002). Specific examples are listed below:
- the shuttle vector and the HHV-7-U21-27- BAC plasmid are recombined via the first homologous region, and the shuttle vector and the HHV-7-U21-27- BAC plasmid are ligated. Resulting in a co-insert.
- the shuttle plasmid is then removed because the replication origin of the shuttle vector is temperature sensitive.
- the co-inserted part is removed. If the second recombination event occurs via the first homologous region, a plasmid is generated that has the same sequence as the HHV-7-U21-27-BAC used for recombination.
- the modified HHV-7-having a foreign gene contained on the shuttle vector The U21-27- BAC plasmid is obtained. If the first homologous region and the second homologous region are approximately the same length, the second recombination event is the second homologous region The probability of occurring in is approximately the same as the probability of the second recombination event occurring in the first region of homology. Therefore, about one-half of the resulting HHV-7-U21-27- BAC plasmid has the same sequence as that used for recombination, and about one-half of the foreign plasmid introduced into the shuttle vector. A plasmid having a gene.
- the recET recombination function derived from prophage Rac is used, or red o derived from Batteriophage ⁇ ;
- 8 recombination function is used, and linear DNA fragments are used.
- Linear DNA has a region homologous to the HHV-7-U21-27-BAC plasmid at both ends. By generating homologous recombination through the homologous region, the desired sequence in the linear DNA fragment can be introduced into HHV-7-U21-27-BAC. When using recET, or red a j8 recombination functions, these recombination functions result in homologous recombination with homologous sequences as long as 25-50 nucleotides, which is It can be used easily.
- the transposon element is used to insert randomly into nucleic acids in E. coli.
- a transposon element containing a desired foreign gene and HHV-7-U21-27-BAC are introduced into E. coli, and the transposon element is randomly inserted into HHV-7-U21-27-BAC.
- the method for preparing recombinant HHV-7 containing a foreign gene containing the above foreign gene is HH-7.
- HIV infects CD4 + T cells. It is known to prevent and treat HIV infection when CD4 + T cells are treated with HHV-7 (Lusso et al., Proc. Natl. Acad. Sci. US A vol. 91, pages 3872-3876, April 1994). This therapeutic effect is brought about by infecting CD4 + T cells with HHV-7 using CD4 on T cells as a receptor. Therefore, by the method of the present invention, it is possible to produce recombinant HHV-7 that retains the ability to infect CD4 + T cells, and to use the HHV-7 for the prevention and epilepsy or treatment of HIV.
- the HIV infection preventing effect of HHV-7 of the present invention can be measured by measuring whether or not V-7 can prevent HIV proliferation ability in target cells.
- target cells used in this measurement include, but are not limited to, cells such as peripheral blood mononuclear cells, CD4 + cells, and Sup-Tl cells. In more detail, for example, it can be measured by the following procedure.
- Target cells are pre-infected with HHV-7 at a MOI (multiplicity of infection) of 0.1, cultured at 37 ° C for 24 to 72 hours, then added with HIV, about 30 minutes to 2 hours Incubate. Then wash the cells thoroughly and reincubate. After 4 days of culture, the HIV-derived antigen released into the culture medium is quantified.
- MOI multipleplicity of infection
- cells infected with HHV-7 are infected with HIV under the same conditions, and the antigen released into the culture medium is measured.
- HIV-derived antigens include, but are not limited to, p24. Since the amount of this antigen serves as an index of HIV growth, if the amount of this antigen is small compared to the control, the HIV infection prevention effect of HHV-7 is demonstrated.
- the effect of HHV-7 treatment of HIV infection of the present invention can be determined by measuring whether HHV-7 suppresses the ability to proliferate HIV in target cells.
- target cells used in this measurement include, but are not limited to, cells such as peripheral blood mononuclear cells, CD4 + T cells, and Sup-T1 cells. More specifically, for example, measurement can be performed by the following procedure.
- Target cells are simultaneously infected with HIV and HHV-7 (MOI between 0.1 and 0.01). After this co-infection, the infected cells are incubated for about 30 minutes to 2 hours, after which the cells are thoroughly washed and incubated again.
- HHV-7 Use cells infected with HIV alone. After 4 days of culture, measure HIV-derived antigen in the culture medium. HIV-derived antigens include, but are not limited to, p24. Since this antigen quantity is an indicator of HIV growth, if the amount of this antigen is small compared to control, the effect of suppressing HIV growth, that is, the effect of treating HIV infection with HHV-7 is demonstrated. .
- an HIV gene silencer gene is introduced into recombinant HHV-7, and the recombinant HHV-7 is administered to HIV patients.
- Infection of HIV-infected CD4 + T cells with recombinant HH V-7 suppresses HIV production, resulting in a therapeutic effect on HIV infection.
- the invention also provides a method for the treatment and Z or prevention of a disease or disorder (eg, infection) by inoculation with an effective amount of a therapeutic agent 'administration of a prophylactic agent to a subject'.
- Therapeutic agent'prevention agent means a composition of the present invention in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier).
- the target “effective amount” in this specification is determined based on such consideration.
- the total pharmaceutically effective amount of a therapeutic agent 'preventive agent administered parenterally per dose is in the range of about 1 g / kg / day to LOmgZkgZ days of patient weight. However, as noted above, this is left to therapeutic discretion. More preferably, for the cell bioactive substance of the present invention, this dose is at least 0. OlmgZkgZ days, most preferably for humans. Between about 0.01 mg / kg / day and about lmgZkgZ day. When administered continuously, typically the therapeutic agent 'prophylactic agent is injected 1 to 4 times a day at a dosage rate of from about 1 ⁇ g ZkgZ time to about 50; To be administered). Intravenous bag solutions may also be used. The duration of treatment required to observe the change and the post-treatment interval at which a response occurs will vary depending on the desired effect.
- Therapeutic agents Preventive agents, oral, rectal, parenteral, intmcistemally, vaginal, intraperitoneal, topical (such as by powder, ointment, gel, infusion, or transdermal patch) ), Administered by mouth or as an oral or nasal spray.
- “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant.
- parenteral refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.
- the therapeutic / prophylactic agent of the present invention is also appropriately administered by a sustained-release system.
- Sustained release therapeutics • Suitable examples of preventives are oral, rectal, parenteral, intmcistemally, vaginal, intraperitoneal, topical (powder, ointment, gel, infusion, or transdermal Can be administered by mouth, orally or as a nasal spray.
- “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant.
- parenteral refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.
- the therapeutic agent'prevention agent is a pharmaceutically acceptable carrier, ie the dosage and concentration used, in the desired degree of purity.
- a pharmaceutically acceptable carrier ie the dosage and concentration used, in the desired degree of purity.
- the formulation preferably contains no other compounds known to be harmful to oxidation and therapeutic / preventive agents.
- the formulation is prepared by contacting the therapeutic / prophylactic agent uniformly and intimately with a liquid carrier or a finely divided solid carrier or both. Then generate if necessary Shape the product into the desired formulation.
- the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as non-volatile oils and ethyl oleate are also useful herein, as are ribosomes.
- the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to recipients at the dosages and concentrations used, such as phosphates, succinates, succinates, acetic acid and other organic acids or their salts.
- a buffer such as ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide (eg, polyarginine or tripeptide); a protein such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as: amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrin; chelating agents such as EDTA Sugar alcohols such as mannitol or sorbitol; Counterions such as sodium; and Z or polysorbate, nonionic surfactants such as poloxamers or PEG is exemplified et be.
- an antioxidant such as ascorbic acid
- a low molecular weight (less than about 10 residues) polypeptide eg, polyarginine or tripeptide
- a protein such as serum albumin, gelatin or immunoglobulin
- Hydrophilic polymers such as
- Any drug to be used for therapeutic administration may be in a state free of organisms other than viruses as an active ingredient, ie, in a sterile state. Aseptic conditions are easily achieved by filtration through sterile filtration membranes (eg, 0.2 micron membranes).
- the therapeutic agent / prophylactic agent is placed in a container having a sterile access port, eg, an intravenous solution bag or vial with a stopper that can be punctured with a hypodermic needle.
- Therapeutic / preventive agents are usually stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or lyophilized formulations for reconstitution.
- a lyophilized formulation a 10 ml vial is filled with sterile filtered 1% (WZV) therapeutic agent • 5 ml of an aqueous prophylactic agent solution and the resulting mixture is lyophilized. Reconstitute the freeze-dried therapeutic / preventive agent with bacteriostatic water for injection to prepare an infusion solution.
- WZV sterile filtered 1%
- the present invention also provides one or more of the therapeutic agent / prophylactic agent of the present invention satisfying one or more components.
- a pharmaceutical pack or kit comprising the container is provided.
- a notice in the form of a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product may be attached to such a container, which notice relates to the manufacture, use or sale for human administration. Represents approval by a government agency.
- therapeutic / prophylactic agents may be used in combination with other therapeutic compounds.
- the therapeutic agent / prophylactic agent of the present invention may be administered alone or in combination with other therapeutic agents / prophylactic agents.
- Preventive agents include chemotherapeutic agents, antibiotics, steroids and non-steroidal anti-inflammatory agents, conventional immune therapeutic agents' preventive agents, etc. Site power in and Z or growth factors, but are not limited to these.
- the combination can be administered, for example, simultaneously as a mixture; simultaneously or concurrently but separately; or over time. This presents the indication that the combined drugs are administered together as a therapeutic mixture, and also the procedure in which the combined drugs are administered separately but simultaneously, for example through separate intravenous lines to the same individual.
- Including. Administration in combination further includes separate administration of one of the compounds or agents given first, followed by the second.
- the therapeutic agent 'preventive agent of the invention is administered in combination with an antiretroviral agent, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, and Z or a protease inhibitor. Is done.
- the therapeutic agent 'prophylactic agent of the invention is administered in combination with an antibiotic.
- Antibiotics that can be used include aminoglycoside antibiotics, polyene antibiotics, penicillin antibiotics, cephem antibiotics, peptide antibiotics, macrolide antibiotics, and tetracycline antibiotics. It is not limited.
- the therapeutic agent 'preventive agent of the invention is administered alone or in combination with an anti-inflammatory agent.
- Anti-inflammatory agents that can be administered together with the therapeutic agent / preventive agent of the present invention include darcocorticoids and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, allylacetic acid derivatives, allylbutyric acid derivatives, allylcarboxylic acid, allylpropion.
- Acid derivatives including, but not limited to, orgoteine, oxaseprol, para-diline, perizoxal, pifoxime, proxazone, proxazole, and tenidap.
- the therapeutic agents' prophylactic agents of the invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation therapy).
- Plasmid PHA-2 was used as distributed by Markus Wagner and Ulrich H. Koszinowski (Adler et al. (2000), J. Virol 74: 6964-74).
- the center of an approximately 4000 bp region covering gene U21, gene U23, gene U24, gene U24a ⁇ gene U25, and gene U26 was selected as the BAC vector insertion site. This is because it was expected that insertion of foreign nucleic acid into such a non-essential region would not adversely affect herpesvirus growth! /.
- Figure 1 shows a schematic diagram of the insertion of the BAC vector into the HHV-7 genome.
- BAC7—E3 GCTTAATTAACATGCTCTGCAA TGCAAGCC
- BAC7—E4 ATGCGGCCGCAAATAG CCTTTGCTCATAGC
- the prepared plasmid pHA-2 / HHV7-U21-27 contains a guanine phosphoribosyltransferase (gpt) gene as a selection marker. And two ⁇ arrays Therefore, the BAC vector sequence sandwiched between ⁇ sequences can be efficiently removed by the action of Cre recombinase. In addition, it is possible to easily confirm cells into which a plasmid containing a BAC vector sequence has been introduced by the fluorescence of green fluorescent protein (GFP).
- GFP green fluorescent protein
- This plasmid was linearized by Notl digestion. 2 ⁇ g of linearized ⁇ — 2 / ⁇ 1 ⁇ 71; 21—27? ⁇ 160 £ 6 1011 unit (Amaxa) was used to transfect the cord blood mononuclear cells cultured in 25cm 2 plastic flasks with electo-poration. One day after transfection, the transfected cells were infected with the herpesvirus KHR strain.
- Recombinant virus was enriched by drug selection with mycophenolic acid and xanthine by the gpt gene.
- SupTl cells were infected with the recombinant virus, and after 6 hours, the cells were collected and circular DNA was extracted.
- Circular DNA recovered from SupTl cells was introduced into E. coli. Escherichia coli was seeded on a plate containing the drug, and DNA was extracted from the mouth that appeared on the plate. Circular virus DNA was extracted from the infected cells by the Heart method (Hirt, (1967) JM Biol 26: 365-9).
- the extracted DNA was introduced into Escherichia coli DH10B by gene mouth porcelain (Bio-Rad) by the electopore position method (0.2 cm cuvette, 2.5 kV) and transformed. This was selected on agar containing 17 g / ml chloramphene-coal to obtain E. coli containing HHV-7U21-27- BAC. It was.
- HHV-7U21-27-BAC was extracted from the cells using NucleoBond PC 100 kit (Mach rey-Nagel). The two obtained clones were each digested with the restriction enzyme EcoRI.
- HHV-7U21-27-BAC DNA (1 ⁇ g) was applied to the cord blood mononuclear cells (5 ⁇ 10 6 to 10 7 cells) cultured in a 25 cm 2 plastic flask using the electoral position method. Therefore, gene transfer was performed. Elect mouth position was performed under the condition of T8 using Amaxa nucleofactor kit according to the manual. After the electoral position, the transfected mononuclear cells were cultured in a medium containing PHA (phytohemagglutinin) for 3-7 days
- umbilical cord blood mononuclear cells were collected and co-cultured with umbilical cord blood mononuclear cells (5 X 10 6 to 10 7 cells) newly stimulated with PHA. At this time, the cells were cultured in a medium not containing PHA, and drugs (MPA, xanthine) were added to the medium. Virus production was observed in 2-3 days after co-cultivation.
- Recombinant adenovirus (AxCANCre) expressing Cre combinase was distributed by Dr. Nei Kawaguchi, Tokyo Medical and Dental University (Tanaka M et al., J. Virol. 2003 Jan; 77 (2): 1 382–1391). Recombinant adenovirus was used to infect cord blood mononuclear cells with MOI 100. After adsorbing the virus for 2 hours, the cells were washed with PBS (—) and cultured in RPMI medium containing 5% FCS. Recombinant human herpesvirus was superinfected with cord blood mononuclear cells 24 hours after infection with recombinant adenovirus.
- Cre recombinase was expressed by recombinant adenovirus, and that 1111 ⁇ -71; 21-27-: 6 8 genomes of 8 AC vector sequences were efficiently excised. From the DNA sequence results of the obtained human herpesvirus, it was confirmed that a BAC vector sequence was excised from HHV-7U21-27- BAC.
- Example 2 [0169] (with characteristics of recombinant herpesvirus)
- Cord blood mononuclear cells were infected with the recombinant virus and cultured for up to 7 days. Thereafter, virus-infected cord blood mononuclear cells were mixed and cultured with SupTl cells. (Mixed by centrifugation at 3000 rpm, 37 ° C, 40 minutes.) When the infected cells were observed under a fluorescence microscope, cells with GFP expression and cytopathic effect could be confirmed. This result indicates that the recombinant herpesvirus of the present invention can be transmitted between cells and retains the proliferation ability (data not shown).
- the method for comparing the growth of recombinant viruses with wild-type viruses is as follows: (1) The expression of viral late proteins in non-infected cells is examined by the fluorescent antibody method. (2) Mixing infected and non-infected cells, examining the expression of late proteins in non-infected cells, and expressing their expression A method of detecting viral growth by examining the infection of non-infected cells with the virus as an index; (3) By examining the expression of GFP in uninfected cells by mixing infected and non-infected cells. A method for detecting viral growth by examining viral infection of uninfected cells using as an index; and (4) Cellular changes caused by viral infection. As an index effects, but include a method of detecting the virus growth, but are not limited to. In the following, the ability to explain the TCID50 method The test method for the recombinant virus of the present invention is not limited to the TCID50 method.
- a herpes virus having a herpes virus genome into which an HIV silencer gene (for example, an antisense nucleic acid for an HIV gene) has been introduced can be easily prepared by the following steps. To do.
- An NF ⁇ BZSpl site of the HIV LTR U3 site is operably linked upstream of the HIV silencer gene, and a region adjacent to the non-essential gene of HHV-7 is linked to both ends of the shuttle vector.
- the shuttle vector and HHV-7U21-27- BAC plasmid (plasmid containing HHV-7 genome and BAC vector sequence) are introduced into E. coli.
- homologous recombination occurs between the HHV-7-U21-27- BAC plasmid and the shuttle vector in E. coli, and the foreign gene (HIV silencer gene) force contained in the shuttle vector is HHV- 7U21-27— This produces a co-insert inserted into the BAC plasmid.
- the shuttle plasmid is removed because the replication origin of the shuttle vector is temperature sensitive.
- the co-inserted portion is removed. If the second recombination event occurs via the first homologous region, a plasmid having the same sequence as the HHV-7U21-27-BAC used for recombination is generated. On the other hand, if the second recombination event occurs via a second homologous region different from the first homologous region, the modification 1111 ⁇ -71 having a foreign gene contained on the shuttle vector; 21 ⁇ 27 ⁇ 8 8 plasmids are obtained.
- the probability that the second recombination event will occur in the second homologous region is that the second recombination event is the first It is almost the same as the probability that occurs in the homologous region. Therefore, about one-half of the resulting HHV-7U21-27-BAC plasmid has the same sequence as that used for recombination, and about one-half of the foreign gene introduced into the shuttle vector. Is a plasmid
- Recombinant HHV-7 virus is prepared using a plasmid containing this HIV silencer. This recombinant virus can be used to treat HIV infection.
- a mutant recombinant herpesvirus incorporating a gene encoding a desired vaccine antigen can be prepared and used as a vaccine.
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CA002565509A CA2565509A1 (en) | 2004-05-06 | 2005-04-28 | Recombinant virus vector for gene introduction in lymphocyte |
AU2005240902A AU2005240902B2 (en) | 2004-05-06 | 2005-04-28 | Recombinant virus vector for gene introduction in lymphocyte |
KR1020067023222A KR101234062B1 (ko) | 2004-05-06 | 2005-04-28 | 림프구 내 유전자 도입용 재조합 바이러스 벡터 |
JP2006512989A JP4796958B2 (ja) | 2004-05-06 | 2005-04-28 | リンパ球細胞へ遺伝子導入するための組換えウイルスベクター |
CN2005800230619A CN1981040B (zh) | 2004-05-06 | 2005-04-28 | 用于向淋巴细胞中导入基因的重组病毒载体 |
EP05736956A EP1743942A4 (en) | 2004-05-06 | 2005-04-28 | RECOMBINANT VIRUS VECTOR FOR GENE INTRODUCTION INTO A LYMPHOCYTE |
KR1020127018909A KR101294238B1 (ko) | 2004-05-06 | 2005-04-28 | 림프구 내 유전자 도입용 재조합 바이러스 벡터 |
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EP (1) | EP1743942A4 (ja) |
JP (2) | JP4796958B2 (ja) |
KR (2) | KR101294238B1 (ja) |
CN (1) | CN1981040B (ja) |
AU (1) | AU2005240902B2 (ja) |
CA (1) | CA2565509A1 (ja) |
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Cited By (2)
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WO2012060025A1 (ja) * | 2010-11-05 | 2012-05-10 | 独立行政法人医薬基盤研究所 | ヒトヘルペスウイルス6 glycoprotein Q1に対する中和抗体の作製とその解析 |
CN106282321A (zh) * | 2015-05-26 | 2017-01-04 | 中山大学 | 由组织snoRNA组成的肝癌复发风险预测标志物及试剂盒 |
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US8642045B2 (en) * | 2003-08-29 | 2014-02-04 | Virus Ikagaku Kenkyusho Inc. | Recombinant virus vector originating in HHV-6 or HHV-7, method of producing the same, method of transforming host cell using the same, host cell transformed thereby and gene therapy method using the same |
US20110189233A1 (en) * | 2004-03-05 | 2011-08-04 | Kazuhiro Nagaike | Recombinant Varicella-Zoster Virus |
GB2426518A (en) * | 2005-05-25 | 2006-11-29 | London School Hygiene & Tropical Medicine | Cyokine from human herpes virus 6 |
EP2471938A3 (en) * | 2005-11-24 | 2013-04-24 | The Research Foundation for Microbial Diseases of Osaka University | Recombinant polyvalent vaccine |
US20110142918A1 (en) * | 2008-06-12 | 2011-06-16 | Phoebus S.R.L. | Anti-angiogenic compositions and therapeutic applications thereof |
US20120220028A1 (en) * | 2009-09-04 | 2012-08-30 | The Research Foundation For Microbial Diseases Of Osaka University | Enhancer for promoter, and use thereof |
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US20120122700A1 (en) * | 2010-11-11 | 2012-05-17 | University Of South Florida | Materials and methods for determining subtelomere dna sequence |
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- 2005-04-28 CN CN2005800230619A patent/CN1981040B/zh not_active Expired - Fee Related
- 2005-04-28 KR KR1020067023222A patent/KR101234062B1/ko not_active IP Right Cessation
- 2005-04-28 CA CA002565509A patent/CA2565509A1/en not_active Abandoned
- 2005-04-28 WO PCT/JP2005/008250 patent/WO2005108581A1/ja active Application Filing
- 2005-04-28 AU AU2005240902A patent/AU2005240902B2/en not_active Ceased
- 2005-04-28 JP JP2006512989A patent/JP4796958B2/ja not_active Expired - Fee Related
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WO2012060025A1 (ja) * | 2010-11-05 | 2012-05-10 | 独立行政法人医薬基盤研究所 | ヒトヘルペスウイルス6 glycoprotein Q1に対する中和抗体の作製とその解析 |
US20140093526A1 (en) * | 2010-11-05 | 2014-04-03 | The Research Foundation For Microbial Diseases Of Osaka University | Preparation of Neutralizing Antibody to Human Herpesvirus 6 Glycoprotein Q1 and Analysis Thereof |
CN106282321A (zh) * | 2015-05-26 | 2017-01-04 | 中山大学 | 由组织snoRNA组成的肝癌复发风险预测标志物及试剂盒 |
Also Published As
Publication number | Publication date |
---|---|
CN1981040B (zh) | 2011-06-22 |
US20090253208A1 (en) | 2009-10-08 |
KR101294238B1 (ko) | 2013-08-07 |
KR20120088004A (ko) | 2012-08-07 |
US7820436B2 (en) | 2010-10-26 |
JPWO2005108581A1 (ja) | 2008-03-21 |
CA2565509A1 (en) | 2005-11-17 |
AU2005240902B2 (en) | 2011-09-15 |
EP1743942A1 (en) | 2007-01-17 |
US8148060B2 (en) | 2012-04-03 |
JP5410477B2 (ja) | 2014-02-05 |
US20110070651A1 (en) | 2011-03-24 |
JP4796958B2 (ja) | 2011-10-19 |
KR101234062B1 (ko) | 2013-02-18 |
JP2011234726A (ja) | 2011-11-24 |
EP1743942A4 (en) | 2010-08-04 |
US20080226677A1 (en) | 2008-09-18 |
AU2005240902A1 (en) | 2005-11-17 |
US20120129263A1 (en) | 2012-05-24 |
KR20060123666A (ko) | 2006-12-01 |
CN1981040A (zh) | 2007-06-13 |
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