WO2006006520A1 - 新規創薬標的の探索方法 - Google Patents
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- WO2006006520A1 WO2006006520A1 PCT/JP2005/012655 JP2005012655W WO2006006520A1 WO 2006006520 A1 WO2006006520 A1 WO 2006006520A1 JP 2005012655 W JP2005012655 W JP 2005012655W WO 2006006520 A1 WO2006006520 A1 WO 2006006520A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/111—Antisense spanning the whole gene, or a large part of it
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Definitions
- the present invention relates to a siRNA expression system that suppresses gene expression by the RNAi effect, a library thereof, and a method for comprehensive gene function analysis and drug discovery target molecule search using the library. . Furthermore, the present invention relates to a gene related to angiogenesis obtained as a result of examination using the library.
- Non-patent document 2 a DNA microarray
- Non-patent document 3 a yeast two-hybrid system
- DNA microarray technology is based on changes in gene expression levels during various intracellular processes.
- the yeast two-hybrid system is a powerful method for detecting direct protein-protein interactions in vivo and identifying genes whose products interact with specific proteins from a cDNA library. provide.
- this method is limited in that the expected interaction does not necessarily reflect its role in the phenomenon and often produces false positive results.
- Non-patent Document 5 a method for exploring gene functions by comprehensively expressing various genes and identifying genes that are specifically expressed in cells that showed the required phenotypic changes.
- RNA interference RNA interference
- RNAi is a double-stranded RNA (hereinafter abbreviated as "dsRNA") consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence. It is a phenomenon that can induce the destruction of the target gene mRNA and suppress the expression of the target gene by introducing it into the cell. RNAi is thus attracting attention as a simple gene knockout method to replace the conventional complicated, low-efficiency gene disruption method by homologous recombination because it can suppress the expression of the target gene.
- dsRNA double-stranded RNA
- Non-patent Document 7 The RNAi was originally found in nematodes (Non-patent Document 7), but is now observed in various organisms such as plants, linear animals, Drosophila and protozoa, as well as nematodes. (Non-patent document 8, Non-patent document 9, Non-patent document 10, Non-patent document 11). In these organisms, it has been confirmed that the expression of the target gene is actually suppressed by introducing dsRNA from a foreign body, and is also being used as a method for creating knockout individuals.
- Non-patent Document 12 Non-Patent Documents
- Reference 13 Non-patent reference 14
- RNAi reactions in vitro require ATP (Non-patent Document 14).
- Non-patent Document 15 Recent studies with synthetic RNA duplexes indicate that each siRNA duplex It was proved to cut (Non-patent Document 15). It became clear that the overhanging 3 ′ end of 2 or 3 nucleotides in the siRNA duplex was necessary for efficient target cleavage (Non-patent Document 15). Such 3 'overhangs are characteristic of the product of the RNase III cleavage reaction, and in cultured Drosophila S2 cells, cleavage of dsRNA into siRNA is a multi-domain RNase III enzyme known as Dicer. (Non-Patent Document 16).
- Non-Patent Document 13 Non-Patent Document 16; Non-patent document 17.
- RNAi provides a way to inactivate target genes and thus provides a powerful tool for studying gene function in C. eleg ans, Drosophila and plants .
- Specific inhibition of gene expression can also be obtained by stable and inducible expression of dsRNA in animals and plants (Non-patent document 18, Non-patent document 19, Non-patent document 10).
- dsRNA stable and inducible expression of dsRNA in animals and plants
- ES embryonic stem
- Non-patent Document 25 RNA and RNAi-related systems exist in mammals.
- RNAi-related proteins such as rde-l, mut-7, and Dicer have been identified (Non-patent document 26, Non-patent document 27, Non-patent document 16).
- An siRNA expression vector has been developed for the purpose of more easily using such siRNA.
- Pol III-based Hl, U6, and tRNA are generally used as promoters for expressing siRNA.
- siRNAs with a hairpin structure length of 21 bases show the highest gene expression suppression
- tRNA the hairpin structure length is around 29 bases.
- the design method of siRNA sequences having a high gene expression suppression effect has not been established, and the most in silico methods have been tried so far, but the certainty is not necessarily high. What is it!
- siRNA expression vectors using a fragment obtained by cleaving any double-stranded DNA non-sequence with a DNA-degrading enzyme as a raw material.
- a method for preparing a rally was reported (Non-patent document 28, Non-patent document 29).
- the individual siRNA expression vector clones contained in this siRNA expression vector library target somewhere in the source of any double-stranded DNA sequence. Therefore, the entire library expresses siRNA comprehensively so as to cover the entire arbitrary double-stranded DNA sequence as a raw material.
- exhaustive search for genes related to a certain phenotypic change that is, drug target genes with high therapeutic effects can be selected from a large number of drug target candidate genes.
- Non-patent Document 30 Non-patent Document 30
- Non-Patent Document 1 Allen K., Nature Biotechnol. 16: 1294, 1998
- Non-Patent Document 2 Fumio Kimizuka et al., Protein Nucleic Acid Enzyme 43: 2004, 1998
- Non-Patent Document 3 Suzuki H. et al, Genome Res. 10: 1758-1765, 2001
- Non-patent document 4 Toshio Kitamura, Cell engineering 19: 893-901, 2000
- Non-Patent Document 5 Leo C. et al., Nature Genetics 27: 23-29, 2001
- Non-Patent Document 6 Beger C. et al., Proc Natl Acad Sci USA 98: 130-135, 2001
- Non-Patent Document 7 Fire, A. et al. Potent and specific genetic interference by douole-stranded RNA in Caenorhabditis elegans Nature 391, 806—811, (1998)
- Non-Patent Document 8 Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358-3 3 (1
- Non-Patent Document 9 Sharp, PA RNA interference 2001. Genes Dev. 15, 485-490 (2001)
- Patent Document 10 Hammond, SM, Caudy, AA & Hannon, GJ Post-transcription al gene silencing by douole-stranded RNA. Nature Rev. Genet. 2, 110-119 (2001)
- Non-Patent Document 11 Zamore, PD RNA interference: listening to the sound of silence. N at Struct Biol. 8, 746—750 (2001)
- Non-Patent Document 12 Tuschl T “et al” Genes Dev. 13: 3191-3197, 1999
- Non-Patent Document 13 Hammond S. M., et al., Nature 404: 293-296, 2000
- Non-Patent Document 14 Zamore P., et al., Cell 101: 25-33, 2000
- Non-Patent Document 15 Elbashir S. M. et al "Genes Dev. 15: 188-200, 2001
- Non-Patent Document 16 Bernstein E. et al., Nature 409: 363-366, 2001
- Non-Patent Document 17 Hammond S. M. et al "Science 293: 1146-1150, 2001
- Non-patent literature 18 Kennerdell and Carthew Nature Biotechnol. 18: 896-898, 2000
- Non-patent literature 19 Tavernarakis N. et al., Nature Genetics 24: 180-183, 2000
- Non-patent literature 20 Billy E. et al. , PNAS 98: 14428-14433, 2001
- Non-Patent Document 21 Paddison P. J. et al "PNAS 99: 1443-1448, 2002
- Non-Patent Document 22 Stark G. R. et al "Annu. Rev. Biochem. 67: 227-264, 1998
- Patent Document 23 Silverman R. H. in Ribonucleases: Structures and Functions, eds. D 'Alessio, G. and Riordan J. F. (Academic, New York) pp.515-551
- Non-Patent Document 24 Clemens M. J. and Elia A., J. Interferon Cytokine Res. 17: 503—524, 1997
- Non-Patent Document 25 Elbashir S. M. et al., Nature 411: 494-498, 2001
- Non-Patent Document 26 Tabara H. et al "Cell 99: 123-132, 1999
- Non-Patent Document 27 Ketting RF et al., Cell 99: 133-141, 1999
- Non-Patent Document 28 Sen, G. et al, Nature Genetics 36: 183-189, 2004
- Non-Patent Document 29 Shirane, D. et al., Nature Genetics 36: 190-196, 2004
- Non-Patent Document 30 Amada RG., Et al., Hum Gene Ther. 13: 2255-2270, 1999 Disclosure of the Invention
- the present invention aims to provide a stem-loop RNA molecule expression vector library capable of exhaustive gene function search, and a comprehensive drug discovery target molecule search method using the library. Furthermore, an object of the present invention is to identify a drug discovery target molecule that is expected to have a high effect by suppressing the expression by this method. Specifically, regarding a target gene that is expected to have a therapeutic effect due to a change in the expression level, a factor that regulates the expression of the target gene in a normal state is identified, and its expression is suppressed. The subject is to control the expression level of the gene. When the factor suppresses the expression of the target gene, the expression of the target gene can be enhanced by suppressing the expression of the factor.
- the factor when the factor enhances the expression of the target gene, it is possible to suppress the expression of the target gene by suppressing the expression of the factor.
- This factor is a novel drug discovery target, and at the same time, siRNA molecules that suppress the expression of the factor may be applicable to drugs with new drug effects.
- a stem-loop RNA molecule expression vector that suppresses the expression of the factor is considered to be applicable to gene therapy.
- the present invention specifically relates to
- composition comprising the DNA according to any one of [1] to [9] or the vector according to [13],
- a method for producing a cell for functional gene search including the following steps (a) to (d):
- a step of introducing into a cell a vector containing a DNA having a structure in which a DNA encoding a target gene expression control region and a promoter and a DNA encoding a marker gene whose cell phenotype changes are functionally connected The functional gene search method according to [18], further comprising:
- a method for confirming the function of the identified gene according to [20], wherein the DNA according to any one of [1] to [9] or the vector according to [13] is used as a cell or experiment A confirmation method including a step of introducing it into an animal
- a polynucleotide comprising the base sequence described in any one of SEQ ID NOs: 1 to 224, [27] 19 bp or more consecutively selected arbitrarily in the base sequence described in any one of SEQ ID NOs: 1 to 224
- a polynucleotide comprising the nucleotide sequence region of
- a protein related to hypoxic stimulation comprising the nucleotide sequence region described in [27] is copied. Polynucleotides to be loaded,
- a vector comprising the polynucleotide according to any one of [26] to [28],
- FIG. L Shows the structure of pHRE—Nl—CMV—CD4 A vector.
- the present invention is a DNA encoding a stem-loop RNA molecule having an RNAi effect in a cell, and a DNA encoding an RNA corresponding to a region selected from an existing base sequence ability arbitrarily selected, Comprehensive factors that control the expression of the target gene, characterized by a structure in which DNA and a complementary sequence are linked so as to face each other across a spacer region and functionally connected to a promoter.
- the present invention relates to an siRNA expression vector library that enables a comprehensive search of gene functions and a comprehensive search method for drug discovery target molecules using the library.
- siRNA is usually a double-stranded RNA molecule of about 20 bp, and is known to have an RNAi effect.
- RNAi RNA interference
- dsRNA double-stranded RNA
- This is a phenomenon that can induce the destruction of the target gene mRNA and suppress the expression of the target gene.
- the siRNA expression vector library that enables a comprehensive search of gene functions in the present invention is performed by a method of producing at least two partial fragments of the DNA having smooth ends from any double-stranded DNA. Prepared. Preferably, a method comprising the following steps (a) to (c) It is.
- step (b) a step of treating the product of step (a) with a single-stranded DNA-specific degrading enzyme to remove the contained single-stranded DNA;
- the siRNA expression vector library capable of exhaustive gene function search in the present invention includes DNA fragments having overlapping DNA regions in two or more fragments prepared as described above. It is characterized by that.
- a DNA fragment is prepared by PCR from double-stranded DNA as a material.
- the double-stranded DNA used in the present invention is preferably derived from genomic DNA, more preferably derived from cDNA.
- the “nick transduction enzyme” in the present invention is preferably DNAsel.
- the system in which the small DNA fragment is prepared is treated with a single-stranded DNA-specific degrading enzyme to remove the single-stranded DNA contained therein.
- a single-stranded DNA-specific degrading enzyme to remove the single-stranded DNA contained therein.
- Exonuclease VII or Exonuclease I can be used.
- a DNA elongation reaction is carried out starting from the 3rd and 3rd ends of the nick site.
- a talenaux enzyme having polymerase activity can be used for example, -5, -3.
- the DNA thus prepared is dephosphorylated by CIP treatment to produce double-stranded DNA having a blunt end.
- an siRNA expression vector that makes it possible to search for a comprehensive gene function in the present invention.
- One library is an inverted repeat (inverted repeat sequence) consisting of a double-stranded DNA and a partial fragment of the DNA as a repeating unit. ) Containing double stranded DNA.
- the method includes the following steps (a) to (f).
- a partial fragment of the DNA having a blunt end is prepared from the double-stranded DNA by the method described above. Manufacturing process,
- step (b) a step of dephosphorylating the 5, end of the DNA fragment of step (a),
- step (c) Ligating the double-stranded DNA dephosphorylated at the 5 ′ end in step (b) with the 5 and the ends of the hairpin-type double-stranded DNA linker A, the dephosphorylated DNA Creating a DNA molecule having a structure in which the linker A is bound to both ends of the double-stranded DNA;
- step (d) DNA is elongated from the 3 'end of the nicked portion of the DNA molecule in step (c) by the 5,3, polymerase activity of the talenou enzyme, and the partial fragment DNA in step (a) and the hairpin A step of producing a DNA molecule having a structure in which a linker A is bound,
- step (e) Ligating the DNA molecule of step (d) with the 5 'end of the double-stranded DNA linker B, and the 3' end of the DNA molecule and the 5 'end of the linker B are bound.
- step (f) Talenow enzyme, 5 and -3, by polymerase activity, the DNA is extended from the 3 'end of the nick site of the DNA molecule in step (e), and the partial fragment in step (a) is repeatedly single-ended.
- step (f) Talenow enzyme, 5 and -3, by polymerase activity, the DNA is extended from the 3 'end of the nick site of the DNA molecule in step (e), and the partial fragment in step (a) is repeatedly single-ended.
- linker A is the BL02 linker.
- the linker used in this method is preferably one having a 5′-terminal phosphate.
- the phosphorylation of the linker may be carried out enzymatically or by adding a phosphate group to the 5′-end during the linker synthesis.
- T4 DNA ligase can be used as long as it has an equivalent function.
- DNA molecule and the 5 'end of a double-stranded DNA linker B are ligated, and the DNA component is ligated.
- a DNA molecule having a structure in which the 3 ′ end of the child is linked to the 5 ′ end of the linker B is prepared.
- Linker B is a DNA having a restriction enzyme site in the DNA region, and is preferably a hairpin double-stranded DNA, but does not necessarily have a hairpin structure.
- a SL02 linker can be used as a hairpin type linker.
- a hairpin fragment is prepared by cutting off the end derived from the hairpin linker (SL02).
- Sacl is preferred for this cut-off.
- the siRNA expression vector library that enables an exhaustive search for gene functions in the present invention is a stem-loop RNA molecule in which a transcription product corresponding to a partial fragment of double-stranded DNA is one strand of the stem. It is prepared by a method for producing an expression vector. Specifically, the method includes the following steps (a) and (b).
- step (b) producing an expression vector having a structure arranged downstream of the promoter so that the inverted repeat sequence of the DNA of step (a) can be transcribed;
- Stem loop is a structure consisting of a double-stranded part (stem) generated by hydrogen bonding between inverted repeats existing on single-stranded RNA and the partial force of the loop sandwiched between them. And is also called a hairpin loop.
- a DNA containing an inverted repeat sequence having the double-stranded DNA partial fragment obtained by the above method as a repeating unit is transcribed downstream of the promoter of the expression vector. Ligation to get. Vector into which the sequence has been inserted If the loop part of the hairpin contains a part called stuffer (BamHI-BamHI), delete it and create a siRNA expression vector library.
- the part called stuffer is determined according to the type of restriction enzyme site to be used, and is not necessarily limited to the BamHI-BamHI fragment as described in the Examples.
- the type, number, position, etc. of the promoter can be arbitrarily determined as long as it can transcribe (express) the DNA construct produced by the method of the present invention.
- examples of the promoter include polll and polIII promoters.
- a polIII promoter suitable for expression of short RNAs such as siRNA can be preferably used.
- examples of the polIII promoter include U6 promoter, tRNA promoter, retroviral LTR promoter, adenovirus VA1 promoter, 5S rRNA promoter, 7SK RNA promoter, 7SL RNA promoter, HI RNA promoter and the like. .
- the above U6 promoter has the ability to add 4 uridine bases to the 3 'and 3' ends of RNA.
- the overhang at the 3 'end is the leading sequence of antisense code DNA and sense code DNA.
- the overhang of the finally generated siRNA 3 ′ end can be freely changed to 4, 3, 2, 1, 0 bases.
- the number of protruding bases at the end can be freely changed.
- polll promoters include cytomegaloinoles promoter, T7 promoter, T3 promoter, SP6 promoter, RSV promoter, EF-1 ⁇ promoter, ⁇ -actin promoter, ⁇ -globulin promoter, SRa promoter. And so on.
- the polll system when used, it is synthesized as RNA of a certain length that is short of a short RNA such as the polIII system. Therefore, when a polll promoter is used, antisense RNA or sense RNA is generated from RNA synthesized as a certain length using means that can cleave RNA such as ribozyme by self-processing. It can also be made.
- a tRNA promoter can be preferably used as the promoter of the present invention.
- the tRNA promoter exists upstream of the gene in prokaryotes, whereas in the eukaryote, it is known to function as a DNA region force 3 ⁇ 4 RNA promoter corresponding to the D- and T-loops of tRNA. Yes.
- the t RNA promoter of the present invention is
- tRNA Val tRNA (tRNA Val ) promoter corresponding to Norin ( Val ) can be preferably used.
- the above "functionally linked" means that the DNA and the tRNA promoter are combined so that the transcription from the tRNA promoter is received and the above hairpin transcription product is generated.
- the position of the tRNA promoter with respect to the DNA is not limited upstream and downstream, but is usually upstream. Further, any DNA sequence may be present between the DNA and the tRNA promoter as long as transcription of the DNA can occur.
- the tRNA by receiving transcription from the tRNA promoter as described above, the tRNA itself is also bound to the stem-loop RNA molecule as a translocation signal to the cytoplasm.
- a terminator can be appropriately provided in the DNA.
- the terminator is not particularly limited as long as it can terminate transcription of the promoter.
- a known terminator such as a sequence that is continuous with A (adenine) base or a sequence that can form a palindromic structure is used. Can be used.
- the DNA of the present invention includes single-stranded DNA and double-stranded DNA.
- the above-mentioned “region arbitrarily selected from existing base sequences” can preferably indicate a region having a length of 20 to 40 bp, for example.
- RNA molecule By receiving transcription from the above-described DNA strength RNA promoter of the present invention, a continuous transcription product (RNA molecule) is generated. Since the DNA of the present invention has an inverted repeat sequence with a spacer region in between, the transcript of the DNA of the present invention also has a structure containing an inverted repeat sequence with the spacer region in between. is doing. An RNA molecule having such a structure usually forms a hydrogen bond between the repetitive sequences, and uses the repetitive sequence as a stem to form a spacer. A stem loop having a region as a loop is formed. In the present specification, an RNA molecule that forms this stem loop is referred to as a “stem loop RNA molecule”. The present invention also includes a stem-loop RNA molecule transcribed from the above DNA of the present invention.
- the present invention also includes siRNA molecules that are produced in the cell from the transcription product of the DNA of the present invention and have an RNAi effect in the cell. Furthermore, the present invention also includes a synthetic siRNA molecule having a sequence corresponding to siRNA produced in the cell from the transcription product of the DNA of the present invention and exhibiting an RNAi effect in the cell.
- the above-mentioned “synthetic siRNA molecule” of the present invention can be appropriately prepared by those skilled in the art based on the method disclosed in the present specification. In this case, if one strand is known, those skilled in the art can easily know the base sequence of the other strand.
- the siRNA of the present invention can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, for synthesis of a desired RNA, it is possible to use a general synthesis contract service.
- RNAi effect means that the metabolite of the stem-loop RNA molecule of the present invention (for example, a product produced by metabolism such as cleavage into an RNA strand) has an RNAi effect. Cases are also included.
- the length of the DNA constituting the spacer region of the present invention is not particularly limited as long as the repetitive sequences adjacent to it can form hydrogen bonds, but usually 1 to 20 bases, preferably 1 to 1 0 base, more preferably 3 to 8 base, and still more preferably 4 to 6 base.
- the base sequence of the DNA constituting the spacer region is not particularly defined and can be any sequence.
- the spacer region is not particularly required, and even if it does not have a spacer region, the DNA of the present invention can be used. include.
- the stem-loop RNA molecule of the present invention has a length force of double-stranded RNA in the stem region usually within 40 bp, preferably 20 to 40 bp.
- DNA of the present invention can be prepared by those skilled in the art using general genetic engineering techniques.
- the DNA of the present invention containing a tRNA promoter is, for example, a well-known oligo Any sequence can be synthesized by nucleotide synthesis.
- the DNA of the present invention can be directly introduced into a chromosome in a cell and expressed in the cell, but in order to efficiently introduce the cell, the DNA is preferably retained in a vector. .
- a vector containing the DNA of the present invention is also included in the present invention.
- the “vector” that can be used here can be selected according to the cell or the like to be introduced. For example, in mammalian cells, for example, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, lentivirus vectors, herpesvirus vectors, alphavirus vectors, EB virus vectors, papilloma winoles.
- Non-viral vectors such as Vinoter vectors such as betaters and foamy winores betaters, cationic ribosomes, ligand DNA complexes, and gene guns (Niitsu Y. et al., Molecular Medicine 35: 1385-1395 (1998) However, it is not limited to these.
- Dumbbell DNA Zaanta MA et al., Gene delivery: a single nuclear localization signal peptide is sufficient to carry D NA to the cell nucleus.Proc Natl Acad Sci US A.
- modified DNA with resistance to nuclease, or naked plasmid can also be used suitably (Liu F, Huang and Improving plasmid L NA mediated liver gene transfer by prolonging its retention in The hepatic vasculature. J. Gene Med. 2001 Nov— Dec; 3 (6): 569—7 6).
- the vector can further hold a selection marker that can select a cell into which the vector has been introduced.
- Selectable markers include neomycin resistance gene, drug resistance marker such as idaromomycin resistance gene, puromycin resistance gene, marker that can be selected using enzyme activity such as galactosidase, or fluorescence emission such as GFP.
- index are mentioned.
- a selection marker that can be selected using a surface antigen such as an EGF receptor or B7-2 as an indicator may be used. By using the selection marker in this way, it is possible to select only cells into which the vector has been introduced, that is, cells into which the vector of the present invention has been introduced.
- a vector increases the retention time in the cell and, depending on the vector, induces chromosomal integration like a retroviral vector. It is possible to supply a stable stem-loop RNA molecule in the cell from the DNA of the invention.
- the “target gene” is, for example, a secretory protein
- the “factor controlling the expression of the target gene” refers to, for example, a protein
- the present invention provides a method for producing a cell in which the expression of a target gene is suppressed, the step of introducing the DNA of the present invention or a vector containing the DNA into the cell, and a cell into which the vector is introduced.
- a production method including the step of selecting.
- the present invention provides a cell carrying the DNA of the present invention or a vector containing the DNA.
- the cell of the present invention is not particularly limited, and a desired cell whose gene expression is to be suppressed can be used.
- the cells of the present invention include mammalian cells. It is preferable that a long dsRNA such as a plant cell that is difficult to maintain stable expression for a long time is also suitable as a cell into which the DNA of the present invention or a vector containing the DNA is introduced.
- a method for introducing the DNA of the present invention or a vector containing the DNA into the cell can be appropriately selected by those skilled in the art depending on the type of the cell.
- the calcium phosphate method (Virology, Vol. 52, p. 456 (1973)), electroporation method (Nucleic Acids Res., Vol. 15, p. 1311 (1987)) , Lipofexion 0. Cli n. Biochem. Nutr., Vol.7, p.175 (198 9)
- infection introduction method by virus Sci.
- a DNA sequence specific to the DNA of the present invention or a vector containing the DNA is probed.
- the DNA can be selected by a known technique such as hybridization or PCR as a primer.
- the expression by the selection marker is used. The type can be selected as an indicator.
- a cell into which the DNA of the present invention or a vector containing the DNA is introduced becomes a knockdown cell in which the expression of a target gene is suppressed.
- the “knock-down cell” includes a cell in which the expression of the target gene is completely suppressed and a cell in which the expression of the target gene is not completely suppressed but reduced.
- a cell has been created by deleting or modifying the target gene or its regulatory region.
- the present invention is simply performed without modifying the target gene on the chromosome.
- a cell with suppressed expression of the target gene can be produced by a simple method.
- the knockdown cells created in this way can be used as research materials for functional analysis of target genes, and as disease model cells in cells whose expression is suppressed by using disease-causing genes as target genes. It becomes possible.
- target gene knockdown animals, disease model animals, etc. are created. It is also possible to do this.
- the present invention also includes the knockdown cell produced by the present invention.
- the present invention also relates to a composition containing the DNA of the present invention or a vector containing the DNA. Since the expression of a desired target gene can be suppressed according to the present invention, the therapeutic or prophylactic effect of the disease can be expected by suppressing the gene expression that causes the disease according to the present invention.
- a target gene that is expected to have a therapeutic effect due to a change in the expression level a factor that regulates the expression of the target gene in a normal state is identified, and its expression is suppressed by suppressing the expression. It is possible to control the expression level. When the factor suppresses the expression of the target gene, it is possible to enhance the expression of the target gene by suppressing the expression of the factor.
- the factor when the factor enhances the expression of the target gene, the expression of the target gene can be suppressed by suppressing the expression of the factor.
- the factor is a new drug discovery target and at the same time There is a possibility that siRNA molecules that suppress the current situation can be applied to drugs with novel medicinal properties. Furthermore, stem loop RNA molecule expression vectors that suppress the expression of the factor are considered to be applicable to gene therapy.
- the composition is useful as a reagent for examining the function of a desired gene.
- a composition to which an appropriate excipient or the like is added can also be used.
- the present invention relates to a vector that expresses a stem-loop RNA molecule.
- a DNA encoding an RNA having an arbitrary sequence ability and a sequence complementary to the DNA are linked so as to face each other with a spacer region in between, and are functionally connected to the RNA promoter.
- DNA having a specific structure is included.
- the transcript expressed from the DNA is referred to as “stem loop RNA molecule” in the present invention.
- stem-loop RNA molecule By introducing this stem-loop RNA molecule into cells, it is possible to search for functional genes. That is, the DNA of the present invention up to the above or a vector containing the DNA can suppress the expression of a specific target gene.
- any gene For example, a gene with unknown function can be used to suppress a gene with unknown sequence and search for a new functional gene.
- the present invention comprises a step of introducing the stem loop RNA molecule expression vector into a cell, a step of selecting a cell into which the vector has been introduced, and a step of analyzing the phenotype of the selected cell.
- a method for searching for a functional gene and a drug discovery target molecule preferably includes a step of analyzing a functional gene based on a stem-loop RNA sequence in a vector sequence in a cell whose phenotype has been changed by phenotypic analysis.
- vectors that can express stem-loop RNA molecules having different sequences can be collected and constructed as a library. By using the library, it becomes possible to search for functional genes more efficiently.
- the stem-loop RNA molecule expression vector can be introduced into cells as described above.
- the following is the stem loop RNA molecule expression vector or live
- the phenotype of the cell is analyzed. This expression type analysis can be performed, for example, by comparing with a phenotype of a cell into which a stem-loop RNA molecule expression vector has not been introduced as a control. This phenotype includes not only what occurs on the cell surface, but also intracellular changes, for example.
- a cell having a changed phenotype is likely to contain a stem-loop RNA molecule capable of suppressing the expression of some functional gene. Therefore, in order to screen for functional genes, for example, probes and primers are constructed based on the sequence of the DNA encoding the stem loop RNA of the stem loop RNA molecule expression vector contained in this cell. Then, functional genes can be cloned by performing hybridization or PCR using these probes and primers. In addition, functional genes can be searched from a database based on the sequence of DNA encoding stem-loop RNA.
- the present invention also provides a method for confirming the function of a gene identified by the functional gene search method.
- the method includes the step of introducing the above-described DNA or vector of the present invention into a cell or experimental animal, and the step of introducing the above-described synthetic siRNA molecule of the present invention into a cell or experimental animal.
- experimental animals include Inu, Rat, Rooster, Muster, Usagi, Pig, Usushi, Horse, Monkey, Hedge, Goat, and Cat.
- the present inventors also provided a partial fragment polynucleotide of a polynucleotide encoding a protein associated with angiogenesis based on hypoxic stimulation.
- the sequences of the above polynucleotides of the present invention are shown in SEQ ID NOs: 1-224.
- sequences complementary to the sequences described in SEQ ID NOs: 1 to 224 are also included in the polynucleotide of the present invention.
- the polynucleotide provided by the present inventors is a partial sequence of a gene related to angiogenesis based on hypoxic stimulation.
- SEQ ID NOs: 1-224 Based on the sequence information of the polynucleotide described above, it is possible to easily isolate a full-length cDNA of a gene related to angiogenesis based on hypoxic stimulation. That is, for example, a cDNA library prepared with various cell strengths using the sequence described in any of SEQ ID NOs: 1 to 224 as a probe is screened by hybridization.
- any of SEQ ID NOs: 1 to 224 as a primer, and using various cDNA libraries and other DNA as saddles to obtain amplification products of a size specific to the primer
- the full length of the cDNA can be obtained by a method of screening a library as an index.
- using the sequence of SEQ ID NO: 1 to 224 as a primer convert mRNA prepared for various cellular forces into single-stranded cDNA, attach an oligomer to the end, and perform PCR.
- RACE It is also possible to obtain a full-length cDNA of a gene related to angiogenesis based on hypoxic stimulation by the method (Frohman, MA et al: Proc. Natl. Acad. Sci.
- the polynucleotide of the present invention is used as a probe for hybridization, usually, a labeled one is used.
- the labeling include labeling with nick translation using DNA polymerase I, end labeling with polynucleotide kinase, and fill-in end labeling with Klenow fragment (Berger SL, Kimmel AR. (1987) Guide to Molecular Cloning Techniques). , Method in Enzymology, Academic Press; Hames BD, Higgins SJ (19 85) Genes Probes: A Practical Approach.IRL Press; Sambrook J, Frits ch EF, Mania tis T.
- the present invention provides a polynucleotide encoding a protein associated with angiogenesis based on hypoxic stimulation comprising the nucleotide sequence set forth in any of SEQ ID NOs: 1-224.
- the polynucleotide includes a full-length cDNA of a gene associated with angiogenesis based on hypoxic stimulation, which can be isolated based on the sequence information set forth in any of SEQ ID NOs: 1-224.
- the polynucleotide of the present invention includes a polynucleotide comprising a continuous 19 bp or more base sequence region arbitrarily selected from the base sequences set forth in any of SEQ ID NOs: 1 to 224.
- the present invention includes siRNA that cleaves the transcript of the polynucleotide of the present invention.
- the present invention includes a polynucleotide that specifically hybridizes under stringent conditions with the polynucleotide comprising the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 224.
- Stringent hybridization conditions are usually about “lxSSC, 0.1% SDS, 37 ° C”, and more severe conditions are about “0.5xSSC, 0.1% SDS, 42 ° C”. More severe conditions are about 0.2xSSC, 0.1% SDS, 65 ° C.
- the polynucleotide of the present invention is an antisense polynucleotide (antisense DNA / RNA; for example, encoding the protein of the present invention) for suppressing the expression of a protein associated with angiogenesis based on the above hypoxic stimulation.
- antisense DNA / RNA for example, encoding the protein of the present invention
- the antisense polynucleotide used in the present invention may suppress the expression of the target gene by any of the actions described above.
- an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of the gene is designed, it will be effective for inhibiting translation of the gene.
- sequences complementary to the coding region or the 3 'untranslated region can also be used.
- a polynucleotide containing an antisense sequence of not only a translation region of a gene but also an untranslated region is also included in the antisense polynucleotide used in the present invention.
- the antisense polynucleotide to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
- the sequence of the antisense polynucleotide is preferably a sequence complementary to the target gene or a part thereof, but may not be completely complementary as long as the expression of the gene can be effectively inhibited.
- the transcribed RNA preferably has a complementarity of 90% or more, and most preferably 95% or more, to the transcript of the target gene.
- the antisense polynucleotide for example, a polynucleotide (e.g., SEQ ID NO: 1 based on the nucleotide sequence of the gene encoding the proteins isolated) of the present invention sequence information [This Hosuhoroteone 1 ⁇ bets (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 16, 3209-21 (1988)).
- Suppression of endogenous gene expression can also be carried out using a polynucleotide encoding a ribozyme.
- ribozymes have various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA.
- Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, (1990) Protein Nucleic Acid Enzymes, 35: 2191).
- the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to base-pair with A at position 9 for activity.
- the ribozyme substrate binding site is designed to be complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA.
- the ribozyme constructed in this manner is considered to cleave a transcript of a polynucleotide encoding a protein related to angiogenesis based on hypoxic stimulation of the present invention. These ribozymes are also included in the present invention.
- the present invention also provides a protein encoded by the polynucleotide of the present invention.
- the protein is a protein associated with angiogenesis based on hypoxic stimulation.
- the protein of the present invention can be produced by any appropriate method. Such proteins include isolated naturally occurring proteins, recombinantly produced proteins, synthetically produced proteins, or proteins produced by a combination of these methods. It is. Means for the production of such proteins are well understood in the art.
- a recombinant protein can be prepared by, for example, introducing a vector into which the polynucleotide of the present invention has been inserted into an appropriate host cell and purifying the protein expressed in the transformant.
- Naturally-derived proteins can be prepared, for example, using a affinity column to which an antibody against the protein is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publisn. Joh n Wiley & Sons Section 16.1-16.19).
- Preparation of an antibody for any protein can be performed by methods well known to those skilled in the art.
- the antibody used for the purification of the facility may be a polyclonal antibody or a monoclonal antibody. It is also possible to prepare the protein of the present invention by in vitro translation or the like.
- the present invention also provides a vector containing the polynucleotide of the present invention, the polynucleotide of the present invention or a host cell carrying the vector, and a method for producing the polypeptide of the present invention using the host cell. .
- the present invention provides a method for producing the protein of the present invention, comprising the steps of culturing the host cell and recovering the host cell or the protein produced by the culture supernatant.
- the vector of the present invention is not particularly limited as long as it stably retains the inserted DNA.
- Escherichia coli is used as a host
- the p Bluescript vector (Stratagene) is used as a cloning vector.
- An expression vector is particularly useful when a vector is used for the purpose of producing the polypeptide of the present invention.
- the expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E. coli, in cultured cells, or in an individual organism.
- pB EST vector manufactured by Promega
- PET vector Invitrogen
- PME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells
- PME18S vector (Mol Cell Biol. 8: 466-472 (1988) for organisms) Etc.) are preferred. Insertion of DNA of the present invention to solid terpolymer in the usual way, for example, restriction enzyme sites ligase using Han, monkey with a line retaining clips and mosquito 3 ⁇ 4 by (Current protocols in Molecular Biology edit. Ausub el et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
- the host cell into which the vector of the present invention is introduced is not particularly limited. Various host cells can be used depending on the purpose. Examples of the cells for expressing the polypeptide include bacterial cells (eg, Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2 Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells.
- bacterial cells eg, Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis
- fungal cells eg, yeast, Aspergillus
- insect cells eg, Drosophila S2 Spodoptera SF9
- animal cells eg, CHO, COS,
- Vector introduction into host cells can be performed by, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipophectamine method ( GIBCO-BRL) and a known method such as microinjection can be used.
- an appropriate secretion signal can be incorporated into the protein of interest. These signals may be endogenous to the protein of interest, or they may be heterologous signals.
- the protein of the present invention can be obtained by recovering a protein that has also produced the above-mentioned host cell or its culture supernatant.
- the protein of the present invention is recovered when the polypeptide of the present invention is secreted into the medium.
- the protein of the present invention is produced in a cell, the cell is first lysed, and then the protein is recovered.
- the ability of recombinant cell culture to recover and purify the protein of the invention also includes ammonium sulfate or ethanol precipitation, acid extraction, ion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction.
- Known methods including action chromatography, affinity chromatography, hydroxynorepatite chromatography, and lectin chromatography can be used.
- the pHRE-Luc force et al. Luciferase gene was excised from the Hindlll-Xbal site, and the EGFP gene excised from the Hindlll-Xbal site from pEGFP-Nl vect or (clontech) was incorporated into that part to construct the pHR E-Nl vector. .
- the CD4 ⁇ gene of pMACS4.1 was excised from the EcoRI-Hindlll site and incorporated into the EcoRI-Hindlll site of the pcDNA3.1 (-) vector (Invitrogen) to construct a pcDNA3.1-CD4 A vector.
- the pcDNA3.1-CD4 A vector was excised from the Nurl-Pvul site, subjected to limited digestion with PvuII, CMV promoter, EGFP-N1 and BGH pol y cut out region containing the (A), was constructed by inserting pHRE-Nl-CMV-CD4 A ve c tor the pHRE- Nl vector (see Figure 1).
- pHRE-Nl-CMV-CD4 A vector and siRNA expression vector library were introduced into HEK293 cells by electroporation. After 48 hours, the cells were collected and stained with anti-human CD4 (Fujisawa Pharmaceutical). About 10 5 positive cells in which EGFP expression was increased by FACSCalibur (BECTON DICKINSON) were collected.
- Plasmid was recovered from the recovered cells using QIAamp DNA / Blood Kit (QIAGEN).
- Hairpin was amplified by PCR from the recovered plasmid.
- the primers are M13-47 (5, -CGCC AGGGTTTTCCCAGTCACGAC-3, Z SEQ ID NO: 225) and M13-RV (5,-agcggataacaat ttcacacagg-3, Z SEQ ID NO: 226), and the enzyme is 9 ° Nm DN A Polymerase ( NEB).
- the PCR product is treated with EcoRI-Sphl, the hairpin fragment is excised by 3.5% PAGE, and gelled with Elution buffer (0.5 M AcONH, 10 mM AcOMg, 1 mM EDTA, 0.1% SDS).
- the purified hairpin was ligated using ptRNA / SS treated with EcoRI-Sphl and Rapid DNA Ligation Kit (Roche).
- E. coli is LB medium (+ Amp) (No. 2 square petri dish) Two plates were plated and cultured at 37 ° C. The next morning, a portion was stored as a glycerol stock, and the remainder was collected using the HiSpeed Plasmid Midi Kit (QIAGEN).
- a diluted glycerol stock was plated, and cultured in LB medium (Amp) at 37 ° C. The colonies were selected and cultured, and then the plasmid was recovered using multiscreen FB (MILLIPORE).
- MILLIPORE multiscreen FB
- the loop was first cut by BamHI (NEB). DYEnamic ET Terminator Cycle sequencing kit (Amersham Bioscience) was used. Ml 3-47 and M13-RV were used as primers, respectively, and the sense and antisense sequences were confirmed. The obtained sequences were searched for genes from a standard database using the NCBI BLAST program.
- evaluation vector including HRE
- cDNA siRNA library Co-transfection of evaluation vector (including HRE) and cDNA siRNA library. Therefore, it is necessary to fuse a reporter gene to HRE into the evaluation vector and to incorporate a gene that is constantly expressed to confirm whether or not a plasmid has been introduced into the cell. Therefore, by using EGFP-N1 as the HRE reporter gene and using a CDV ⁇ incorporated into the CMV promoter to check whether the plasmid was introduced into the cell, the evaluation vector (pHRE-Nl-CMV -CD4 A vector) (see Fig. 1).
- siRNA expression vector library and pHRE-Nl-CMV-CD4 A vector were co-transfected by electroporation in HEK293 cells, and the expression of EGFP increased compared to the control! It was collected by.
- the plasmid was recovered from the recovered cells, and the hairpin fragment was amplified by PCR and cloned.
- the cloned plasmid was reintroduced into the cultured cells and EGFP expression was increased. Cells were collected by sorting. Hirpin was amplified by PCR and cloned.
- VHL von-Hippel Lindau syndrome
- the present invention provides a stem-loop RNA molecule expression vector library that enables comprehensive gene function searches, and a comprehensive drug discovery target molecule search method using the library, Furthermore, by this method, it has become possible to identify drug discovery target molecules that are expected to be highly effective by suppressing their expression. Furthermore, regarding the target gene that is expected to have a therapeutic effect due to changes in the expression level, the factor that regulates the expression of the target gene under normal conditions is identified, and the expression level of the gene is suppressed by suppressing its expression. The possibility of being able to control was shown. When the factor suppresses the expression of the target gene, the expression of the target gene can be enhanced by suppressing the expression of the factor.
- the factor when the factor enhances the expression of the target gene, the expression of the target gene can be suppressed by suppressing the expression of the factor.
- Specific factors were identified that indirectly increased the expression of genes that enhance angiogenesis downstream of HRE by suppressing the expression.
- the factor is a novel drug discovery target, and at the same time, siRNA molecules that suppress the expression of the factor may be applicable to pharmaceuticals having a novel medicinal effect.
- a stem-loop RNA molecule expression vector that suppresses the expression of the factor is considered to be applicable to gene therapy.
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WO2007149033A1 (en) | 2006-06-22 | 2007-12-27 | Astrazeneca Ab | Substituted isoindoles as bace inhibitors and their use |
WO2014098637A1 (en) | 2012-12-21 | 2014-06-26 | Instituto Superior Técnico | Reactive aqueous emulsions for composite coatngs |
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WO2003020931A2 (en) * | 2001-09-01 | 2003-03-13 | Galapagos Genomics N.V. | Sirna knockout assay method and constructs |
WO2004009796A2 (en) * | 2002-07-24 | 2004-01-29 | Immusol Incorporated | Single promoter system for making sirna expression cassettes and expression libraries using a polymerase primer hairpin linker |
WO2005056750A2 (en) * | 2003-12-11 | 2005-06-23 | Quark Biotech, Inc. | Inversion-duplication of nucleic acids and libraries prepared thereby |
WO2005059157A2 (en) * | 2003-12-11 | 2005-06-30 | The Board Of Trustees Of The Leland Stanford Junior University | METHODS AND COMPOSITIONS FOR USE IN PREPARING HAIRPIN RNAs |
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WO2003020931A2 (en) * | 2001-09-01 | 2003-03-13 | Galapagos Genomics N.V. | Sirna knockout assay method and constructs |
WO2004009796A2 (en) * | 2002-07-24 | 2004-01-29 | Immusol Incorporated | Single promoter system for making sirna expression cassettes and expression libraries using a polymerase primer hairpin linker |
WO2005056750A2 (en) * | 2003-12-11 | 2005-06-23 | Quark Biotech, Inc. | Inversion-duplication of nucleic acids and libraries prepared thereby |
WO2005059157A2 (en) * | 2003-12-11 | 2005-06-30 | The Board Of Trustees Of The Leland Stanford Junior University | METHODS AND COMPOSITIONS FOR USE IN PREPARING HAIRPIN RNAs |
Non-Patent Citations (1)
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WO2007149033A1 (en) | 2006-06-22 | 2007-12-27 | Astrazeneca Ab | Substituted isoindoles as bace inhibitors and their use |
WO2014098637A1 (en) | 2012-12-21 | 2014-06-26 | Instituto Superior Técnico | Reactive aqueous emulsions for composite coatngs |
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