WO2005056792A1 - Long double-stranded rna for interference with lowered interferon response - Google Patents

Abstract

A double-stranded RNA for interference comprising an antisense RNA being complementary to a sequence in the neighborhood region of a target gene or a sequence in any of regions in mRNA of the target gene and a sense RNA homologous with the above sequence which is hybridizable with the above-described antisense RNA in a cell, wherein the above-described antisense RNA and the above-described sense RNA form double-strands and the double-stranded moiety has a length consisting of 50 or more bases. Use of this long double-stranded RNA for interference makes it possible to highly efficiently induce RNAi with a lowered cytotoxicity without resort to attempts to determine the part to be used as a sequence complementary to the antisense RNA. By introducing a base substitution or bulge into such a long double-stranded RNA for interference, the effects of the RNAi can be further enhanced and interferon response can be lowered.

Description

 Specification

 Long, double-stranded RNA for interference with reduced interferon response

 Technical field

 The present invention relates to a double-stranded RNA for long-term interference, a vector for expressing the double-stranded RNA for long-term interference, a method for producing knockdown cells using the long-stranded double-stranded RNA for interference, and the like. . More specifically, the double-stranded portion of the double-stranded RNA for interference has a length of 50 bases or more, a vector for expressing the double-stranded RNA for interference in cells, and a vector containing the vector. The present invention relates to an RNA expression system, a cell carrying the same, and the like.

 Background art

 [0002] RNA interference (hereinafter abbreviated as "RNAi") is a double-stranded RNA comprising sense RNA having homologous sequence power to target gene mRNA and antisense RNA having a sequence complementary thereto. (Hereinafter, abbreviated as "dsRNA") 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 cells or the like. Since RNAi can suppress the expression of target genes in this way, it can be used as a simple gene knockout method instead of the conventional complicated and inefficient method for gene disruption by homologous recombination, or as an application to gene therapy. Attracting attention. Such RNAi was first discovered in C. elegans (Non-Patent Document 1), but is now observed not only in C. elegans but also in various organisms such as plants, nematodes, Drosophila and protozoa. (Non-patent document 2, Non-patent document 3, Non-patent document 4, Non-patent document 5). In these organisms, it has been actually confirmed that introduction of foreign dsRNA suppresses the expression of the target gene, and is being used as a method for creating knockout individuals.

 [0003] In mammalian cells, as in other organisms, induction of RNAi has been attempted by introducing extracellular dsRNA. Then, the introduced dsRNA activated the host cell's defense mechanism against virus infection, etc., thereby inhibiting protein synthesis and observing RNAi.

[0004] Recently, Tuschl et al. Replaced long dsRNAs as used in other organisms with 21- or 3-nucleotide single-stranded 3, 21-length overhangs having overhangs. Is introduced into mammalian cells by introducing a short interfering RNA (short interfering RNA, hereinafter abbreviated as “siRNA”) of 22 nucleotides (including counting overhangs) into mammalian cells. Has been reported (Non-Patent Document 6, Non-Patent Document 7).

 Thus, it has been found that siRNA can be used as a powerful tool for functional analysis and expression control of genes beyond ribozymes and antisense oligonucleotides, and improvements are being made day and night. In particular, as the primary sequence of genes has been almost determined in many species including humans, the use of siRNA as the most powerful tool for comprehensive and efficient analysis of gene functions in living organisms has attracted attention. I have.

Non-Special Publication 1: Fire, A. et ai. Potent and specific genetic interference by

double-stranded RNA in Caenorhabditis elegans.Nature 391, 806-811, (1998) Non-Patent Document 2: Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358-363 (1999)

Non-Patent Document 3: Sharp, PA RNA interference 2001.Genes Dev. 15, 485-490 (2001) Patent Document 4: Hammond, SM, Caudy, AA & Hannon, GJ Post—transcriptional gene silencing by double-stranded RNA.Nature Rev. Genet. 2, 110-1119 (2001) Non-Patent Document 5: Zamore, PD RNA interference: listening to the sound of silence.Nat Struct Biol. 8, 746-750 (2001)

Non-Patent Document 6: Elbashir, S. M et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 411, 494-498 (2001)

Non-Patent Document 7: Caplen, N.J. et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.Proc Natl Acad Sci U S A 98, 9742-9747 (2001)

Non-Patent Document 8: Ohkawa, J. & Taira, K. Control of the lunctional activity of an antisense RNA by a tetracycline— responsive derivative of the human U6 snRNA promoter.Hum Gene Ther. 11, 577—585 (2000)

Non-Patent Document 9: Zanta MA et al., Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus.Proc Natl AcadSci US A. 1999 Jan 5; 96 (1): 91-6)

 Non-Patent Document 10: Liu F, Huang L. Improving plasmid DNA-mediated liver gene transfer by prolonging its retention in the hepatic vasculature. J. Gene Med. 2001 Nov— Dec; 3 (6): 569— 76

 Disclosure of the invention

 Problems to be solved by the invention

 [0006] By using RNAi to suppress the expression of any gene at the mRNA level, the function of that gene can be analyzed. In addition, as an application to the medical field, disease In addition to elucidating the causative gene, it may be possible to suppress the growth of RNA viruses and the expression of disease-related genes.

 [0007] In chemical synthesis of RNA, the larger the number of bases is, the more expensive it is compared to the DNA synthesis, and the like. Therefore, in mammalian cells, the number of bases in the double-stranded RNA portion performed by Tuschl et al. An siRNA with a size of 191-21 (2 to 23 bases in the count including the overhang, because the overhang of 2 bases is included on the 3rd side of the antisense RNA and sense RNA) is used. However, with these base numbers, the effect of suppressing gene expression differs depending on the target sequence. In other words, the expression suppression effect differs depending on which site of the target gene whose expression is to be suppressed is complementary to the antisense RNA, and gene expression can be effectively suppressed depending on the target site. It may be hardly suppressed depending on the force part. For this reason, an effective target site must be considered in advance for practical application.

[0008] On the other hand, it has been said that RNAi is unlikely to occur in mammalian cells when RNA having a long base number (30 bp or more) is used. One of the causes is thought to be the activation of two pathways called the interferon response. Interferon response refers to a phenomenon in which when extra-cellular force is introduced into a long dsRNA of 30 bp or more, cells produce interferon as in the case of virus infection, and the translation of the entire gene is inhibited. Interferon enhances transcription of the gene encoding dsRNA-dependent protein kinase (PKR) and promotes PKR autophosphorylation and kinase activity. The activated PKR phosphorylates elF-2α, thereby inhibiting the translation of the entire gene. Due to such an interferon response, mammalian cells into which long dsRNA has been introduced have a problem that the specificity of the RNA interference effect is reduced and apoptosis is also induced, thereby increasing cytotoxicity.

[0009] Thus, in applying RNAi to gene function analysis and gene therapy, it is desired to develop siRNA that effectively suppresses gene expression regardless of the target site, but has low cytotoxicity. Therefore, the present invention relates to a vector for expressing such double-stranded RNA for interference and siRNA, and a method for producing a knockdown cell of a functional gene. Means for solving the problem

 [0010] The inventors of the present application have conducted intensive studies in view of the above problems, and as a result, the target specificity is sufficiently high! We have developed dsRNA which has a long number of bases but does not easily cause interferon response. In other words, they found that RNAi with reduced interferon response can be induced by using a ροΙΠΙ-based promoter to generate intracellular dsRNA with a double-stranded portion of 50 bases or more. Furthermore, by introducing a bulge II mutation into a dsRNA having a double-stranded portion of 50 bases or more, RNAi activity is increased and interferon response is significantly reduced as compared to those without a bulge II mutation. I was strong.

 [0011] Furthermore, the present inventors have found that long dsRNA having a double-stranded portion of 50 bases or more induces RNAi not only at the post-transcriptional level but also at the transcriptional level. Specifically, it has been found that dsRNA is induced not only in the mRNA of the target gene but also in the peripheral portion of the target gene (eg, promoter portion and transcription regulatory region), thereby inducing DNA methylation to inhibit transcription.

 That is, the present invention provides an antisense RNA complementary to a sequence in a region around a target gene or a sequence in a region in an mRNA of a target gene, and the sequence hybridizing intracellularly to the antisense RNA. A double-stranded interference RNA comprising a sense RNA homologous to the antisense RNA and the sense RNA, wherein the antisense RNA and the sense RNA form a double strand, and the length of the double strand forming portion is 50 bases or more; For example, an RNA for double-stranded interference characterized by having 50 to 300 bases, preferably 100 bases is provided.

Examples of the sequence of the peripheral region of the target gene include, for example, a promoter portion and a transcription regulatory region. Transcription control regions include transcription regulators including enhancers and repressors. The nodal region and its neighboring region can be exemplified.

 [0013] Such a double-stranded RNA for interference is obtained by substituting one or more bases with another base in the antisense RNA or the sense RNA in the double-stranded region, Preferably, one or more bases in the sense RNA are replaced with other bases. If one or more bases are replaced in the sense RNA, cytosine (C) is replaced with peracyl (U), adenine (A) is replaced with guanine (G), or adenine (A) is inosine. Substitution to (I) is preferred. Such base substitutions can be guided by deamination.

 [0014] The double-stranded RNA for interference of the present application may contain one or more bulges due to insertion or deletion of a base in the antisense RNA or the sense RNA in the double-stranded region. The position of the bulge, for example, is preferably contained in positions other than the 20th to 23rd bases from the end of the double strand, or between the 119th base from the end of the double strand. It is preferable that the first bulge is included, and the next bulge is included for each of the 125th bases counted from the position of the bulge. Such bulges are preferably introduced into sense RNA.

 The double-stranded RNA for interference of the present application may further include a loop between the antisense RNA and the sense RNA.

[0015] The present invention also relates to a vector for expressing a double-stranded interfering RNA in a cell, comprising a sequence of a peripheral region including a promoter of a target gene or a region of any region in mRNA of a target gene. An antisense code DNA encoding an antisense RNA complementary to the sequence; a sense code DNA encoding a sense RNA homologous to the sequence hybridizing to the antisense RNA intracellularly; the antisense code DNA; A vector comprising one or more promoters for expressing the antisense RNA and the sense RNA from the sense code DNA, respectively, wherein the antisense RNA and the sense RNA have a double-stranded structure. And the length of the double-chain forming portion is 50 bases or more, for example, 50-300 bases, preferably 50-100 bases. To provide that vector. Examples of the sequence of the peripheral region of the target gene include a promoter portion and a transcription regulatory region. [0016] The vector of the present application can be used even if one or more bases in the antisense code DNA or the sense code DNA in the region of the DNA encoding the RNA forming the double strand are replaced with other bases. More preferably, one or more bases in the sense code DNA are replaced by other bases. When one or more bases are replaced in the sense code DNA, the substitution of cytosine (C) with thymine (T) or the substitution of adenine (A) with guanine (G) is not possible. preferable. Such substitution of the base can be introduced by deamination.

 [0017] The vector of the present application may generate an interference double-stranded RNA containing one or more bulges due to insertion or deletion of a base in an antisense RNA or a sense RNA in a double-stranded region. Good. The position of the bulge, for example, is preferably contained in positions other than the 20th to 23rd bases from the end of the double strand, or the first bulge is located between the 119th base from the end of the double strand. It is preferable that the following bulges are contained for each of the fifth to fifth bases counted from the position force of the bulge. Such bulges are preferably introduced into sense RNA!

 The vector of the present application may generate a double stranded RNA for interference further comprising a loop between the antisense RNA and the sense RNA.

 As the promoter contained in the vector of the present application, a ροΙΠΙ promoter can be used, for example, a pοΙΠΙ system selected from the group consisting of U6 promoter, tRNA promoter, HI promoter, 7SK promoter, 7SL promoter and 5SrRNA promoter. A promoter can be used.

Further, the present application relates to an expression system for expressing RNA for double-stranded interference in a cell, wherein the sequence of a peripheral region including a promoter of a target gene or the sequence of any region in mRNA of a target gene is present. A first vector comprising an antisense coding DNA encoding an antisense RNA complementary to the antisense RNA, a first promoter for expressing the antisense RNA from the antisense coding DNA, and the antisense RNA. A second vector comprising a sense code DNA encoding a sense RNA homologous to the sequence that hybridizes in a cell, and a second promoter for expressing the sense RNA from the sense code DNA. , With the antisense RNA The sense RNA forms a duplex, and the length of the duplex forming portion is 50 bases or more, for example, 50 to 300 bases, preferably 50 to 100 bases. I do. Examples of the sequence of the peripheral region of the target gene include a promoter portion and a transcription regulatory region.

 [0019] The expression system of the present application generates an interference double-stranded RNA in which one or more bases are replaced by another base in the antisense RNA or the sense RNA in the double-stranded region. Is also good.

 The expression system of the present application may be modified such that one or more bases in the antisense code DNA or the sense code DNA in the region of the DNA encoding the RNA form a double strand and are replaced with another base. More preferably, one or more bases in the sense code DNA are replaced by other bases. In the case where one or more bases are substituted in the sense code DNA, substitution of cytosine (C) with thymine (T) or substitution of adenine (A) with guanine (G) is preferable. . Such substitution of the base can be introduced by deamination.

 [0020] The expression system of the present application generates an interference double-stranded RNA containing one or more bulges due to insertion or deletion of a base in an antisense RNA or a sense RNA in a double-stranded region. It may be something. The position of the bulge, for example, is desirably included in positions other than the positions 20 and 23 from the end of the double strand, It is preferable that each of the 125th bases counted from the position of the bulge contains the following residues. Such bulges are preferably introduced into sense RNA.

 As the first and second promoters included in the expression system of the present application, ροΙΠΙ promoters can be used, and include, for example, a U6 promoter, a tRNA promoter, a HI promoter, a 7SK promoter, a 7SL promoter, and a 5SrRNA promoter. ΡοΙΠΙ promoters can also be used, for which group strength is also selected.

 Further, the present invention provides a cell holding the above-mentioned vector or the above-mentioned expression system. Plant cells and animal cells are preferred as such cells.

The present invention also provides a composition comprising the above-mentioned vector or the above-mentioned expression system. This As such a composition, a pharmaceutical composition is preferred.

 Further, the present invention provides a method for producing a cell in which the expression of a target gene is suppressed, comprising a step of introducing the vector or the expression system into a cell, and a step of selecting a cell into which the vector or the expression system has been introduced. I will provide a.

[0022] The present invention further provides an antisense RNA complementary to a sequence in a region around the target gene or an mRNA in the target gene, which is complementary to a sequence in any region, and an antisense RNA of the target gene in a cell. A double-stranded RNA for interference comprising a sense RNA homologous to the region to be hybridized and having a length of at least 50 bases in a double-stranded portion formed by the antisense RNA and the sense RNA; There is provided a method for suppressing the expression of a target gene, which comprises the step of introducing the above vector into a cell.

 The invention's effect

 In [0023] the present invention, as described above, by using a long dsRNA, without considering whether to advance any part position sequence complementary to the antisense RNA and, and low!, RNAi cytotoxic Can be brought.

 In addition, since the vector or the expression system of the present invention can synthesize siRNA in a cell without using chemical synthesis, the expression suppressing effect is shorter in advance as in the case of using siRNA. In addition, low-toxicity siRNA can be produced at low cost without specifying the site. Using such a vector or expression system of the present invention, gene therapy can be performed by analyzing the function of a target gene, suppressing the expression of a virus, suppressing the expression of a disease-causing gene, and the like. Furthermore, using the vector or system of the present invention, a knockdown cell, a knockdown animal or a plant which can be used as a research material for functional analysis of a target gene can be produced. When the siRNA of the present invention is introduced into plant cells, it can be used not only for functional analysis but also for breeding. In particular, there has been no case in which the expression of plant cells has been suppressed by introducing mutations so far, and it can be a powerful means as a more efficient method of suppressing the expression of plant cells.

 Brief Description of Drawings

FIG. 1 is a diagram showing a vector that expresses a long dsRNA under the control of a U6 promoter and dsRNA generated from the vector. FIG. 2 is a graph comparing the suppression of expression by long dsRNA expressed by the U6 promoter.

 [FIG. 3] FIG. 3 shows the results of a Western blot comparing interferon responses in HeLa S3 cells with long dsRNA expressed by the U6 promoter.

 [FIG. 4] FIG. 4 shows the results of a Western blot comparing interferon responses in HeLa S3 cells by long dsRNA expressed by a tRNA promoter.

 FIG. 5 shows long dsRN expressed by U6 promoter having various bulge structures.

6 is a graph comparing expression suppression by A.

 FIG. 6 is a graph showing a comparison between a 21 bp or 20 bp siRNA and a 50 bp siRNA in suppressing hepatitis C virus.

 FIG. 7 is a graph showing the effect of E-cadherin siRNA targeting the E-cadherin promoter on E-cadherin mRNA expression.

 [Fig. 8] Fig. 8 is a view showing the effect of substitution of a DNA sequence from C to T by the deamination method.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0025] The first aspect of the present invention relates to a double-stranded RNA for interference that does not have a limited target sequence and has low cytotoxicity. The double-stranded RNA for interference comprises an antisense RNA complementary to a sequence of a region around the target gene or a sequence of any region in the mRNA of the target gene, and the sequence hybridizing intracellularly to the antisense RNA. A double-stranded interfering RNA comprising a sense RNA homologous to the antisense RNA and the sense RNA, wherein the double-stranded portion has a length of 50 bases or more. For example, 50 to 300 bases, preferably 50 to 100 bases. Examples of the peripheral region of the target gene include a promoter region and a transcription regulatory region.

[0026] Further, in the interference double-stranded RNA, one or more bases are replaced with other bases in the antisense RNA or the sense RNA in the double-stranded region, and the target sequence It may contain a base that is mismatched with the base. This substitution may be introduced into the antisense RNA or the sense RNA, or may be introduced into both the antisense RNA and the sense RNA, but it is preferable that the substitution be introduced into the sense RNA. Also, If a substitution has been introduced into the sense RNA, the cytosine (C) force can be replaced by uracil (U), the adenine (A) can be replaced by guanine (G), or the adenine (A) force can be reduced to inosine (I). Replacement is particularly preferred. This base substitution can be introduced by deamination.

 Further, the double-stranded RNA for interference of the present invention may contain one or more bulges in the double-stranded strand. Here, the bulge refers to a double-stranded RNA formed by antisense RNA and sense RNA, in which one or more corresponding bases are not present in one strand. A bulge can be formed by deleting one or more bases from one RNA strand of a double-stranded RNA, or inserting one or more bases. A bulge can be formed in either or both antisense RNA and sense RNA that form a duplex. Preferably, a bulge is formed in the sense RNA.

 It is presumed that the double-stranded RNA for interference containing a bulge of the present invention is processed in a cell to have an siRNA-like structure and a single-stranded microRNA (miRNA) -like structure.

 [0027] It is desirable to introduce several bulges into the double-stranded portion, which is presumed to be a cleavage site at the time of siRNA generation, avoiding the vicinity of the 20th to 23rd bases. For example, the bulge is introduced by counting the first bulge at the terminal strength of the dsRNA double-stranded portion, introducing it between the 119th base, and introducing the next bulge every 125 bases. As high as or less than that without, the cytotoxic effect can be reduced while maintaining the expression suppressing effect.

 Further, a loop (linker) may be included between the antisense RNA and the sense RNA. This loop may be included between the 3 'end of the antisense RNA and the 5' end of the sense RNA, or may be included between the 3 'end of the sense RNA and the 5' end of the antisense RNA. You can also. As long as the sequence of the loop does not inhibit dsRNA formation, the number of bases of the loop in any sequence is not particularly limited. For example, a loop of about 420 bases or a loop of several hundred bases can be used as long as it does not inhibit dsRNA formation!

[0028] Further, the double-stranded RNA for interference of the present application is not only a double-stranded portion formed between the antisense RNA and the sense RNA, but also on the 3 'side of the antisense RNA and the sense RNA. It may have an overhang of 1-4 bases, preferably 2-3 bases. Overhangs are single-stranded overhangs that do not form a duplex. Since this overhang has low specificity with the target gene, it is not always necessary to have a sequence complementary to or the same (homologous to) the sequence of the target gene. Further, as long as the effect of suppressing the expression of the target gene by the siRNA can be maintained, for example, a low molecular weight RNA may be provided at a protruding portion at one end. Such small RNAs include, for example, natural RNA molecules such as tRNA, rRNA or viral RNA, and artificial RNA molecules.

[0029] As another aspect of the present invention, there is provided a vector for expressing a double-stranded RNA for interference, which has no limited target sequence and low cytotoxicity. This vector contains an antisense-encoding DNA encoding an antisense RNA that is complementary to the sequence of the peripheral region including the promoter of the target gene or the sequence of any region in the mRNA of the target gene, and hybridizes intracellularly with the antisense RNA. A sense code DNA encoding a sense RNA homologous to the sequence, and one or more promoters for expressing the antisense RNA and the sense RNA from the antisense code DNA and the sense code DNA, respectively. Wherein the antisense RNA and the sense RNA form a double strand, and the length of the double-stranded portion is 50 bases or more, for example, 50 to 300 bases, preferably 50 bases. — 100 bases.

[0030] In this vector, the antisense-encoding DNA and the sense-encoding DNA can be expressed under the control of separate promoters. As a result, the antisense RNA and the sense RNA are formed as separate RNA strands. Then form a duplex between complementary base pairs. On the other hand, the antisense code DNA and the sense code DNA may be expressed under the control of the same promoter. As a result, the antisense RNA and the sense RNA are formed as the same RNA strand. In this case, a loop sequence can be included in the vector so that a loop (linker) is included between the antisense RNA and the sense RNA. A loop may be included between the 3 'end of the antisense RNA and the 5' end of the sense RNA, as described above, or may be included between the 3 'end of the sense RNA and the 5' end of the antisense sense RNA. Talk about things. [0031] As yet another aspect of the present invention, there is provided an expression system for expressing a double-stranded RNA for interference without limiting the target sequence and having low cytotoxicity. This expression system is an expression system for expressing RNA for double-stranded interference in cells, and is complementary to the sequence of the peripheral region including the promoter of the target gene or the sequence of any region in the mRNA of the target gene. A first vector comprising an antisense coding DNA encoding a functional antisense RNA, a first promoter for expressing the antisense RNA from the antisense coding DNA, and a cell comprising the antisense RNA. And a second vector comprising a sense code DNA encoding a sense RNA homologous to the sequence hybridizing within, and a second promoter for expressing the sense RNA from the sense code DNA. The antisense RNA and the sense RNA form a duplex, and the length of the duplex forming portion is 50 bases or more, for example, 50 bases. 300 bases, preferably 50 to 100 bases.

 [0032] As described above, one double-stranded RNA for interference generated from the vector or the expression system of the present application forms one double-stranded antisense RNA or sense RNA within the region. These bases may be replaced by other bases, and may contain bases that are mismatched with bases in the target sequence. The double-stranded RNA for interference generated from the vector or the expression system of the present application contains one or more bulges due to insertion or deletion of a base in a double-stranded portion formed by antisense RNA and sense RNA, Good. These replacements and bulges can be configured as described above.

[0033] The promoter used in the vector and the expression system of the present invention is not particularly limited as long as it can control transcription of a base sequence of 50 base pairs or more. Examples of a promoter capable of controlling the transcription of a base sequence of 50 bases or more include a ροΙΠ promoter or a ροΙΠΙ promoter. Examples of the ροΙΠΙ promoter include the U6 promoter, HI promoter, 5S rRNA promoter, tRNA promoter, 7SL promoter, 7SK promoter, retroviral LTR promoter, and adenovirus VA1 promoter. Among them, U6 promoter, HI promoter and tRNA promoter are preferable. ροΙΠ promoters include Cytomegalo Winores promoter, Τ7 promoter, Τ3 promoter, SP6 promoter, RSV Promoter, EF-1α promoter, β-actin promoter, γ-globulin promoter, SRa promoter and the like.

 [0034] "dsRNA" refers to a range of double-stranded RNAs that do not exhibit toxicity in mammalian cells.

 In the present application, even if the double-stranded portion is a dsRNA of 50 bases or more, it is not necessary to introduce the dsRNA from outside the cell as in the conventional case. It was confirmed that the cytotoxicity decreased. Desirable length of dsRNA is that the length of the double-stranded portion formed between antisense RNA and sense RNA is 50 bases or more, preferably 50 bases to 300 bases, more preferably 50 bases or less. Not less than base and not more than 100 bases. However, even if the double-stranded portion has a length of 301 bases or more, it is not intended to be excluded from the scope of the present invention as long as it does not exhibit cytotoxicity such as an interferon response and an RNA interference effect can be obtained. Not something.

 In the present invention, “antisense RNA” is an RNA comprising a sequence in a peripheral region such as a promoter of a target gene or a sequence complementary to a sequence in any region in mRNA of a target gene. It is thought that this antisense RNA binds to the mRNA of the target gene and causes RNAi at the post-transcriptional level, or induces RNA methylation at the sequence around the target sequence to induce RNAi at the transcriptional level. I have. Antisense RNA does not need to have a nucleotide sequence that is completely complementary to a specific sequence in the peripheral region of the target gene or a specific region in the mRNA of the target gene. A part of the sequence may contain an unmatched part.

[0036] The "sense RNA" is a sequence complementary to the antisense RNA, that is, a base sequence homologous (same) as a specific sequence in the peripheral region of the target gene or a specific region in the mRNA of the target gene. And an RNA that anneals to complementary antisense RNA to generate siRNA. The sense RNA can form a double-stranded RNA with an antisense RNA that does not need to have a completely homologous (same) nucleotide sequence as a specific sequence in the peripheral region of the target gene or a specific region in the mRNA of the target gene. As long as a part of the base sequence may contain an unpaired portion such as bulge substitution. Conventionally, these antisense RNA and sense RNA can be chemically synthesized by an RNA synthesizer. In many cases, in the present invention, not only a chemical synthesis but also a DNA encoding an antisense RNA (antisense code DNA) and a DNA encoding a sense RNA (sense code DNA) in a vector are expressed in cells.

 [0037] The "target gene" is a gene that is suppressed by the expression RNA of the gene, and can be arbitrarily selected. As the target gene, for example, a gene whose sequence is known but whose function is to be clarified, a gene whose expression is considered to be a cause of a disease, and the like can be suitably selected. Further, by targeting a gene encoding a disease-related gene such as an oncogene or a virus protein as a target gene, it can be used as a pharmaceutical composition for gene therapy. If the target gene is known to have at least 15 bases or more that can bind to a part of its mRNA sequence, that is, one strand of siRNA (antisense RNA strand), it is possible to determine the genomic sequence. Genes that are not disclosed can be selected. Therefore, a gene, such as EST (Expressed Sequence Tag), which has a part of mRNA that is known but whose total length is not known, can also be selected as a “target gene”.

 [0038] The vectors of the present invention include a tandem-type interference double-stranded RNA expression vector having a promoter upstream of the antisense code DNA and the sense code DNA, and an antisense DNA and a sense code DNA. A stem-loop type interference double-stranded RNA vector in which units arranged in the opposite direction on the same DNA strand and connected by a linker between these DNAs are connected downstream of one promoter .

 [0039] In the tandem-type interference double-stranded RNA expression vector, the antisense RNA and the sense RNA can be expressed as separate RNA strands and then annealed to obtain double-stranded RNA. The siRNA generated from the stem-loop type siRNA expression vector has a stem-loop structure in which a linker is used as a loop and sense RNA and antisense RNA on both sides are paired (stem structure). Then, the loop portion is cleaved by processing by an intracellular enzyme, and siRNA can be generated. In this case, the length of the stem portion, the length of the linker (loop), the type, and the like can be configured as described above.

[0040] In the expression system of the present invention, antisense code DNA and sense code DNA are separated. Vector. In this case, a promoter is included 5 ′ upstream of each of the antisense coding DNA and the sense code DNA. In this case, the antisense RNA and the sense RNA can be expressed as separate RNA strands and then annealed to obtain double-stranded RNA.

[0041] In order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added at the 3 'end of each strand (antisense RNA coding strand and sense RNA coding strand). It is preferable to provide each. As this terminator, an array in which four or more τ (thymine) bases are continuous can be used.

 [0042] In any case, a sequence capable of promoting the transcription of the promoter may be provided at the 5 'end. Specifically, in the case of the tandem type, transcription of the promoter force is carried out at the 5 ′ end of each of the antisense coding DNA and the sense code DNA, and in the case of the stem loop type, at the 5 ′ end of the above unit. By providing a sequence that can be promoted, the production of double-stranded RNA for interference may be efficiently performed. A transcript from such a sequence may be used in a state where it is added to the double-stranded RNA for interference, provided that it does not hinder the expression of the target gene by the double-stranded RNA for interference. In case of affecting trimming, it is preferable to perform trimming using a trimming means such as ribozyme.

 [0043] Further, the invention of the present application is an interference wherein the length of the double-stranded portion formed from the antisense RNA and the sense RNA is 50 bases or more, preferably 50-300 bases, more preferably 50-100 bases. Cells containing a vector for expressing a double-stranded RNA for use, an individual living organism having the cells, and a method for producing cells in which the target gene expression is suppressed by introducing the vectors into the cells. are doing. Those skilled in the art can appropriately carry out the method for producing a cell holding the vector or the system for expressing the double-stranded RNA for interference of the present invention or the method for producing an individual organism having the cell based on ordinary knowledge in the technical field. It is possible to do. The present invention will be described with reference to the following examples, which are not intended to be limited to the specific methods described in the examples.

 Example

 [Example 1: RNAi by long dsRNA expressed by U6 promoter]

Under the control of the U6 promoter as shown in Fig. 1, a vector that expresses long! Inhibition of luciferase gene expression was confirmed.

HeLa S3 Itoda spores were seeded at a density of 2.5 × 10 ⁴ per well in a 48-well plate. After 24 hours, the plasmid was transfected with 12 ng of Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, luciferase activity was measured using Dual Luciferase Assay Kit (Promega). From left to right, lanes 1 to 3 are negative control, lane 4 is a 50 bp dsRNA expression vector with a mutation, lane 5 is a fully complemented 50 bp dsRNA expression vector, and lane 6 is a 100 bp dsRNA expression with a mutation. The vector, lane 7, is a transfected 100 bp dsRNA expression vector that is completely complementary.

The vectors used as negative controls, cl pU6ic, c2-pU6ic50AS and c3-pU6icl00AS, do not express dsRNA! ヽ A vector containing the U6 promoter and the resulting RNA sequence is is there. The RNA sequence that also produces pU6i50S, a 50 bp dsRNA expression vector with mutations, is GCCUUUAGGAUUAUAAG

UGAAGGCUUUU (SEQ ID NO: 2), the RNA sequence resulting from the fully complementary 50 bp dsRNA expression vector pU6i50m-1 is GCCUUCAGGAUUACAAGAUU

 GGCUUUU (SEQ ID NO: 3), RNA derived from pU6il00Sl, which is a lOObp dsRNA expression vector into which a mutation has been introduced, is GAUUUCGGGUUGUCUUGAUGUAUG.

UAAGACGACUCGAAAUCUUUU (SEQ ID NO: 4), the RNA sequence resulting from pU6il00m, a fully complementary lOObp dsRNA expression vector, is GAUUUCGAGUCGU

FIG. 2 shows the results. Long dsRNA (duplex, Obp, lOObp) transcribed under the U6 promoter suppresses the expression of luciferase in HeLa cells, and those with mutations introduced in the dsRNA are completely targeted. Complementary to the sequence, it was revealed that RNAi activity was increased as compared to dsRNA.

 Example 2: Interferon response in HeLa S3 cells by long dsRNA expressed by U6 promoter

 To determine whether long dsRNA expressed by the U6 promoter induces an interferon response, phosphorylation of PKR and phosphorylation of elF2a were examined by Western blot.

2.1 × 10 ⁵ HeLa S3 cells were seeded per well in a 12-well plate, and after 24 hours, the plasmid was transfected using Lipofectamine 2000 (Invitrogen). 48 hours after the transfection, proteins were detected by Western blot.

 [0048] PKR antibody is PKR (K-17): sc-707, Santa Cruz Biotechnology, Inc., el F2 antibody is eIF2 '(FL-315): sc-11386, Santa Cruz Biotechnology, Inc, phosphorylated PKR The antibody used was Phospho-PKR (Thr451) Antibody, the Cell Signaling Technology, and the phosphorylated eIF2α antibody was Phospho-eIF2a (Ser51) Antibody, Cell Signaling Technology.

 The results are shown in Figure 3. mock was treated with the transfection reagent alone, IFN was treated with 1 000 U / ml of interferon α (1-2396, Interferon, Human, Leukocyte, Sigma Chemical Co.) together with the transfection reagent. A vector containing a U6 promoter that does not express dsRNA was transfected. The resulting RNA sequence is as shown in SEQ ID NO: 1.

[0049] 21—mut is a dsRNA having a mutated double-stranded portion of 21 bp (SEQ ID NO: 6: GUGCG CACUUUU), 50-mut: Mutated 50 bp dsRNA (SEQ ID NO: 2), 50-match: double-stranded portion completely complementary to the target sequence 50 bp dsRNA (SEQ ID NO: 3), 100-mut: Mutated Introduced lOObp dsRNA (SEQ ID NO: 4), 100-match is a lOObp dsRNA completely complementary to the target sequence (SEQ ID NO: 5). The vector to be expressed is transfected.

[0050] long dsRNA transcribed intracellularly by U6 promoter, also those containing mutations in the duplex portion, even one that fully complementary to the target sequence, both phosphorylation is suppressed in PKR and ELF2 _a It was shown that it did not induce an interferon response. It was shown that long dsRNA having a double-stranded portion having a mutation in the double-stranded portion and having a length of 50 bp and 100 bp was particularly effective in suppressing phosphorylation of PKR and elF2a.

 [Example 3: Interferon response in HeLa S3 cells by long dsRNA expressed by tRNA promoter]

 To determine whether long dsRNA expressed by the tRNA promoter induces an interferon response, phosphorylation of PKR and phosphorylation of elF2a were examined by Western blot.

2.1 × 10 ⁵ HeLa S3 cells were seeded per well in a 12-well plate, and after 24 hours, the plasmid was transfected using Lipofectamine 2000 (Invitrogen). 48 hours after the transfection, proteins were detected by Western blot. PKR Antibody Puma PKR (K-17): sc-707, Santa Cruz Biotechnology, Inc., eIF2a Antibody Puma e IF2a (FL-315): sc-11386, Santa Cruz Biotechnology, Inc, Phosphorido The PKR antibody was Phospho-PKR (Thr451) Antibody, Cell Signaling Technology, and the phosphorylated eIF2a antibody was Phospho-eIF2a (Ser51) Antibody, Cell Signaling Technology.

FIG. 4 shows the results. mock is the transfection reagent only, IFN is 1000 U / ml wing ~~ Feron α (1-2396, Interieron, Human, LeuKocyte, Sigma Chemical Co.,) was added together with the transfection reagent, control was dsRNA Express This is a vector containing the tRNA promoter, and the resulting RNA is ACCGUUGG

 (SEQ ID NO: 7).

 50-match is a dsRNA with a double-stranded portion completely complementary to the target of 50 bp (SEQ ID NO: 8:

 CACUUUGAAUCUUGUAAUCCUGAAGGCUUUU), 100—mut is a mutation-introduced double-stranded lOObp dsRNA (ACCGUUGGUUUCCGUAGUGUA

 CUAUACAUUAAGACGACUCGAAAUCUUUU: SEQ ID NO: 9), 100-mate is a dsRNA whose lOObp double-stranded part (ACCGUUGGUUUC

Transfection of vectors expressed by tRNA promoter It was done.

[0053] long dsRNA transcribed intracellularly by tRNA promoter also those containing mutations in the duplex portion, even one that fully complementary to the target sequence, both phosphorylation is suppressed in PKR and ELF2 _a It was shown that it did not induce an interferon response. In particular, it was shown that long dsRNA having a double-stranded portion having a mutation in the double-stranded portion and having a lOObp particularly effectively inhibited PKR induction and PKR phosphorylation.

 [Example 4: RNAi by long dsRNA expressed by U6 promoter having bulge structure]

 The effect of suppressing the expression by the position of bulge was examined. Under the control of the U6 promoter as shown in FIG. 1, suppression of luciferase gene expression was confirmed using a vector expressing a long dsRNA containing a bulge structure.

HeLa S3 Itoda spores were seeded at a density of 2.5 × 10 ⁴ per well in a 48-well plate. After 24 hours, the plasmid was transfected with 12 ng of Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, luciferase activity was measured using the Dual Luciferase Assay Kit (Promega). The plasmid can be either a DNA sequence encoding dsRNA without residue (50S) or a DNA sequence encoding dsRNA containing three types of bulges (50SV1, 50SV2, and 50SV), shown on the left side of Figure 4. Was used. 50SV1 has a bulge inserted near the 10th or 30th base from the 5 'end, which is a position presumed not to interfere with siRNA production (GCCUUUAGGAUU

号 12)、 50SVは、 siRNA生成時の切断個所と推定されている 5'末端から 21から 23 番目塩基付近および 15番目の塩基前後にバルジを入れたもの（GCCUUUAUGG AAUCUUGUAAUCCUGAAGGCUUUU：配列番号 13)である。 UGUAAUCCUGAAGGCUUUU: SEQ ID NO: 11), 50SV2 is a bulge near the 21st to 23rd bases from the 5 'end, which is estimated to be a cleavage site during the generation of siRNA in SV2.

No. 12), 50SV has a bulge inserted around the 21st to 23rd bases and around the 15th base from the 5 'end, which is estimated to be the cleavage site at the time of siRNA generation (GCCUUUAUGG AAUCUUGUAAUCCUGAAGGCUUUU: SEQ ID NO: 13).

 [0055] The results are shown on the right side of FIG. From these results, it was found that, as in 50SV1, several bulges were introduced avoiding the 21st to 23rd bases from the 5 'end of the double-stranded portion presumed to have been cut at the time of siRNA generation. No bulge was introduced by counting the 5'-terminal force of the dsRNA double-stranded portion between the 1st and 20th bases and then introducing the next bulge every 125 bases It has been shown that the cytotoxic effect can be reduced while maintaining the same high expression suppression effect as that of.

 [Example 5: Suppression of hepatitis C virus using 50 bp mhRNA vector (1)]

 Seeger strain, Wakita strain Plasmids pSeeger ~ luc and pWakita-luc, which are plasmids having a gene obtained by fusing a partial sequence of hepatitis C virus (HCV) to a luciferase gene, were constructed. 293T cells were transfected with 50ng of pSeeger-luc or pWakita-luc, 200ng of siRNA expression vector, and 30ng of pRL-RSV using Lipofectamine plus (Invitrogen), and 24 hours later, Bright-Glo Luciferase Assay was performed. Luciferase activity was measured on a System (Promega). As the siRNA expression vector, either one expressing a 51 bp siRNA (U6-51) or one expressing a 21 bp siRNA (U6-21) was used downstream of the U6 promoter. As a negative control, transfection was performed using only the U6 vector (Mock) instead of the siRNA expression vector. The DNA sequence transcribed by the U6 promoter is as follows.

 [0057] 21 bp-A; 5'-GAA TCC CGG CTG CGT CCC AGT TAT ACA AG A GAC TGG GAC GCA GCC GGG ATT TTT-3, (SEQ ID NO: 14)

[0058] 21 bp—B; 5, GCT GCG TCC CAG TTG GAC TTA TTC AAG AG A TAA GTC CAA CTG GGA CGC AGC TTT TT-3, (SEQ ID NO: 15)

[0059] 50 bp; 5, AAA CTT ACT CTA ATC TCG GTT GTG TCC TAG T TG GGC TTA TTC AAG AGA TAA GTC CAA CTG GGA CGC AGC CGG GAT TGG AGT GAG TTT GAG CTT GGT CTT TT

T-3 '(SEQ ID NO: 16) [0060] The results are shown in the left graph of FIG. The 21 bp siRNA vector (21bb-A and 21bp-B) for the sequence derived from the strain Seger, compared to the sequence derived from the Wakita strain having the mutation, The 50 bp siRNA vector (51 bp) showed a high effect.

 [Example 6: Suppression of hepatitis C virus using 50 bp mhRNA vector (2)]

HuH-7 cells infected with HCV carrying the luciferase gene were established. 5 × 10 ³ HCV-infected HuH-7 cells were seeded on a 96-well plate. On the next day, the 20 bp and 50 bp hairpin RNA expression vectors were transfected using Lipofectamine 2000, and after 58 and 88 hours, the reluciferase activity was measured using a Bright-Glo luciferase assay system (Promega). As a negative control, a U6 vector alone (Mock) and p53 [a corresponding 50 bp hairpin RNA vector (p53-50 bp)] were used.

 The DNA sequence transcribed by the U6 promoter is as follows.

 [0062] HCV-20bp; 5'-GTC TTG TAG ATT GTG TAT TAT AGA ATT ACA TCA AGG GAG ATT GAT GCA CGG TCT ACG AGA CT T TTT 3 '(SEQ ID NO: 17)

 [0063] p53—50 bp; 5, —CAT TAC ATT GGA GGA TTC CAG TGG TGA T CT ATT GGG GCG GAG TAG CTT TGG TGT GCT GTC CCA AAG CTG TTC CGT CCC AGT AGA TTA CCA CTG GAG TCT TCC AGT GTG ATG TTT TT 3 '(SEQ ID NO: 18)

 [0064] HCV—50 bp; 5, GAG TGT TCT GGG AGG TTT CGT AGA TCG

TGT ATC GTG AGT ACA AGT TCT AAG TGT GCT GTC CTT AGG ATT TGT GCT CAT GAT GCA CGG TCT ACG AGA CC T CCC GGG GCA CTC TTT TT-3, (SEQ ID NO: 19)

 [0065] The results are shown in the graph on the right side of FIG. The 50 bp siRNA vector (HCV-50 bp) was shown to suppress the growth of hepatitis C virus more rapidly and more effectively than the 20 bp siRNA vector (HCV-20 bp).

[Example 7: Effect of siRNA on E-cadherin targeting E-cadherin promoter on mRNA expression of E-cadherin] As a peripheral region of the E-cadherin gene, suppression of the expression of the E-cadherin gene by an siRNA targeting the promoter region of the E-cadherin gene was examined. The siRNA used was for 10 sites in the promoter region (indicated by Sitel-10 at the top of FIG. 7). The ability to transfect any of Sitel-10 siRNA into MCF-7 cells, or transfected all 10 Sitel-10 siRNA into MCF-7 cells. Oligofectamine (Invitrogen) was used for the transfusion. After transfection, total RNA was detected by Northern blotting and normalized by the amount of actin mRNA.

 The results are shown in the graph of FIG. Regardless of the transfection of any of Sitel-10 against the promoter region, suppression of E-force doherin expression was observed as compared to the control. When all 10 siRNAs of Sitel-10 were transfected, synergistic suppression of expression was observed. Subsequent experiments showed that this repression was due to methylation of the targeted promoter sequence, resulting in repression of transcription from the promoter (data not shown). Also in this case, it was shown that the duplex portion was longer than the conventional siRNA, and the use of siRNA resulted in high efficiency and suppression of transcription.

[Example 8: Replacement of DNA sequence from C to T by deamination]

 As a method for introducing a mutation into the sense strand of DNA, deamination was examined and its efficiency was confirmed.

 The EGFP gene was used as the DNA for the deamination treatment. The treatment for DNA-stranding was performed using strandase (Novagen) according to the attached protocol. All the products obtained in one reaction were directly used for the deamination treatment. The demineralization treatment was performed according to the attached protocol using CHEMICON International's CpGenome ™ DNA Modification Kit. After the deamination treatment, the product was amplified by PCR. For confirmation of the deamination, the product of the deamination treatment amplified by PCR was subjected to TA-cloning (pGEM-Teasy Vect or Systems of PROMEGA), and the nucleotide sequence of each 10 clones was confirmed.

Fig. 8 shows the results. All bases that were C before the deamination treatment were replaced with T. Had been. In other words, it was shown that C can be replaced with T with very high efficiency by deamination.

Claims

The scope of the claims  [1] an antisense RNA complementary to a sequence in a region around a target gene or a sequence in a region in mRNA of the target gene;  A sense RNA homologous to the sequence of the target gene;  A double-stranded interference RNA comprising:  A double-stranded interference RNA, wherein the antisense RNA and the sense RNA form a double strand, and the length of the double strand-forming portion is 50 bases or more. [2] The RNA for double-stranded interference according to claim 1, wherein the peripheral region of the target gene is a promoter region or a transcription regulatory region. [3] The RNA for double-stranded interference according to claim 1 or 2, wherein the length of the double-stranded portion is 50 to 300 bases. [4] The RNA for double-stranded interference according to claim 3, wherein the length of the double-stranded portion is 50-100 bases. [5] The method according to claim 1, wherein one or more bases are replaced by other bases in the antisense RNA or the sense RNA in the region that forms a double strand. One of four 2. The RNA for double-stranded interference according to item 1. [6] The double-stranded structure according to claim 5, wherein one or more bases are replaced by another base in the sense RNA in the region forming the double-stranded structure. Interfering RNA. [7] Nucleotide substitution in sense RNA is cytosine (C) force ゥ Racyl (U) substitution, adenine  7. The RNA for double-stranded interference according to claim 6, wherein the substitution of (A) with guanine (G) or the substitution of adenine (A) with inosine (I) is also performed. [8] The double-stranded interference R according to claim 7, wherein the substitution of the base is introduced by deamination. [9] The antisense RNA or the sense RNA in the region which forms a duplex and contains one or more bulges due to insertion or deletion of a base. The RNA for duplex interference according to any one of 8 above. [10] The RNA for duplex interference according to claim 9, wherein the bulge or the substitution is contained at a position other than the position of the 20th to 23rd bases from the end of the duplex. [11] The first bulge is included between the 119th and the 19th base from the end of the double strand, and the next bulge is included for each 125th base counted from the position of the bulge. The RNA for double-stranded interference according to claim 9. [12] The RNA for double-stranded interference according to claim 9-11, wherein the bulge is included in sense RNA. [13] The method of claim 11, further comprising a loop between the antisense RNA and the sense RNA. 2. The RNA for double-stranded interference according to claim 1. [14] A vector for expressing the RNA for double-stranded interference in a cell,  An antisense coding DNA encoding an antisense RNA complementary to a sequence in the peripheral region of the target gene or a sequence in any region in the mRNA of the target gene; and a sense RNA homologous to the sequence of the target gene. One or more promoters for expressing the antisense RNA and the sense RNA from the sense code DNA, the antisense code DNA and the sense code DNA, respectively;  A vector comprising  A vector, wherein the antisense RNA and the sense RNA form a duplex, and the length of the duplex forming portion is 50 bases or more. [15] The vector according to claim 14, wherein the peripheral region of the target gene is a promoter region or a transcription regulatory region. [16] The vector according to claim 14 or 15, wherein the length of the double-stranded portion is 50 to 300 bases. [17] The vector according to claim 16, wherein the length of the double-stranded portion is 50-100 bases.  [18] The claim, wherein one or more bases in the antisense code DNA or the sense code DNA in the region of the DNA encoding the RNA forming the duplex are replaced by other bases. The vector according to 14-17. [19] The claim, wherein one or more bases are replaced by another base in the sense code DNA in the region of the DNA encoding the RNA by forming a duplex. See section 18 On the vector.  [20] The vector according to claim 19, wherein the base substitution in the sense code DNA is substitution of cytosine (C) with thymine (T) or substitution of adenine (A) with guanine (G). [21] The vector according to claim 20, wherein the substitution of the base is introduced by deamination. [22] The antisense RNA or the sense RNA in the region which forms a double strand and contains one or more bulges due to insertion or deletion of a base. 22. The vector according to any one of 21. [23] The vector according to claim 22, wherein the bulge or the substitution is contained at a position other than the 20th to 23rd bases from the end of the double strand. [24] The first bulge is included between the 119th and the 19th base from the end of the duplex, and the next bulge is included for each 125th base counted from the position of the bulge. A vector according to claim 22.  [25] The vector according to any one of claims 22 to 24, wherein the bulge is contained in sense RNA. 26. The method according to claim 14, further comprising a loop between the antisense RNA and the sense RNA. 5 !, the slip force Vector according to 1. [27] The vector according to any one of claims 14 to 26, wherein the promoter is a ροΙΠΙ promoter.  [28] The vector according to claim 27, wherein the polIII promoter is also selected from the group consisting of a U6 promoter, a tRNA promoter, an HI promoter, a 7SK promoter, a 7SL promoter and a 5SrRNA promoter.  [29] An expression system for expressing RNA for double-stranded interference in a cell, An antisense coding DNA encoding an antisense RNA complementary to a sequence in the peripheral region of the target gene or a sequence in any region in the mRNA of the target gene; and for expressing the antisense RNA from the antisense coding DNA. A first vector comprising a first promoter, a sense code DNA encoding a sense RNA homologous to the sequence of the target gene, and a second vector for expressing the sense RNA from the sense code DNA. Promoter A second vector containing one and  Comprising  An expression system, wherein the antisense RNA and the sense RNA form a duplex, and the length of the duplex forming portion is 50 bases or more. 30. The expression system according to claim 29, wherein the peripheral region of the target gene is a promoter region or a transcription regulatory region. [31] The expression system according to claim 29 or 30, wherein the length of the double-stranded portion is 50 to 300 bases. [32] The expression system according to claim 31, wherein the length of the double-stranded portion is 50 to 100 bases.  [33] The claim wherein one or more bases in the antisense code DNA or the sense code DNA in the region of the DNA encoding the RNA forming a duplex are replaced by other bases. Item 33. The expression system according to any one of Items 29 to 32.  [34] The claim, wherein one or more bases are replaced by another base in the sense code RNA in the region of the DNA encoding the RNA by forming a duplex. Expression system according to item 33.  35. The expression system according to claim 34, wherein the substitution of a base in the sense code DNA is a substitution of cytosine (C) with thymine (T) and a substitution of adenine (A) with guanine (G). [36] The expression system according to claim 35, wherein the substitution of the base is introduced by deamination. [37] The antisense RNA or the sense RNA in the region which forms a duplex and contains one or more bulges due to insertion or deletion of a base. 36 of V, the expression system according to item 1. 38. The expression system according to claim 37, wherein the bulge or the substitution is included at a position other than the 20th to 23rd bases from the end of the double strand. [39] The first bulge is included between the 119th and the 19th base from the end of the double strand, and the next bulge is included for each 125th base counted from the position of the bulge. An expression system according to claim 37. [40] The expression system according to any one of [37] to [39], wherein the bulge is contained in sense RNA.  41. The method according to claim 29, wherein the first and second promoters are ροΙΠΙ promoters. The expression system according to item 1. 42. The expression system according to claim 41, wherein the polIII promoter is selected from the group consisting of a U6 promoter, a tRNA promoter, an HI promoter, a 7SK promoter, a 7SL promoter, and a 5SrRNA promoter. [43] The vector according to any one of claims 14 to 28 or any one of claims 29 to 42 A cell holding the expression system according to claim 1. [44] The cell according to claim 43, which is a plant cell. [45] The cell according to claim 43, which is an animal cell. [46] The cell according to claim 45, which is a mammalian cell.  [47] A vector according to any one of claims 14 to 28 or any one of claims 29 to 42 A composition comprising the expression system of claim 1. [48] The composition according to claim 47, which is a pharmaceutical composition. [49] A method for producing a cell in which the expression of a target gene is suppressed,  Introducing the vector according to any one of claims 14 to 28 or the expression system according to any one of claims 29 to 42 into a cell,  Selecting cells into which the vector or the expression system has been introduced.  [50] The method according to the above, comprising an antisense RNA complementary to a sequence in a region in a region around the target gene or an mRNA in the target gene, and a sense RNA homologous to the sequence of the target gene. A target gene comprising a step of introducing into a cell one or more vectors expressing a double-stranded interfering RNA having a length of 50 bases or more in a double-stranded portion formed by the antisense RNA and the sense RNA; A method for suppressing the expression of