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WO2008044787A1 - Composition pour le traitement du cancer à l'aide de dref comme cible moléculaire - Google Patents

Composition pour le traitement du cancer à l'aide de dref comme cible moléculaire Download PDF

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Publication number
WO2008044787A1
WO2008044787A1 PCT/JP2007/070031 JP2007070031W WO2008044787A1 WO 2008044787 A1 WO2008044787 A1 WO 2008044787A1 JP 2007070031 W JP2007070031 W JP 2007070031W WO 2008044787 A1 WO2008044787 A1 WO 2008044787A1
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Prior art keywords
dref
seq
cancer
gene
cell
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Japanese (ja)
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Hirotaka Osada
Takashi Takahashi
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Nagoya University NUC
Aichi Prefecture
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Nagoya University NUC
Aichi Prefecture
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Priority to JP2008538780A priority Critical patent/JP5176035B2/ja
Publication of WO2008044787A1 publication Critical patent/WO2008044787A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention relates to a composition for cancer treatment that targets the human cell growth regulator DREF or DREF gene as a molecular target. 'Ming
  • DREF is a Drosophila transcription factor isolated by Hirose et al., which recognizes and binds to a DNA sequence called DRE-mot if in one sequence of a gene promoter and induces the expression of that gene (non-patent literature). 1).
  • Drosophila it is reported that PCNA (prol iferating cell nuclear antigen) transcription is induced in the S phase, and that several gene forces related to cell proliferation are reported to be regulated by DREF. Yes.
  • a human homologue of Drosophila DREF was cloned and sequenced by Ohshima et al., And it was reported that human DREF was involved in the induction of histone HI gene (Non-patent Document 2). At this time, the expression of the human DREF gene is reduced by the two siRNAs generated against the DREF CR1 domain, thereby suppressing the histone HI gene expression (Non-patent Document 2).
  • the human DREF gene is a gene that was found by Esposito et al. (Non-patent Document 3) from a cDNA library derived from a human non-inducible male teratocarcinoma cell line Tramp (Ac-like transposable element; putative Ac-like transposable element; Chromosome: 'X, Y; Location: Xp22.33; Ypll).
  • DREF is one of the factors involved in cell cycle progression by knocking down the human DREF gene, and that expression of liposomal protein (RP) is reduced by knockdown. It was pointed out that it plays a role of stimulating cell proliferation through gene regulation (Non-patent Documents 4 and 5). - So far, human DREF has been suggested to be associated with cell proliferation ability S, but no detailed functional analysis has been performed in relation to cancer therapy.
  • Non-Patent Document 4 Sano, Y. et al., Abstracts of the Molecular Biology Society of Japan (issued on November 25, 2005), lecture number 1P-0873, 2005
  • Refractory cancers such as lung cancer and esophageal cancer are resistant to general chemotherapy, and effective means that can be expected to cure other than surgery have not been established. For this purpose, it is necessary to develop molecular targeted therapeutics targeting genes involved in cancer onset, propagation and metastasis, and to establish treatments aimed at cure of cancer.
  • An object of the present invention is to provide a therapeutic composition for cancer.
  • Another object of the present invention is to provide a method of screening for a therapeutic agent for cancer.
  • the present inventors have found that human DREF is a target for cancer treatment, and have completed the present invention. .
  • DREF was discovered as a transcription factor of Drosophila, and it was later shown that its homolog exists in humans.
  • the present inventors have now found that human DREF is not substantially expressed in normal cells, but is significantly expressed in cancer cells, and based on this, DREF is predicted to be a molecular target for cancer treatment. It was demonstrated that suppression of DREF expression actually leads to cancer suppression and cell death. Therefore, the present invention has the following features.
  • the present invention provides an RNAi nucleus against DREF, which suppresses the in vivo function of human cell growth control factor DREF or DREF gene in cancer cells or cancer tissues.
  • a cancer therapeutic composition comprising an acid, an antisense nucleic acid, or an antibody.
  • DREF is an abbreviation for DRE (DNA replication-related element) -binding factor named by Hirose et al., Also known as ZBEDl (zinc finger, BED-type containing 1).
  • ZBEDl zinc finger, BED-type containing 1
  • DREF human DREF
  • DREF is a protein comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence comprising a deletion, substitution or addition of one or several amino acids in the amino acid sequence.
  • DREF gene the open reading frame (0RF) nucleotide sequence encoded by an amino acid sequence of SEQ ID NO: 1, nucleotide sequence and more specifically shown in position 202 to 2 28 6 of SEQ ID NO: 2 Including. Both amino acid and nucleotide sequences of DREF are registered with GenBank as Accession number NMJ304729.
  • DREF in the present invention includes an amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1.
  • Variants having in vivo functions equivalent to DREF for example, polymorphic variants, splice variants, degenerate variants, mutants, etc.
  • the DREF gene in the present invention is SEQ ID NO: nucleotide sequence shown in 2 (position 202 to 228 6th) 95% or more, preferably 97% or more, more preferably 98% or more, including the most favorable 'Mashiku nucleotide sequence having 99% or more identity And variants similar to those described above having an in vivo function equivalent to DREF after translation.
  • the DREF gene is used with the intention of encompassing not only exons, but also introns, 3 'untranslated regions and 5' untranslated regions. '
  • the suppression of in vivo function is an effect of suppressing cell proliferation, cell cycle arrest and / or cell death in cancer cells.
  • in vivo function refers to the action of DREF in cancer cells, such as cell proliferation, cell cycle progression, and the like.
  • RNAi nucleic acid or antisense nucleic acid is capable of cleaving DREF raRNA or suppressing its function.
  • RNAi is so-called RNA interference, and is a gene having a sequence homologous to DNA or double-stranded RNA (dsRNA) added from the outside. (For example, Chisato Ushida, Protein Nucleic Acid Enzyme Vol. 46 (No. 10), pp. 1381-1386, 2001).
  • the target RNA is mRNA or pre-mRNA encoded by the gene, and the target mRNA is degraded or suppressed in expression by RNAi.
  • Other RNAi nucleic acids that can be used in the present invention also include miRNA (microRNA). miRNA binds to DREF mRNA 3, the untranslated region and suppresses DREF gene expression. The size of these RNAi nucleic acids is 19-30 nucleotides long, preferably 19-25 nucleotides long, more preferably 19-23 nucleotides long.
  • the “antisense nucleic acid” used in the present invention includes an RNA sequence complementary to the whole or a part of DREF mRNA, DNA encoding the RNA sequence, or a vector containing the DNA sequence. .
  • vector refers to a regulatory sequence such as a promoter, poly T sequence, replication origin, terminator, etc., in addition to the DNA encoding the target siRNA or shRNA or DNA encoding the antisense RNA. , Selection markers, etc. can be included.
  • the siRNA or shRNA, or antisense RNA is produced in the cell by the expression of the DNA.
  • the RNAi nucleic acid or antisense nucleic acid is derived from a nucleotide sequence encoding the CR2 domain or CR3 domain of DREF (SEQ ID NOs: 3 and 4, respectively).
  • a preferable nucleotide sequence in the present invention is, for example, in the sequence of SEQ ID NO: 2 of the DREF gene, from position 1,300 to 1,500 (from position 1099 when “A” of the start codon ATG of 0RF is the first position) I 2 "position), 1,900 to 2,000 position (positions 1699 to 1799 when 0RF start codon ATG" A "is first position).
  • CR refers to a highly conserved region in the sequence comparison between human DREF and Drosophila DREF. Such areas (Amino acid sequence; nucleotide sequence) is CR1 (positions 4 to 140 of SEQ ID NO: 1; positions 21 to 621 of SEQ ID NO: 2), CR2 (positions 377 to 506 of SEQ ID NO: 1; 1330 to 1719 of SEQ ID NO: 2) ) And CR3 (positions 541 to 688 of SEQ ID NO: 1; positions 1822 to 2265 of SEQ ID NO: 2), and the identities are 27.7%, 29.2%, and 21.1%, respectively (Ohshima, N. et al., J. Biol. Chem. 278: 22928-38, 2003).
  • RNAi nucleic acids in the present invention include any one of the DNA sequences of SEQ ID NOs: 5 to 15, 19, 22, 25 and 28 or an RNA sequence corresponding to the DNA sequence.
  • DNA represents deoxyribonucleic acid
  • RNA represents ribonucleic acid.
  • a more preferable vector in the present invention includes the DNA sequence of any one of SEQ ID NOs: 20, 21, 23, 24, 26, 27, 29 and 30.
  • the composition of the present invention can comprise an antibody against DREF that inhibits the in vivo function of DREF.
  • Any antibody can be used in the present invention as long as such a function can be suppressed.
  • Preferred antibodies are DREF CR1 domain, CR2 domain or CR3 domain (SEQ ID NO: 138, 133 or 134, respectively), or a functional domain related to DNA binding of DREF in the CR1 or CR3 domain (eg, SEQ ID NO: 140 or 141), It is an antibody that binds to the epitope.
  • the term epitope refers to a site to which an antibody on an antigen molecule binds, and usually the number of amino acids in such a site is 8 or more, preferably 10 or more. It may consist of a target sequence or a discontinuous sequence. Therefore, the antibody of the present invention can immunologically bind to an epitope composed of 8 or more, preferably 10 or more amino acids in the amino acid sequence of DREF.
  • 'Preferred antibodies against DREF that can be used in the present invention are human antibodies, humanized antibodies or fragments thereof.
  • the present invention further includes adding a candidate drug to a medium containing human cultured cancer cells, and screening a drug that suppresses the expression or translation of the DREF gene in vitro.
  • a method for screening is provided.
  • Cancer cells that can be used in the present invention are cancer cells from cancer cell lines, biopsy. including.
  • the latter cancer cells are derived from a patient and can be used to search for an effective drug for the patient.
  • the type of cancer cell is not particularly limited, but cancer cells in which DREF is expressed in cancer cells but hardly or not in normal cells are desirable.
  • Candidate drugs that can be used in the present invention may be any of small molecules to macromolecules, and include low molecular organic compounds, peptides, proteins, oligosaccharides, polysaccharides, lipids, and the like.
  • screening can be performed using as an index the suppression of cancer cell growth, cell cycle arrest and / or cell death induction. Such an effect is not substantially observed in normal tissues or cells because DREF is hardly or not expressed in normal tissues or cells.
  • Figure 1 shows the domain structure of the human DREF / ZBED1 gene (Xp22.33 & Ypll), and the RNAi site and sequence.
  • Figure 2 shows the results of RT-PCR analysis of the RNAi effect.
  • Figure 3 shows the results of Western blot analysis of the RNAi effect.
  • Figure 4 shows the cell cycle analysis results.
  • Figure 5A shows the cell cycle analysis results.
  • Figure 5B shows the cell cycle analysis results.
  • Figure 5C shows the cell cycle analysis results.
  • FIG. 6 A shows the results of cell proliferation ability analysis.
  • B shows the results of cell proliferation ability analysis.
  • FIG. 7 shows the results of immunostaining for 7 H2AX (Phospho-H2AX) and Phospho-ATM (Serl981).
  • Figure 8 shows the TUNEL analysis results.
  • FIG. 9 shows the results of cell death and cell cycle analysis.
  • FIG. 10 shows cell death (mitotic catastroph) in M phase.
  • Figure 11 shows the gene expression analysis results of siDREF-, # 4 in ACC-LC-91. 1 2, O showing expression of DREF in lung cancer patient specimens
  • Figure 13 shows the DNA sequence of the RPL17 gene (A; SEQ ID NO: 137), which is thought to be associated with cancer growth, among the genes that are suppressed by DREF-RNAifH, and the transcriptional activation of the RPL17 gene by DREF.
  • Mechanism (B) is shown.
  • the present invention includes an RNAi nucleic acid, an antisense nucleic acid or an antibody against DREF, which suppresses the in vivo function of a human cell growth regulator DREF or DREF gene in cancer cells or cancer tissues.
  • a composition is provided.
  • DREF is expressed in cancer cells or tissues, but is not substantially (or little or not) expressed in normal cells or tissues.
  • the present inventors suppressed cancer cell growth, cell cycle arrest, cell death induction by suppressing (including blocking or inhibiting) DREF gene expression or protein translation in cancer cells or tissues. It was found that such an event occurred. ⁇
  • Cancers to be treated by the present invention are not limited to cancers capable of expressing DREF, such as lung cancer, esophageal cancer, knee cancer, stomach cancer, liver cancer, colon cancer, thyroid cancer, prostate cancer, Includes bladder cancer, kidney cancer, skin cancer, gall cancer, brain cancer, breast cancer, ovarian cancer, cervical cancer, testicular cancer, lymphoma, melanoma, sarcoma, osteosarcoma, etc.
  • Preferred cancers are lung cancer, esophageal cancer, breast cancer, knee cancer and the like. ',
  • the amino acid sequence of DREF which is the target of the present invention, and the nucleotide sequence of the DREF gene are known, for example, as SEQ ID NO: 1 and SEQ ID NO: 2 (open reading frame (0RF): positions 202 to 228 ⁇ ), respectively. Both DREF amino acid and nucleotide sequences are registered in GenBank as Accession number ⁇ _004729.
  • the present invention also includes suppressing the in vivo function of a DREF or a mutant of the DREF gene, for example, a mutant based on mutation, polymorphism, alternative splicing, degeneracy and the like. ⁇
  • DREF in the present invention is a protein comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence containing one or several amino acid deletions, substitutions or additions in the amino acid sequence.
  • DREF in the present invention is In vivo function equivalent to DREF, comprising an amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1. The variant which has is also included.
  • the DREF gene in the present invention corresponds to the nucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 1, more specifically the nucleotide sequence shown in SEQ ID NO: 2, particularly DREF 0RF at positions 202 to 2286. Contains nucleotide sequence.
  • the DREF gene in the present invention is a DNA further comprising a nucleotide sequence containing a deletion, substitution or addition of one or several nucleotides in the nucleotide sequence shown at positions 202 to 2286 of SEQ ID NO: 2.
  • the DREF gene in the present invention has 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence shown in positions 202 to 2286 of SEQ ID NO: 2. And a variant having an in vivo function equivalent to DREF after translation. '
  • severe means an integer of about 10 or less, such as 9, 8, 7, 6, 5, 4, 3, 2.
  • DREF has in vivo functions such as cell proliferation and cell cycle progression, but the present invention makes it possible to suppress the in vivo functions of DREF in cancer and lead to cancer degeneration and cell death.
  • examples of the agent that suppresses the expression or protein translation of the target DREF gene include RNAi nucleic acid, antisense nucleic acid and antibody against DREF. Each of these drugs will be described more specifically below.
  • RNAi nucleic acid usable in the present invention is a continuous 19 to 30 nucleotide sequence derived from the nucleotide sequence of the DREF gene (consisting of exons, introns, 3 ′ untranslated region and 5 ′ untranslated region) or the nucleotide sequence of DREF mRNA. It is a DNA or RNA containing nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.
  • RNAi methods that can be used in the present invention include 1) direct introduction of short double-stranded RNA (siRNA) into cells, or 2) incorporation of short-hairpin RNA (shRNA) into various expression vectors, Introduce the vector into the cell, or 3) Insert a short double-stranded DNA corresponding to siRNA between the promoters into a vector with two promoters aligned in the opposite direction to express s iRNA. This includes methods such as preparation and introduction into cells.
  • siRNA short double-stranded RNA
  • shRNA short-hairpin RNA
  • the RNAi nucleic acid includes s iRNA, shRNA or miRNA that enables cleavage of DREF mRNA or suppression of its function.
  • the RNAi nucleic acid may also be a DNA encoding these RNAs or a vector containing the DNA, which, when used in vivo, makes it possible to produce siRNA, shRNA or miRNA.
  • RNAi nucleic acid is a continuous 19 to 30 nucleotides, preferably 19 to 25 nucleotides derived from nucleotide sequences (SEQ ID NOs: 3 and 4, respectively) encoding the CR2 domain or CR3 domain of DREF.
  • Further preferred CR2 domain or CR3 domain target sites are, for example, positions 1,300 to 1,500 in the sequence of SEQ ID NO: 2 of the DREF gene (0RF open-start codon ATG “A” is the first Position 1099 to 1299), 1,900 to 2,000 (0RF start codon ATG
  • RNAi nucleic acid that can be used in the present invention is, for example, the DNA sequence of any of the following SEQ ID NOs: 5 to 15, 19, 22, 25, and 28, or the RNA sequence corresponding to the DM sequence (that is, the DNA sequence) Array with T in U replaced by U).
  • the number after the sequence is the position of the start site on 0RF of DREF (however, the start codon of 0RF
  • CAACCACCACCTCATGCTGGAG 1139 (SEQ ID NO: 7)
  • Particularly preferred sequences are SEQ ID NO: 19 (RNAi # 3 (positions 1136 to 1156)), SEQ ID NO: 22 (RNAi # 4 (positions 1718 to 1738) :), SEQ ID NO: 25 (RNAi # 5 (positions 1245 to 1265)) ), And SEQ ID NO: 28 (RNAi # 6 (positions 1737 to 1757)), or an RNA sequence corresponding to the DNA sequence (that is, a sequence in which T in the DNA sequence is replaced with U).
  • the number range shown in parentheses together with the SEQ ID No. is when the base number “A” (position 2 ) of the start codon ATG of DREF 0RF is numbered as the first in the nucleotide sequence of SEQ ID No. 2. Represents the position of. ⁇
  • RNAi nucleic acid for DREF is the DNA sequence of SEQ ID NO: 33-132 below, or the RNA sequence corresponding to that DNA sequence (ie, a sequence in which T in the DNA sequence is replaced with U).
  • CAAGAGCAAGGTGTGGAAGTATT 63 (SEQ ID NO: 33)
  • CAGATCGCCTACTCCGGAA 157 (SEQ ID NO: 40)
  • AGATCGCCTACTCCGGAAACA 158 (SEQ ID NO: 41)
  • GTCCTACCACCTGGAGAAGAAC 188 (SEQ ID NO: 2)
  • CTGCGAGTTCGTCAAGAGCAAC 224 (SEQ ID NO: 44) GCGAGTTCGTCAAGAGCAA 227 (SEQ ID NO: 45) CGTGCTGGGCCTCATCTGCGAG 386 (SEQ ID NO: 46) CCCAGCCTCCATCGTGGACGAG 416 (SEQ ID NO: 47) CAGCCTCCATCGTGGACGA 419 (SEQ ID NO: 48) CACCTTCAAGGTGCTGCTGAAG 440 (SEQ ID NO: 49) CCTTCAAGGTGCTGGA 491 (SEQ ID NO: 52) CTCTACCAAGGCCATCCCTGAG 503 (SEQ ID NO: 53) CGTCCGGGAGGTGATCCTGAAG 536 (SEQ ID NO: 54) TCCGGGAGGTGATCCTGAA 538 (SEQ ID NO: 55) CCGGGAGGTGATCCTGAAGGAG 539 (SEQ ID NO: 56) GGGAGGTGATCCTGAAGGA GT GG (GT ACC GG (SEQ ID NO:
  • GCTCTATGAGGTCTTCATCGAG 749 (SEQ ID NO: 68) TTCGGGGCCACCACCAACTATGG 793 (SEQ ID NO: 69) CACCAACTATGGCAAGGACATC 803 (SEQ ID NO: 70) CCAACTATGGCAAGGACAT 806 (SEQ ID NO: 71) AGGACATCGTGAAGGCGTGCT 818 (SEQ ID NO: 72) CTGCCGCAAACTGGTGGAGTAC 941 (SEQ ID NO: 73) GCCGCAAACTGGTGGAGTACT 944 (SEQ ID NO: 74)
  • CAAGGAGCTTTCCAAGACCTA 1356 (SEQ ID NO: 94)
  • ACGCCCGAGATCGACATGTTTCT 1384 (SEQ ID NO: 96)
  • CAGAAGTACTGGTGCGTGA 1816 (SEQ ID NO: 117
  • GGACGAGCAGGTGTTTCTGTAT 1922 (SEQ ID NO: 119
  • the RNAi nucleic acid is a continuous 19-30 nucleotides, preferably 19-25, from the sequence of the mRNA or variant thereof encoded by the DRSF gene comprising the nucleotide sequence of SEQ ID NO: 2.
  • An siRNA comprising a nucleotide, more preferably a 19-23 nucleotide sense strand sequence and its complementary sequence, the antisense strand sequence.
  • the siRNA can suppress the expression of the DREF gene or mRNA in vivo or in vitro.
  • a preferred example of the corresponding DNA sequence comprising the siRNA is a contiguous 19-30 nucleotide, preferably 19-25 nucleotides derived from the nucleotide sequence encoding the CR2 domain or CR3 domain of DREF (SEQ ID NOs: 3 and 4, respectively).
  • the DNA sequence comprising 19 to 23 nucleotides more preferably, the nucleotide sequence of SEQ ID NO: 2, positions 1,300 to 1,500 (0RF starting codon ATG “A” is the first position 1999 to 1299), 1,900 to 2,000 (from 1699 to 1799 if the 0RF start codon ATG “A” is the first), 19-30 DNA sequences comprising nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, more preferred examples are shown by SEQ ID NOs: 5-15, 19, .22, 25, 28, 33-132 DNA sequence.
  • the sequence of the DREF RNAi nucleic acid is not limited to the above sequence, and for example, in the nucleotide sequence of SEQ ID NO: 2 as long as it suppresses the in vivo function of the DREF gene, the corresponding mRNA, or a variant thereof.
  • the sequence may be different from the sequence of, for example, 1 to 3 bases. '
  • the siRNA of the present invention can be synthesized using a well-known chemical synthesis technique based on the DREF sequence that is the target of siRNA as exemplified above.
  • it can be synthesized chemically using an automated DNA (/ RNA) synthesizer using DNA synthesis technology such as solid phase phosphoamidite method, or a siRNA-related contract synthesis company (for example, Funakoshi Corporation) It is also possible to synthesize by outsourcing to companies such as Dharmacon and Ambion.
  • the siRNA of the present invention is a duplex that is a precursor thereof. It can also be derived from RNA (shRNA) via processing by the intracellular RNase Dicer.
  • shRNA is a double-stranded RNA having a loop between the sense strand sequence and the antisense strand sequence of siRNA, preferably from 1 to 6, preferably 2 to 4 poly U at its 3 ′ end. Including overhangs.
  • shRNA is processed into siRNA by a dicer belonging to the RNase III family, and then siRNA is made into a single strand, and its antisense strand RNA is complexed with a molecule having RNA cleavage activity, etc.
  • RISC RNA- Induced Silencing Complex
  • both the above siRNA of the present invention and its precursor shRNA can be used as an active ingredient of the composition of the present invention.
  • the selected RNAi nucleic acid does not exhibit a so-called off-target action in clinical use.
  • Off-target action refers to the action of suppressing the expression of another gene that is partially homologous to the RNAi nucleic acid used in addition to the target m gene.
  • the candidate RNAi nucleic acid has the ability to confirm that there is no cross-reaction using a gene chip in advance, or there is no sequence identity using a database such as GenBank (NCBI) Or it is necessary to confirm that there is little.
  • GenBank NCBI
  • human DREF is not substantially expressed in normal cells in humans, but is significantly expressed in cancer cells, so it is expected that the problem of aftergetting can be avoided.
  • RNAi nucleic acid of the present invention When introducing the RNAi nucleic acid of the present invention into a patient's body, it is preferable to use a vector capable of directly injecting the nucleic acid into the affected area or capable of expressing the nucleic acid.
  • siRNA or vectors can be complexed with ribosomes such as lipofectamine, lipofectin, selfectin, other positively charged ribosomes (eg, positively charged cholesterol), or microcapsules, and this complex can be used.
  • ribosomes such as lipofectamine, lipofectin, selfectin, other positively charged ribosomes (eg, positively charged cholesterol), or microcapsules
  • the cell membrane of mammals is negatively charged, positively charged ribosomes are preferably used.
  • the positively charged ribosome-nucleic acid complex is taken up into the cell by engineered cytosis.
  • the nucleic acid can be further transferred to the cytoplasm or nucleus.
  • the therapeutic RNAi nucleic acid of the present invention can be encapsulated in nanoparticles having a particle size of about 500 nm or less.
  • nanoparticles include hollow nanoparticles formed from, for example, hepatitis B virus envelope L particles, and nucleic acids are encapsulated in the particles by electroporation. This nucleic acid-encapsulated particle can be delivered to the liver when administered into the blood (T. Yamada et al., Nature Biotech 21 (8): 885-890, 2003) and may be useful in the treatment of liver cancer.
  • the nucleic acid when encapsulating the therapeutic RNAi nucleic acid of the present invention in a liposome, the nucleic acid is treated with protamine sulfate to cause condensation to form a nucleic acid-protein complex, which is then encapsulated in a positively charged lipid or polymer micelle.
  • protamine sulfate when encapsulating the therapeutic RNAi nucleic acid of the present invention in a liposome, the nucleic acid is treated with protamine sulfate to cause condensation to form a nucleic acid-protein complex, which is then encapsulated in a positively charged lipid or polymer micelle.
  • Liposome-nucleic acid complexes can be obtained, for example, by the reverse phase evaporation method (F: Szoka et al., Biochim. Biophys. Acta, 601: 559, 1980), Bonoretex shaking method, Calcium fusion-EDTA chelate method (Yasufumi Kaneda, Experimental Medicine 22 ⁇ No. 14 (extra number), pages 14-152, 2004).
  • F reverse phase evaporation method
  • Bonoretex shaking method Bonoretex shaking method
  • Calcium fusion-EDTA chelate method Yasufumi Kaneda, Experimental Medicine 22 ⁇ No. 14 (extra number), pages 14-152, 2004.
  • the microinjection method is a method in which a nucleic acid is directly microinjected into a cell with a fine injection needle.
  • the viral vector method is a method of introducing a nucleic acid into a cell using a viral vector, in which a recombinant virus incorporating an expression unit that expresses siRNA, shRNA or miRNA is prepared, and the cell is infected. It is a method of expressing in cells. Examples of viruses include adenovirus, adeno-associated virus, retrovirus and the like.
  • a nucleic acid that can be used in the present invention includes an expression vector comprising a DNA sequence encoding an RNAi nucleic acid such as siRNA or shRNA under the control of a promoter.
  • an expression vector is a hairpin vector.
  • This vector includes DNA encoding a hairpin RNA in which the sense strand RNA sequence and the antisense strand RNA sequence are covalently bonded via a single-stranded loop sequence, wherein the DNA is intracellularly contained.
  • the hairpin RNA is formed by transcription and processed by a dicer.
  • a vector that forms the siRNA is formed by transcription and processed by a dicer.
  • Short hairpin RNA as a siRNA precursor transcribed from vector DNA should have an overhang consisting of 3 to 4 U at the end of its antisense strand.
  • sense strand RNA and antisense strand RNA can be more stable against nuclease degradation.
  • dicer There is one endogenous dicer in humans, which has the role of converting long dsRNA and precursor miRNA into siRNA and mature miRNA, respectively.
  • loop sequence according to the present invention examples include 5'-UUCAAGAGA-3 '(SEQ ID NO: 135), 5'-CUUCCUGUCA-3' (SEQ ID NO: 136), 5'-UUCCAG-3 '(SEQ ID NO: 137), etc. However, it is not limited to these.
  • RNAi # 3, RNAi # 4, RNAi # 5 and RNAi # 6, which are particularly preferred among the RNAi nucleic acids of the present invention, are shown in SEQ ID NOs: 20 and 21, respectively.
  • the underline indicates the sequence corresponding to the sense strand and antisense strand of siRNA.
  • RNAi # 3 oligo SEQ ID NOS: 20 and 21, respectively: '
  • RNAi # 4 oligo SEQ ID NOS: 23 and 24, respectively:
  • RNAi # 5 oligo SEQ ID NOS: 26 and 27, respectively:
  • si5F 5, -TACCCCACCATCAGCATGGTCTTCCTGTCAACCATGCTGATGGTGGGGTACTTTTTG-3 '
  • RNAi # 6 oligo SEQ ID NO: 29 and 30 respectively:
  • si6F 5-( si6R: 3,-The expression vector of the present invention can contain a promoter on the 5th side of the hairpin DNA.
  • promoters are pol III promoters, eg, U6 promoter or HI promoter from human or mouse, pol II promoter, or cytomegaloinoles promoter.
  • Another example of the expression vector of the present invention is a tandem vector.
  • This vector comprises a DNA sequence encoding the sense strand RNA sequence constituting the siRNA and a DNA sequence encoding the antisense strand RNA sequence, and a promoter at the 5 ′ end of each strand, DNA comprising a poly T sequence linked to the 3 'end of each strand, wherein the DNA hybridizes with the sense strand RNA and the antisense strand RNA after transcription in the cell to form the siRNA It is a vector.
  • a promoter in a tandem vector is a pol HI promoter, such as a human or mouse-derived U6 promoter or HI promoter, or a site megaloinoles promoter.
  • Poly T ⁇ ⁇ ⁇ IJ A poly T sequence consisting of ⁇ 6, preferably 1-5 T, such as 4 or 5 poly T sequences.
  • tandem vector of the present invention can be introduced into a cell, transcribed into RNA corresponding to the sense strand and the antisense strand, and hybridized with each other to produce the desired siRNA.
  • the hairpin type and tandem type vector is a plasmid vector or a winores vector.
  • the plasmid vector may be prepared using the methods described in the Examples below or the methods described in the literature, or commercially available vector systems such as the piGENE TM U6 vector and the piGENE TM Hl vector (Takarabai Bio Inc.). Company) ( ⁇ ⁇ R.
  • a plasmid vector is a drug resistance gene (eg, neomycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene) in addition to the DNA sequence and promoter encoding the siRNA of the present invention. It may contain transcription termination sequences, unique restriction sites or multiple cloning sites, and replication origins.
  • drug resistance gene eg, neomycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene
  • virus vector for example, an adenovirus vector, an adeno-associated virus vector, a lentiwinores betater, a retrowinoles vector (such as a leukemia virus vector), a herpes virus vector, or the like can be used.
  • the virus vector is preferably of a type lacking self-replicating ability, for example, so as not to cause disease when used in humans.
  • a self-replication ability-deficient adenovirus vector lacking the E1 gene and E3 gene eg, pAdeno- ⁇ from Invitrogen
  • Construction of viral base click terpolymer can utilize literature methods (U.S. Patent No. 525 2 4 ⁇ 9 No., and International Publication W094 / 13788). ',
  • the plasmid vector of the present invention forms a complex with a positively charged liposome such as lipophectamine, lipofectin, self-actin, or positively charged cholesterol, and is introduced into the patient's body in a force-pellated state.
  • a positively charged liposome such as lipophectamine, lipofectin, self-actin, or positively charged cholesterol
  • viral vectors can be introduced into cells by introducing them into affected areas and infecting cells (L. Zender et al., Proc. Natl. Acad. Sci. USA (2003), 100: 77797-7802; H. Xia et al., Nature Biotech.
  • adenovirus vectors or adeno-associated virus vectors can introduce genes into various cell types with very high efficiency. Since this vector is also not incorporated into the genome, its effect is transient and is considered to be safer than other viral vectors.
  • the sense strand sequence and the antisense strand sequence constituting the siRNA are covalently linked via a single strand loop sequence.
  • the hairpin RNA is encoded, and the hairpin RNA is formed by transcription in a cell and processed by a dicer to form the siRNA.
  • DNA encoding siRNA incorporated into the vector of the present invention comprises a DNA sequence encoding a sense strand sequence constituting siRNA and a DNA sequence encoding an antisense strand sequence, and A promoter is linked to the 5 'end of each strand, and a poly T sequence is linked to the 3' end of each strand, and the sense strand RNA and the antisense strand RNA hybridize after transcription in the cell.
  • the siRNA is formed.
  • Another nucleic acid as an active ingredient of the composition of the present invention is an antisense nucleic acid.
  • This antisense nucleic acid is an RNA sequence containing a sequence complementary to the mRNA sequence corresponding to the DREF gene containing the nucleotide sequence of SEQ ID NO: 2 (positions 202 to 2286), or a partial sequence thereof, or a sequence. Either the nucleotide sequence of number 2 (positions 2 to 2286 ), or a partial sequence thereof, and DNA containing a complementary sequence. ,
  • the partial sequence is a sequence consisting of about 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, or 250 or more and a full length of, for example, 50 to 150 nucleotides in the DREF gene or mRNA sequence. Can be included.
  • antisense nucleic acid nucleotides In addition to natural nucleotides, antisense nucleic acid nucleotides, antisense nucleic acid nucleotides, antisense nucleic acid nucleotides, antisense nucleic acid nucleotides, antisense nucleic acid nucleotides, antisense nucleic acid nucleotides, antisense nucleic acid nucleotides
  • modified nucleotides having groups such as (fluorine, chlorine, bromine or iodine), methyl, carboxymethyl or thio groups.
  • Antisense nucleic acids can be synthesized using well-known DNA / RNA synthesis techniques or DNA recombination techniques.
  • the vector DNA containing the sequence of SEQ ID NO: 2 is mirrored, and the target sequence is amplified by polymerase chain reaction (PCR) using primers that squeeze the sequence to be amplified. If necessary, it can be cloned into a vector to produce antisense DNA.
  • PCR polymerase chain reaction
  • the DNA having the amplified target sequence thus obtained is inserted into a vector, the vector is introduced into a eukaryotic or prokaryotic cell, and the transcription system is used for antisense.
  • RNA can be obtained.
  • the above-described virus vector or plasmid vector can be used.
  • the antisense nucleic acid of the present invention is DNA or RNA, it can suppress translation into protein by binding to DREF mRNA.
  • the antisense nucleic acid may be encapsulated in a liposome such as a positively charged ribosome as described above, or the antisense nucleic acid may be, for example, a strong pol II or It may be incorporated into a vector (the above-mentioned plasmid or viral vector) so as to be under the control of the pol III promoter. 13
  • the present invention further provides a composition for treating cancer comprising an antibody or a fragment thereof that suppresses the in vivo function of a DREF protein comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof.
  • the target protein of the present invention is a protein encoded by the DREF gene, and is a protein involved in human cancer cell proliferation and cell cycle progression. Therefore, inhibition or suppression of the function of the protein expressed in cancer cells can result in suppression of cancer growth and cell death.
  • a drug for this purpose is an antibody against the DREF protein or part thereof.
  • the antibody against DREF is an antibody against the CR1 domain, CR2 domain or CR3 domain of DREF (SEQ ID NO: 135, 133 or 134, respectively), or a portion thereof.
  • An example of a particularly preferred antibody is an antibody against a DRE (DNA replication-related element) binding region on DREF.
  • the DNA-binding domain of DREF is called “BED zinc finger” and has a protein structure with one zinc ion. This domain is part of CR1 and contains the 20th to 74th sequence (SEQ ID NO: 140) of the DREF amino acid sequence of SEQ ID NO: 1.
  • Antibodies to the domain containing this domain of DREF can inhibit DREF DNA binding and consequently inhibit DREF's in vivo function.
  • Zinc finger amino acid sequence (SEQ ID NO: 140):
  • Another domain associated with DNA binding is a part of CR3 of DREF, and includes the 571st to 651th sequence (SEQ ID NO: 141) of the DREF amino acid sequence of SEQ ID NO: 1.
  • This domain is an activator's family (hAT element superf amily) Called the dimerization domain. Antibodies to the region of DREF containing this domain inhibit DREF dimer formation, resulting in inhibition of DREF DNA binding and inhibition of DREF in vivo function.
  • amino acid sequence and nucleotide sequence of the following CR1, CR2, and CR3 domains are used to make (poly) peptides using known peptide synthesis techniques or DNA recombination techniques. Can be synthesized to produce the desired antibody.
  • EDSNNHHLMLEASEWATIEGLVELLQPFKQVAEIV EVIAKELSKTYQETPEIDMFLNVATFLDPRYKRLPFLSAFERQQVENRVVEEAKGLLD CR2 nucleotide sequence (SEQ ID NO: 3): GTGGTGGAAGAGGCCAAGGGCCTGCTGGAC
  • Such antibodies include polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab, F (ab ') 2 , scFv, Fv, two These include bispecific antibodies and synthetic antibodies.
  • the characteristics and structure of the antibody are described in Toru Otsuki et al., Experimental Medicine Vol. 22 No. 14 (Extra) 125-: 130, 2004, Yodosha (Tokyo, Japan). Can be used for.
  • Preferred antibodies suitable for use in the present invention are human or humanized antibodies, particularly human or humanized monoclonal antibodies, that cause little or no side effects due to anaphylaxis.
  • the antibody class and subclass may be of any type.
  • the type of antibody, IgG ⁇ IgM ⁇ IgE ⁇ IgD, IgA include IgG had I g G 2, IgG 3, IgG 4, IgA have IgA 2.
  • An antibody may also be derivatized by pegylation, acetylation, glycosylation, amidation, and the like.
  • Human antibodies can be generated by, for example, phage display library (pharge display library).
  • Human antibody-producing mice contain, for example, human antibody-producing genes in human artificial chromosomes After introducing the human chromosome fragment, using the microcell method, for example, an artificial chromosome is integrated into the genome of a mouse embryonic stem cell, transplanted into the uterus of a foster parent mouse, a chimeric mouse is born, a male or female chimeric mouse is bred, or a chimeric mouse And a wild-type mouse of the same species, for example, by producing a homozygous offspring mouse that contains a human antibody gene and is therefore capable of producing a human antibody (for example, Table 02/092812, International W0 98/24893, International W0 96/34096, etc.).
  • This human antibody-producing transgenic mouse is immunized with the target DREF protein of the present invention as an antigen, and then the spleen is removed and fused with this spleen cell and mouse myeloma cell by conventional techniques to form a hybridoma.
  • the desired monoclonal antibody can be prepared (G. Kohler and C. Milstein, Nature 256: 495-497, 1975).
  • DNA encoding the antibody of interest is screened from a library of immunoglobulin genes obtained directly from untreated human lymphocytes, and phage particles are placed between this DNA and the antibody chain.
  • phage particles are placed between this DNA and the antibody chain.
  • To establish a physical association which involves enriching affinity-screened phage that present antibodies with affinity for the target.
  • an antibody having a binding affinity for a target can be synthesized in a large amount by an ordinary method (for example, JP 20013-527832).
  • a humanized antibody can be obtained, for example, by binding a human IgG complementarity determining region (CDR) produced from a mouse immunized with human DREF protein to human IgG.
  • CDR complementarity determining region
  • Such humanized antibodies can be produced using genetic recombination techniques. Techniques for humanizing antibodies are described, for example, in US Pat. Nos. 6639055 and 5530101. '' In the present invention, DREF protein variants include all naturally occurring variants in human individuals, such as variants based on polymorphisms or mutations, or selective Includes variants by splicing.
  • the variant has a sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence of SEQ ID NO: 1, and has an in vivo function involved in cancer cell growth.
  • “several” means 10 or less, 8 ⁇ or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 Means individual.
  • the variant comprises a sequence having% identity of 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of SEQ ID NO: 1.
  • '' 0/0 identity may be determined based on the possible known BLAST program introduction of gaps (BL ASTX and BLASTN) In a sequence alignment (e.g. SF Altschul et al, J. Mol. Bio 215: 403-410, 1990, etc.).
  • the antibody or fragment thereof of the present invention can be used as a therapeutic agent for cancer alone or in a form to which a drug such as an anticancer drug (chemotherapeutic agent, radioactive substance, etc.) is bound.
  • Anti-cancer agents include, but are not limited to, chemotherapeutic agents such as taxol, cytarabine, cisplatin, etoposide, doxonorevicin, daunonorevicin, vinblastine, paclitaxe ⁇ , and radioactive metals such as radioactive indium, technetium, and ytterbium.
  • Binding of the anticancer agent to the antibody can be performed by a method including binding of the anticancer agent to any site of the antibody constant region, for example, by covalent bonding through a linker or by coordinate bonding with a metal ion.
  • Delivery of the antibody or fragment thereof to the patient is usually appropriate, either alone or in the form of the antibody or fragment thereof encapsulated in liposomes (preferably positively charged liposomes), microcapsules or nanoparticles. It can be administered by a parenteral route (for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, topical administration, etc.) in combination with an appropriate carrier, excipient or diluent. '1.4 Composition
  • compositions of the invention can be used to treat patients with cancer, in particular cancers that express DREF.
  • cancers include, but are not limited to, lung cancer, esophageal cancer, sputum cancer, stomach cancer, liver cancer, colon cancer, thyroid cancer, prostate cancer, bladder cancer, kidney cancer, skin cancer, gall cancer, brain tumor Breast cancer, ovarian cancer, cervical cancer, testicular cancer, lymphoma, melanoma, sarcoma, osteosarcoma, etc.
  • More preferred cancers include lung cancer, esophageal cancer, breast cancer, vaginal cancer and the like.
  • the dose of the nucleic acid in the composition of the present invention is not limited to the following when converted to siRNA molecules or antisense nucleic acid molecules, but is 1 ⁇ 100 to 100 ⁇ ⁇ ⁇ , preferably 10 ⁇ to 50 ⁇ per dosage unit. More preferably, it is 100 ⁇ to 20 °.
  • the dose of the antibody or fragment thereof in the composition of the present invention is not limited to the following, but is about 1 to about 100 mg / ml, about 5 to about 70 mg / ml, about 10 to 50 mg per dosage unit. / ml.
  • the above dose or dose may vary depending on the patient's condition, age, gender, severity, etc., and the dose or dose should be determined at the discretion of a specialist.
  • the composition of the present invention usually contains a pharmaceutically acceptable carrier, excipient or diluent such as sterile water, physiological saline, buffer solution, non-aqueous liquid (eg almond oil, vegetable oil, ethanol, etc.), etc. Can be included.
  • the composition further comprises a pharmaceutically acceptable stabilizer (for example, amino acids such as methionine), a preservative (methyl P-hydroxybenzoate, sorbic acid), an isotonic agent (for example, sodium chloride), an emulsifier ( For example, lecithin, gum arabic), a suspending agent (for example, a cellulose derivative) and the like can be contained. .
  • a pharmaceutically acceptable carrier for example, amino acids such as methionine
  • a preservative methyl P-hydroxybenzoate, sorbic acid
  • an isotonic agent for example, sodium chloride
  • an emulsifier for example, lecithin, gum arabic
  • a suspending agent for example, a
  • compositions of the present invention can be administered parenterally, for example intravenously, intraperitoneally, intramuscularly. Administration, subcutaneous administration, local administration, etc. Local administration includes direct injection into the affected area under surgery or endoscopy, etc.
  • administration includes direct injection into the affected area under surgery or endoscopy, etc.
  • One to several doses of the composition of the present invention to the patient at time intervals such as 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, 8 months, 1 year or 2 years Can be administered separately.
  • RNAi nucleic acid, antisense nucleic acid, or antibody of the present invention When the RNAi nucleic acid, antisense nucleic acid, or antibody of the present invention is applied to cancer, particularly lung cancer, it can induce cell death together with cell cycle arrest strongly in lung cancer cells. In particular, in some lung cancer cells, induction of cell death in M phase called mitotic catastroph was observed. On the other hand, in normal lung-derived cell lines, cell cycle arrest was mild compared to lung cancer.
  • the cancer treatment method targeting DREF of the present invention can be used as a tailor-made medicine optimal for cancer patient cases by combining with the screening method for therapeutic agents described below.
  • the present invention further suppresses the expression or translation of the DREF gene by adding a candidate drug to a culture medium containing human cultured cancer cells or a human cell line in which the DREF gene has been transfected so that it can be strongly expressed (normal or non-normal).
  • a method for screening a drug for treating cancer comprising screening a drug in vitro.
  • drug screening is performed by inhibiting cancer cell growth, cell cycle arrest, and / or cell death in cancer cells or mammalian cells that strongly express DREF (eg, humans, mice, etc., preferably humans). Can be used as an index.
  • DREF eg, humans, mice, etc., preferably humans.
  • the method of the present invention comprises preparing a human cultured cancer cell or a cell line that strongly expresses DREF, culturing the cell in the presence of a candidate drug, and suppressing the expression of the DREF gene or mRNA. Or screening candidate drugs for cell proliferation inhibition, cell cycle arrest and Z or cell death induction of the cell or cell line.
  • the human cultured cancer cells include known cancer cell lines, cancer cells derived from biopsies from cancer patients, etc., and can be used in the present invention.
  • cancer cell lines are all lung cancer cell lines such as ACC-LC-91 (H. Osada et al., Mol. Carcinog., 2005, 44: 233-241), A549 (ATCC, Rockvill, MD), PC10 (Immuno-Biological Research Institute, Gunma, Japan), Calu6 (ATCC, Rockville, MD).
  • a cell line that strongly expresses DREF can be prepared, for example, as follows.
  • a lentivirus was prepared by inserting the DREF cDNA into a lentivirus vector (eg CSII-CMV-MCS-IRES2-Bsd vector (obtained from Dr. Hiroyuki Miyoshi of RIKEN (Kodama, Japan)).
  • a lentivirus vector eg CSII-CMV-MCS-IRES2-Bsd vector (obtained from Dr. Hiroyuki Miyoshi of RIKEN (Kodama, Japan)
  • Blasticidin Invitrogen
  • the growth rate can be measured by MTT Atsey etc. using TetraColorOne TM (Seikagaku, Tokyo, Japan) etc.
  • DREF strong expression cell lines eg BEAS2B- The DREF strain was observed to increase growth rate about 3 times compared to the control strain after 6 days of culture.
  • the degree of suppression of DREF gene or raRNA expression can be determined by a comparison experiment with a control to which no candidate drug is added.
  • the expression level is determined by a well-known method (for example, phenol / chloroform / isoform) from cancer cells or DREF strongly expressing cell lines.
  • RT-PCR reverse transcriptase-PCR
  • For synthesized cDNA, quantitative RT-PCR method, hybridization method using fluorescently or radioactively labeled probe eg Northern hybridization, Southern hybridization, DNA microarray, tissue microarray, etc.
  • fluorescently or radioactively labeled probe eg Northern hybridization, Southern hybridization, DNA microarray, tissue microarray, etc.
  • the expression level is determined by measuring the intracellular level of the protein encoded by the DREF gene (SEQ ID NO: 1) by immunoassay using an antibody against the protein or a fragment thereof, Western hyperprecipitation method, tissue staining method, etc. It can be determined indirectly by measurement.
  • the probe is a nucleotide sequence of SEQ ID NO: 2 (positions 202 to 2286) or a sequence complementary thereto, or a sequence thereof, for example, about 20 or more, about 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 A DNA having a sequence consisting of 250 or more nucleotides.
  • the probe is preferably a labeled probe bound with a fluorescent or radioactive label.
  • Fluorescent labels include, for example, fluoresamine, rhodamine, their derivatives, Cy3, Cy5, etc.
  • Radiolabels include, for example, radioactive phosphorus or thio atoms.
  • Hybridization can be performed under low hybridization, medium hybridization or high hybridization conditions, preferably under high hybridization conditions.
  • the hybridization temperature can generally be 5 to 10 ° C lower than the melting temperature (Tm).
  • Tm melting temperature
  • Candidate agents include, but are not limited to, small molecules, peptides, polypeptides, proteins, nucleosides, oligonucleotides, polynucleotides, nucleic acids (DNA or RNA), and the like.
  • An immunoassay is an analysis method that uses an antigen-antibody reaction.
  • an enzyme-linked antibody method for example, ELISA
  • a fluorescent antibody method for example, a solid phase method
  • a homogenous method for example, a sandwich method, a piotine / avidin system, etc.
  • the solid phase for example, a commercially available plastic plate (for example, a 96-well plate made of polystyrene) can be used.
  • the candidate The drug can be identified as a cancer therapeutic.
  • Quantitative RT-PCR is performed, for example, by PCR using mRNA or poly A (+) RNA in the vertical form in the presence of thermostable polymerases such as Taq polymerase and using a PREF derived from the DREF gene sequence. Can do.
  • the size of the primer is about 15-30 nucleotides, preferably 17-25 nucleotides.
  • the expression level of the DREF gene is determined relative to the expression level of the housekeeping gene with no expression fluctuation.
  • Candidate drug screening can also be performed by examining cell growth inhibition, cell cycle arrest, or cell death in human cultured cancer cells, or DREF strongly expressing cell lines.
  • Cell proliferation can be measured by the following method. On the next day after gene transfer, repopulate the cells and select with puromycin / zg / ml) for 2 days, then continue with puromycin (0.5 ⁇ g / ml) for 10 days. Then, replace with TetraColorOne TM (Seikagaku, Tokyo, Japan) 5% medium and incubate at 37 ° C for 1 hour. The medium is then collected and removed. 0D450nm is measured using a reader (in this case, OD630nm is used as a control), and the number of living cells is determined (colorimetric assay). 0 After that, the cells are fixed with methanol, and then 5% Gierasa (Sigma-Aldrich) Stain with aqueous solution.
  • Cell cycle and cell death can be measured by the following methods.
  • the cells were observed with a stereomicroscope BHT-323 (Olympus), and the cells were interphase (G9 / G1, S, G2), prophase, metaphase, metaphase, late phase / terminal phase (Anaphase / telophase) ; ⁇ (mi tot ic catastroph ⁇ ), measure by apoptosis.
  • RNAi nucleic acid A group of genes whose expression changes when DREF gene expression is suppressed by RNAi nucleic acid was analyzed using a microarray. ⁇
  • Human cancer cell line Human lung cancer cell line (ACC-LC- 91 or A549), were cultured in 5% C0 2 presence with 5% fetal calf serum (Invitrogen) added RPMI 1640 medium (Sigma), it was used in the following experiments.
  • Human normal airway epithelial cell lines (BEAS2B or HPL1) are 1% fetal bovine serum (Invitrogen;), 5 ⁇ g / ml bovine insulin (Sigma), 5 ⁇ g / ral human transferrin (Sigma), with 0. 1 mu M hydrocortisone (S i gma) ⁇ Pi 0. 2 nM preparative RHO de thyronine (S i gma) was added a Ham F12 medium (Sigma) and cultured in 5% C0 2 the presence of the following Used in the experiment.
  • RNAi-vector gene transfer into cells For DREF expression vector and RNAi-vector gene transfer into cells (tra.nsf ect ion), seed 3 x 10 5 cells in a 3.5 cm culture dish and mix DNA and Lipofectamine 2000 (Invitrogen) the next day. And added to the medium. '' When a vector with a puromycin-resistant gene such as RNAi vector is transfected, puromyc in dihydrochlori de (Sigma-Aldrich, St (Louis, M0) 2 g / ml medium was added, and after 2 days of selection, the transfected cells were collected, and RNA and protein samples were prepared or FACS analysis was performed.
  • the DREF expression vector was obtained by inserting the 0RF portion of the DREF cDNA obtained by RT-PCR into the expression vector pcDNA3 (Invitrogen). Dr. Fumiko Hirose (Hyogo Prefectural University, Hyogo, Japan) More distributed.
  • RNAi sites # 1, # 2 as a control (# 1) and four new RNAi sites (# 3, # 4, # 5 and # 6) ) DNA oligonucleotide was prepared by requesting Greiner Japan (Tokyo, Japan). Its base sequence is as follows (note that the number at the RNAi site represents the position number of the nucleic acid residue when the base “A” of the start codon in DREF 0RF is the first position).
  • RNAi # l (SEQ ID NO: 16)
  • H1F1 H1R1:
  • si3F 5 '-CAACAACCACCACCTCATGCCTTCCTGTCAGCATGAGGTGGTGGTTGTTGCTTTTTG-3'
  • RNAi # 4 (SEQ ID NO: 22)
  • RNAi # 5 (SEQ ID NO: 25)
  • RNAi # 6 (SEQ ID NO: 28)
  • Plasmid vector with puromycin resistance gene (pCMV-puro) [Note] This vector The filter was prepared by cutting off the unnecessary part 1. Okb length from Clontech's plasmid pIRESpuro2 (5.2 kb length).
  • an RNAi vector containing the mouse U6 gene promoter, a promoter transcribed by RNA polymerase III was prepared (pU6-puro) (Osada., .. et al., Cancer Res. 65: 10680-5, 2005).
  • an RNAi betater inserted with the human HI gene promoter was prepared (pHl-RNApuro).
  • RNAi # l was inserted after digestion of pHl-RNApuro with Bglll-Hindlll.
  • RNAi # 3 to # 6 were inserted after pU6-puro was digested with Apal, treated with T4 DNA polymerase, and digested with EcoRI.
  • RNA was recovered from the transfected cells using the RNeasy Mini Kit (Qiagen).
  • RT-PCR was performed using the One-step RT-PCR kit (Qiagen) according to the attached instruction manual, and the RT-PCR product was subjected to TBE-agarose gel electrophoresis to examine the expression of the DREF gene ( Figure 2). ).
  • DREF F1 5, -TGGCAAGGACATCGTGAAGG (positions 813 to 832 of hDREF 0RF; SEQ ID NO: 135)
  • DREF R1 5 '-GACAGCAACAACCACCACCT (1132 to 1151 of hDREF 0RF; SEQ ID NO: 136)'
  • DREF protein was detected using an antibody against HA-tag / myc-tag to which DREF-cDNA was added.
  • the cells after gene transfer are lysed with 4% SDS solution and heat-treated, and then the protein is electrophoresed on SDS-PAGE and Immobilon using the semi-dry electrotransfer system Ti ⁇ ns-Blot SD cell (Bio-Rad). -Transferred to Millipore.
  • the membrane is overlaid with anti-HA-tag antibody (Santa cruz) and anti-myc-tag antibody (9E10, Santa cruz), washed and then overlaid with secondary antibody (Cell Signaling) bound with horseradish peroxidase, and ECL Western DREF protein was detected by Blotting Detection Reagents (GE Healthcare).
  • FACS analysis ⁇ - The cells after gene transfer are suspended in IGEPAL (registered trademark) CA-630 (0.5% Sigma-Aldrich), Propidiura iodide (20 ⁇ g / ml, Sigma-Aldrich), PBS solution, and DNA in the naked nucleus is transferred. After staining, the amount of DNA was quantified using FACSCalibur (Becton-Dickinson), and the cell cycle (G0 / G1, S, G2 / M) was analyzed using the cell cycle analysis software ModFit (BD Biosciences).
  • the cells were interphase (Go / Gl, S, G2), early cell division (Prophase), metaphase (Metaphase), late phase / terminal (Anaphase / telophase), and dead cells (mitot i (? catastroph and apoptos is)).
  • Hiroyuki Miyoshi of RIKEN BioResource Center (Wako City, 'Saitama, Japan) from Lentiviral Systems (pENTR4-Hl, CS-RfA-EG, pCAG-HIVgp, pCMV-VSV-G -Receive distribution of RSV- Rev).
  • the U6 prmoter-RNAi-oligo part of the pU6-puro-RNAi vector is inserted into the pENTR4-HI vector and then incorporated into the CS-RfA-EG vector using LR clonase (Invitrogen).
  • CDNA is synthesized from 250 ng of the obtained 36 hour RNA sample using MMLV-RT (Agilent Technologies, Palo Alto, Calif.) And an oligo dT primer to which a T7 promoter sequence is added.
  • Figure 1 shows the domain structure, RNAi site and sequence of the human DREF / ZBED1 gene.
  • the RNAi system was constructed (pCMV-puro-siDREF # l to # 6)
  • the DREF-RNAi system was transfected into a human lung cancer cell line, and then the transfected cells were selected by puromyc in treatment for 24-48 hours.
  • the lentivirus vector was used for RNAi, and a short hairpin type.ol igo was inserted into the lentivirus vector in the same manner, and the gene was introduced into 293T cells according to a standard method. lentivirus particles expressing short hairpin RNA were collected and added to the cell culture for analysis.
  • Figure 2 shows the results of RT-PCR analysis of the RNAi effect. Introduce RNAi vector pCMV-puro-s iDREF # l, # 3, # 4, '# 5, # 6 into lung cancer cell A549, and after puromycin treatment for 48 hours, collect cells, extract RNA, RT- DREF expression was examined by PCR. Compared to the control vector that does not express DREF-siRNA (pCMV-puro_) U6blank, all pCMV-puro-siDREF vectors showed decreased expression due to RNAi.
  • pCMV-puro_ DREF-siRNA
  • Figure 3 shows the results of Western blot analysis of the RNAi effect.
  • a DREF full-length cDNA expression vector and a DREF full-length cDNA expression vector that had undergone a silent mutation in the RNAi sites of # 3 and # 4, respectively, and became RNAi resistant.
  • DREF Changes in the expression of DREF protein were examined by Western blot. For detection, an anti-HA polyclonal antibody, which is an antibody against HA-tag inserted at the N-terminus of DREF cDNA, was used. siDREF # 3 In # 4, both wild-type DREF proteins showed a marked decrease. DREF (# 3mut, # 4mut) with # 3 and # 4 silent mutations was resistant to RNAi.
  • Figure 4 shows the results of cell cycle analysis (1).
  • s iDREF- # 3 and # 5 showed a decrease in the S phase, and a trend toward cell cycle arrest (G1 arrest) at the G1-S transition stage.
  • G1 decreased significantly, G2 / M increased, and cell cycle arrest at a very strong G2 / M step was observed.
  • a slight increase in G2 / M was also seen in # 3, and # 3 was thought to cause a strong G1 stop and a relatively mild G2 / M stop.
  • Figures 5A, 5B, and 5C show the results of cell cycle analysis (2).
  • pCMV-puro-siDREF, # 3, # 4 (and U6blank) were introduced into several lung cancer cell lines, and the cell cycle was analyzed.
  • ACC-LC-172 as in ACC-LC-91, # 3 causes a strong G1 arrest and a relatively mild G2 / M arrest, while # 4 shows a strong G2 / M cell cycle arrest. It was.
  • Calu6 'ACC-LC-319 also showed a tendency to stop G2 / M in # 4.
  • A549- # 4 'PC10- # 3 and-# 4 showed a decrease in S and a tendency to stop G1.
  • cell cycle arrest was hardly observed in the normal lung-derived cell lines BEAS2B and HPL1.
  • 6A and 6B show the results of cell proliferation ability analysis. Puromycin-selection cell number after 10 days was analyzed with MTT assay (using cell proliferation kit). s iDREF- # 3 and # 4 showed a much stronger cell growth inhibitory effect than '4, and # 4 was considered to be stronger. # 1 had only a weak antiproliferative effect.
  • FIG. 7 shows the results of staining with yH2AX (Phospho- ⁇ 2 ⁇ ) and Phospho-ATM (Serl981).
  • yH2AX Phospho- ⁇ 2 ⁇
  • Phospho-ATM Phospho-ATM
  • Figure 8 shows the TUNEL analysis results. Induction of apoptosis (cell death) was examined by TUNEL analysis to detect DNA fragmentation. Compared to U6blank, apoptosis induction was seen at # 3 and # 4. Apoptosis frequency was different between # 3 and # 4, and # 3 was almost the same in 4 strains of A549, PC10, ACC-LC-91 and ACC-LC-172, but in # 4, A549 It is almost the same as PC10, and ACC-LC-91 is almost the same as ACC-LC-172, and ACC-LC-91 showed significantly greater apoptosis induction than A549 (some data not shown) ).
  • FIG. 10 shows cell death (mitotic catastroph) in M phase.
  • Phospho-histon H3 (H3-P) and ⁇ H2AX which are phosphorylated in M phase, were immunostained to investigate the relationship between DNA damage and cell death induction and the cell cycle.
  • ACC-LC-91 showed that most of ⁇ 3- ⁇ positive ⁇ phase cells were ⁇ 2 ⁇ -positive, indicating that cell death was induced by DNA damage in the ⁇ phase.
  • FIG. 11 shows the results of gene expression analysis of siDREF-# 3 and # 4.
  • ACC-LC-91 was infected with siDREF # 3 and siDREF # 4 lentivirus to induce RNAi.
  • SAS StatView
  • DREF expression was observed, whereas in normal lung, DREF expression was absent.
  • Fig. 13 shows the DNA sequence (A) of RPL17 gene, which is considered to be related to cancer growth, among the genes that are suppressed by DREF-RNAi # 4, and the mechanism of transcriptional activation by DREF of RPL17 gene.
  • (B) is shown.
  • DREF binds to this sequence to induce RPL17 DREF expression, and DREF-RNAi # 4 reduces DREF expression, so it is thought that RPL17 gene expression also decreases.
  • RNAi induction 11 are shown in Table 1 (# 4) and Table 2 (# 3). These genes whose expression changes at an early stage of RNAi induction (36 hours) are directly regulated by DREF, and are likely to be cell groups that cause cell cycle arrest and cell death induction. It is considered to be a target.
  • SRRMl / SRtnl60 is a co-activator of splicing and induces CD splicing abnormalities in an oncogene RAS-dependent manner (Mol Cell Biol. 2006 Jan; 26 (1): 362-70). It also binds to the oncogene TLS / FUS (Exp Cell Res. 2003 Feb 15; 283 (2): 184-95). The association with cancer development is strongly suggested.
  • a gene family with TO40 domains involved in various processes such as signal transduction, apoptosis induction, pre-mRNA maturation, and cytoskeleton formation.
  • HBV hepatitis B virus
  • Oif icial symbol S ⁇ RS16 and Name: splicing factor, ar'ginine / serine-rich 16 (suppressor-of-white- apricot homolog, Drosophila)
  • Su thigh ary Controls splicing before becoming mature mRNA after gene transcription.
  • Oxf icial Symbol ZBED1 and Name: zinc finger, BED-type containing 1 Gene aliases: ALTE; TRAMP; KIAA0785
  • CTDSP1 Carboxy-terminal domain, RNA polymerase II, polypeptide A
  • Gene aliases SCP1; NLIIF (nuclear LIM interac tor-interacting factor) A phosphatase that regulates the activity of RNA polymerase II that transcribes the gene. It controls the expression of a wide range of gene groups.
  • RNA helicase with a DEAD box domain that regulates RNA conformation. Involved in the control of a wide range of functions related to RNA, such as the initiation of translation into proteins and splicing.
  • This gene family is a gene family with a WD40 domain involved in various processes such as signal transduction, apoptosis induction, pre-mRNA maturation, and cytoskeleton formation.
  • TLR Toll-like receptor
  • TNF receptor TNF receptor
  • TNF-R / TNFRSF1A TNF receptor
  • EGRl is a transcriptional regulatory molecule of C2H2_zinc-finger type that regulates the expression of genes involved in differentiation and cell division It is thought to be involved in cancer development.
  • ITPKA inositol 1, 4, 5-trisphosphate 3 kinase
  • IP3 is an intracellular signal concentration of calcium that is an important signal pathway ITPKA phosphorylates this IP3 to inositol 1, 3, 4, 5-tetrakisphosphate (IP (4)) and suppresses the increase in the strength concentration. It is deeply involved in the regulation of intracellular signals.
  • TRAF7 and Name: TNF receptor-associated factor 7 Gene aliases: RFWD1; RNF119; MGC7807; DKFZp586I021
  • Oii'icial Symbol DVLl; Name Name: dishevelled, dsh nomolog 1 (Drosophila) Summary: Onset of cancer 'Wnt- -catenin signaling pathway that has been reported to be involved in growth, suppresses -catenin degradation and Wnt --Increase catenin signal. 'Relevant to cancer development is strongly suggested.
  • EGR1 is a C2H2-zinc-finger transcriptional regulatory molecule that regulates the expression of genes involved in differentiation and cell division and is thought to be involved in cancer development.
  • CtBPl C-terminal binding molecule of adenovirus oncogene E1A.
  • This CtBP gene family (CtBPl'CtBP2) is a target gene when the tumor suppressor gene ARF induces apoptosis induction independent of p53. Overexpression of CtBP induces p53-independent apoptosis induction of this ARF. Inhibits (Mol Cell Biol. 2006 Mar; 26 (6): 2360-72 ⁇ ). Therefore, this CtBPl is considered an oncogene.
  • SWI / SNF complex that regulates chromatin structure and regulates gene expression. It also functions as a co-activator for nuclear hormone receptors such as androgen receptors that promote prostate cancer growth. It is deeply involved in the regulation of gene expression and essential for prostate cancer growth (Mol Cell Bi. L. 2005 Mar; 25 (6): 2200-15). The association with cancer development is strongly suggested.
  • This is a substrate of the representative kinase-type oncogene Src family and functions by binding to PKD2, the gene responsible for polycystic kidney disease. It is suggested to be associated with cancer development.
  • One of the biosynthetic enzymes of purine nucleic acid which is a material for DNA, and is related to cell growth.
  • the SUV39H family (SUV39H1, SUV39H2) gene induces the methylation of histone H3-lysine9, which induces heterochromatin formation. It binds to RB2 / pl30 of the typical tumor suppressor gene Retinoblastoma (RB) family and promotes transcriptional regulation by RB2. It is suggested to be associated with cancer development. ,
  • CEBPD CCAAT / enhancer binding protein
  • This gene is upregulated in B-cell malignant lymphoma and is induced by interleukin-4. It is suggested to be related to the onset of tumors such as lymphoma.
  • the cancer treatment targeting DREF according to the present invention has a very strong suppression of cell proliferation, induction of cell death in M phase, and G2 / M phase without substantially affecting normal tissues. It has a very strong cell cycle arresting effect in cancer and is very effective for cancer regression and cell death. ,
  • the present invention enables cancer treatment using DREF as a molecular target. That is, since the therapeutic agent of the present invention is very effective for cancer regression and cell death, it can be effectively used for the treatment of cancer expressing DREF.

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Abstract

L'invention porte sur une composition pour le traitement du cancer, caractérisée par le fait qu'elle contient un acide nucléique ARNi, un acide nucléique anti-sens ou un anticorps dirigé contre DREF, qui inhibe la fonction in vivo d'un régulateur de croissance de cellules humaines, de DREF ou du gène DREF dans une cellule cancéreuse ou un tissu cancéreux. L'invention porte également sur un procédé de criblage pour un agent pour traiter le cancer en utilisant, comme indice, l'inhibition de l'expression du gène DREF ou de la traduction de la protéine DREF ou l'inhibition de la croissance cellulaire, l'arrêt du cycle cellulaire et/ou l'induction de la mort cellulaire dans une cellule cancéreuse humaine mise en culture ou une lignée cellulaire à expression élevée de DREF.
PCT/JP2007/070031 2006-10-06 2007-10-05 Composition pour le traitement du cancer à l'aide de dref comme cible moléculaire Ceased WO2008044787A1 (fr)

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CN118910050A (zh) * 2024-07-19 2024-11-08 威海市立医院 一种靶向沉默ALIX基因表达的shRNA、慢病毒表达载体和应用

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SANO Y. ET AL.: "Tensha Inshi hDREF ha Saibo Shuki Shinko ni Kanyo suru", DAI 28 KAI ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN KOEN YOSHISHU, 2005, pages 278, XP003021395 *
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Cited By (11)

* Cited by examiner, † Cited by third party
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US12066429B2 (en) 2016-05-10 2024-08-20 National University Corporation Tokyo Medical And Dental University Method for inhibiting the expression of inflammation promoting factors
WO2019093502A1 (fr) * 2017-11-09 2019-05-16 国立大学法人 東京医科歯科大学 Inhibiteur de l'expression de facteurs favorisant le cancer, méthode de criblage pour principe actif de celui-ci, cassette d'expression utile dans ladite méthode, médicament de diagnostic et méthode de diagnostic
CN111315882A (zh) * 2017-11-09 2020-06-19 国立大学法人东京医科齿科大学 癌症促进因子表达抑制剂、其有效成分的筛选方法、对该方法有用的表达盒、诊断药和诊断方法
JPWO2019093502A1 (ja) * 2017-11-09 2021-02-25 国立大学法人 東京医科歯科大学 がん促進因子発現抑制剤、その有効成分のスクリーニング方法、該方法に有用な発現カセット、診断薬、及び診断方法
JP7376873B2 (ja) 2017-11-09 2023-11-09 国立大学法人 東京医科歯科大学 がん促進因子発現抑制剤、その有効成分のスクリーニング方法、該方法に有用な発現カセット、診断薬、及び診断方法
IL274456B1 (en) * 2017-11-09 2024-04-01 Univ Nat Corp Tokyo Medical & Dental Inhibitor of the expression of cancer-promoting factors, screening method for active ingredient thereof, expression cassette useful in said method, diagnostic drug, and diagnostic method
IL274456B2 (en) * 2017-11-09 2024-08-01 Univ Nat Corp Tokyo Medical & Dental Inhibitor of the expression of cancer-promoting factors, screening method for active ingredient thereof, expression cassette useful in said method, diagnostic drug, and diagnostic method
CN111315882B (zh) * 2017-11-09 2024-09-24 国立大学法人东京医科齿科大学 癌症促进因子表达抑制剂、其有效成分的筛选方法、对该方法有用的表达盒、诊断药和诊断方法
US12253515B2 (en) 2017-11-09 2025-03-18 National University Corporation Tokyo Medical And Dental University Method for inhibiting the expression of cancer-promoting factors
CN109777800A (zh) * 2017-11-15 2019-05-21 信雅生物科技（苏州）有限公司 一种能够特异性抑制ZBED1基因的siRNA的构建方法及其应用
CN118910050A (zh) * 2024-07-19 2024-11-08 威海市立医院 一种靶向沉默ALIX基因表达的shRNA、慢病毒表达载体和应用

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