WO2007010840A1 - MULTIPLE shRNA EXPRESSION VECTOR - Google Patents

Abstract

A multiple shRNA expression vector for use in the inhibition of the expression of a target gene, which carries multiple shRNA-producing units each of which can produce shRNA. In the vector, each of the shRNA-producing units comprises DNA encoding shRNA ligated downstream to a promoter, the DNA encoding the shRNA is composed of antisense DNA encoding antisense RNA which has a sequence complementary to mRNA of the target gene and sense DNA encoding sense RNA having a sequence hybridizable with the antisense DNA, and both of the antisense DNA and the sense DNA are ligated to each other through linker DNA.

Description

 Specification

 Multiple shRNA expression vector

 Technical field

 [0001] The present invention relates to a vector that produces shRNA capable of suppressing the expression of a target gene, and more particularly to a vector that produces a single vector force that produces a plurality of shRNAs targeting a plurality of genes.

 Background art

 [0002] RNA interference (hereinafter referred to as "RNAi") is one of the great discoveries of life science in recent years (Non-Patent Documents 1, 2, and 3). However, despite various improvements made to design effective short interfering RNA (hereinafter “siRNA”) duplexes (Non-Patent Documents 4 and 5), still low transfer It is difficult to induce efficient RNAi using the efficiency and method (Non-patent Document 6). The development of siRNA systems expressed from vectors is an innovative advance (Patent Document 1, Non-Patent Documents 7 and 8), and the following (1) to (3) when using conventional oligo siRNAs: We can overcome problems listed in

 (1) The knockdown efficiency of oligo siRNA duplexes depends greatly on the transfection efficiency of the host cell line. In order to obtain a sufficient knockdown effect, it is necessary to carry out transfection under a large amount and under optimum conditions. Therefore, the time for optimization and the cost of mass synthesis of oligo RNA are also high.

 (2) Second, due to the transient nature of the oligo siRNA duplex, it is difficult to silence target genes with long half-lives.

 (3) Third, most transfection methods are cytotoxic, which makes it difficult to observe important phenotypic changes such as cell death, apoptosis and cell proliferation.

 [0003] When using the oligo siRNA shown above, the problem is that using a siRNA expression vector, which is DNA, and establishing a stable knockdown cell line by expressing the siRNA, these problems Katsutsuki can be β.

[0004] In addition, several labor-intensive techniques for creating large-scale RNAi libraries were introduced. Force S (9, 10), they are costly and time consuming, and optimization is required to stably knock down multiple genes to similar levels in the same cell. In addition to the purpose of screening large-scale RNAi libraries, there is a demand for the development of a multiple knockdown method that can stably and simultaneously suppress a large number of target genes without using multiple types of selectable markers.

 [0005] Recent studies have reported that short hairpin (hereinafter abbreviated as “sh”) type RNA dicer substrates are effective for inducing RNAi ( Non-patent document 11). From this result, it is expected that sh-type RNA expression vectors will become the mainstream tool for RNAi analysis in the future.

 [0006] Patent Literature l: WO 03/046186 Nonfret

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

 Non-Patent Document 3: Hannon, G.J. & Rossi, J.J.Unlocking the potential of the human geno me with RNA interference.Nature 431, 371-378 (2004).

 Patent Document 4: Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex.Cell 115, 199-208 (2003).

 ^^ Patent Literature 5: Reynolds, A. et al. Rational siRNA design for RNA interference. Nat Biotechnol 22, 326-330 (2004).

 Non-Patent Document 6: Dykxhoorn, DM, Novina, CD. & Sharp, PA Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4, 457-467 (2003). Non-Patent Document 7: Miyagishi, M & Taira, K. U6 promoter-driven siRNAs with four uridi ne 3 overhangs efficiently suppress targeted gene expression in mammalian cells. N at Biotechnol 20, 497—500 (2002).

Patent Document 8: Brummelkamp, TR, Bernards, R. & Agami, R. A system for stable ex pression of short interfering RNAs in mammalian cells. Science 296, 550-553 (2002). Non-Patent Document 9: Miyagishi, M. & Taira, K. Strategies for generation of an siRNA expre ssion library directed against the human genome.Oligonucleotides 13, 325-333 (200 3).

 Special Review 10: Downward, J. Use of RNA interference libraries to investigate oncog enic signaling in mammalian cells.Oncogene 23, 8376-8383 (2004).

Non-Patent Document 11: Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nat Biot echnol 23, 227-231 (2005)

 Special Review 12: Imamura, T. et al. Proteomic analysis of the TGF— beta signaling pat hway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 e xpression. Biochem Biophys Res Commun 318, 289-296 (2004)

Non-Special Terms 13: Jazag, A. et al. Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growt h factor-beta. Oncogene (2004).

Non-Patent Literature Literature l4: Zanta MA et al "Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci US A. 19 99 Jan 5; 96 (1): 91— 6 )

Non-Special Terms 15: Liu F, Huang L. Improving plasmid DNA-mediated liver gene transfe r by prolonging its retention in the hepatic vasculature. J. Gene Med. 2001 Nov— De c; 3 (6): 569-76

Non-Patent Document 16: Zawel, L. et al. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell 1, 611—617 (1998).

 Special Review 17: Ijichi, H. et al. Systematic analysis of the TGF— beta— Smad signaling pathway in gastrointestinal cancer cells. Biochem Biophys Res Commun 289, 350—35 7 (2001)

Non-Patent Document 18: Ijichi, Tsuji et al. Smad4—independent regulation of p21 / WAFl by tran sforming growth factor-beta. Oncogene 23, 1043-1051 (2004).

Non-Patent Document 19: Seoane, J., Le, HV, Shen, L., Anderson, SA & Massague, J. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioolasto ma cell proliferation.Cell 117, 211— 223 (2004). Non-Patent Document 20: Jackson, AL et al. Expression profiling reveals off- target gene regulation by RNAi. Nat Biotechnol 21, 635-637 (2003).

Non-Patent Document 21: Bridge, AJ, Pebernard, S., Ducraux, A "Nicoulaz, AL & Iggo, R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 3 4, 263-264 (2003)

Non-Patent Document 22: Pardali, K. et al. Role of Smad proteins and transcription factor Spl in p21 (Wafl / Cipl) regulation by transforming growth factor-beta.J Biol Chem 275, 29244-29256 (2000)

Non-Patent Document 23: Kretschmer, A. et al. Differential regulation of TGF-beta signaling t hrough Smad2, Smad3 and Smad4. Oncogene 22, 6748—6763 (2003).

Non-Patent Document 24: Major, M.B. & Jones, D.A.Identification of a gadd45beta 3 'enhancer that mediates SMAD3— and SMAD4— dependent transcriptional induction by transfo rming growth factor beta.J Biol Chem 279, 5278—5287 (2004).

Non-Patent Document 25: Siegel, P.M. & Massague, J. Cytostatic and apoptotic actions of TG

F-beta in homeostasis and cancer.Nat Rev Cancer 3, 807-821 (2003)

Non-Patent Document 26: Takaku, K. et al. Gastric and duodenal polyps in Smad4 (Dpc4) knoc kout mice. Cancer Res 59, 6113-6117 (1999)

Non-patent document 27: Ashcroft, GS et al. Mice lacking Smad3 show accelerated wound he aling and an impaired local inflammatory response. Nat Cell Biol 1, 260- 26b (1999). Non-patent document 28: Shin, I "Bakin, AV , Rodeck, U., Brunet, A. & Arteaga, CL Trans forming growth factor beta enhances epithelial cell survival via AM— dependent regul ation of FKHRL1. Mol Biol Cell 12, 3328-3339 (2001)

Non-Patent Document 29: Haley, B. & Zamore, P.D.Kinetic analysis of the RNAi enzyme comp lex. Nat Struct Mol Biol 11, 599-606 (2004)

Disclosure of the invention

Problems to be solved by the invention

In the sh-type expression vector, the present invention is a multi-type gene comprising a large number of shRNA expression cassettes targeting a large number of genes or targeting a single gene. The challenge is to develop a knockdown system.

 Means for solving the problem

 [0008] In developing a system capable of expressing a large number of shRNAs from a single vector, the present inventors tried to simultaneously perform functional analysis of the signal transduction system of Smad-TGF- | 8. The present inventors have already reported the functional analysis of Smad4 using a short hairpin RNA expression vector to analyze complex signal transduction pathways (Non-Patent Documents 12 and 13), but other Smad genes. At the same time, we thought that further functional analysis could be performed.

 [0009] As a result of diligent efforts, the present inventors have succeeded in developing a system capable of producing sh-type RNAs targeting multiple genes and succeeding in developing a system capable of simultaneously suppressing the Smad family. did. The present invention is based on these results, and is specifically as follows.

 [0010] 1. A multiple shRNA expression vector carrying a plurality of shRNA generation units capable of generating shRNA for suppressing the expression of a target gene in the vector,

 The shRNA generator is configured by connecting DNA encoding shRNA downstream of the promoter,

 The DNA encoding the shRNA is constituted by linking an antisense DNA having a sequence complementary to the target gene and a sense DNA comprising a sequence capable of pairing with the antisense DNA with a linker DNA. Multiple shRNA expression vector.

 2. The multiplex shRNA expression vector according to 1 above, wherein the sense DNA contains a mismatch sequence that does not complement the antisense DNA.

 3. The multiplex shRNA expression vector according to 2 above, wherein the mismatch sequence is contained in 2-55% of the sense DNA.

 4. The multiplex according to 2 above, wherein the mismatch sequence on the sense DNA is formed by any one of substitution, deletion, insertion, addition, or a combination thereof in relation to the corresponding base on the antisense DNA. shRNA expression vector.

5. The multiple shRNA expression vector according to 4 above, wherein the substitution is performed by changing C on the sense DNA to T or G to A. 6. The multiple shRNA expression vector according to 1 above, wherein the vector is a vector that induces transient expression or a vector that induces stable expression.

 7. The multiple shRNA expression vector according to 1 above, wherein the plurality of shRNA generation units supported in the vector correspond to any one of the following (1) to (3) or a combination of two or more thereof.

 (1) The same shRNA generation unit targeting the same site of a single gene can be used multiple times

(2) Consists of multiple shRNA generation units targeting two or more different sites of a single gene

 (3) Consists of multiple shRNA generation units targeting two or more different genes

8. The multiple shRNA expression vector according to 1 above, wherein the promoter is a polIII promoter.

 9. A method for producing a multiple shRNA expression vector from a circular structure first shRNA expression vector having a first shRNA generation unit and a circular second shRNA expression vector having a second shRNA generation unit as raw materials. ,

 Similarly, the first and second shRNA expression vectors are provided with recognition digestion sites a and b for restriction enzymes A and B, respectively, upstream and downstream of the first and second shRNA generating units, respectively. And the second shRNA expression vector is provided with a recognition digestion site c of restriction enzyme C that exists only in the a-b region on the side not containing the shRNA generation unit, and the restriction enzymes A, B, and C are all different, A and B are restriction enzymes that generate an end that can be connected to A and B, and an end that cannot be connected to the digested end of C. The a and b sites are the first, In conditions that exist as a single location in the second shRNA expression vector:

 A method comprising the following steps (1) to (4):

 (1) digesting the first shRNA expression vector with restriction enzymes A and C,

 (2) digesting the second shRNA expression vector with restriction enzymes B and C,

 (3) Step of mixing the digested fragments of (1) and (2) above and performing ligation

(4) The a-c fragment containing the first shRNA generating unit of the first shRNA expression vector and the bc fragment containing the second shRNA generating unit of the second shRNA expression vector were connected. Recovering the circular structure

 10. The method according to 9 above, wherein restriction enzymes A, B, and C are restriction enzymes that generate sticky ends.

 11. The method according to 9 above, wherein the restriction enzymes A and B can form a sequence different from both a and b when the generated ends are connected to each other.

 12. The first and second shRNA expression vectors are provided with a selection marker, and a site c is provided in the selection marker.

 10. The method according to 9 above, comprising a step of selecting with the selection marker after the step (3).

 13. A vector for the production of a multiple shRNA expression construct,

 A promoter,

 A cloning region into which DNA encoding shRNA provided downstream of the promoter can be inserted;

 With one or more selection markers,

 A recognition digestion site a of restriction enzyme A is provided upstream of the promoter, a recognition site b of restriction enzyme B is provided downstream of the cloning region,

 Within one selectable marker is a restriction enzyme C recognition digestion site c,

 A and B are restriction enzymes that can generate connectable ends, and the restriction enzyme C is a restriction enzyme that can form a stump that cannot be connected to the digestive ends of A and B, and The a and b parts are provided as a single part.

 vector.

 14. The vector according to 13 above, wherein restriction enzymes A, B, and C are restriction enzymes that generate sticky ends.

 15. The vector according to the above 14, wherein the restriction enzymes A and B can form a sequence different from both a and b when the generated ends are connected to each other.

 16. A cell having the vector according to any one of 1 to 8 above.

 17. A non-human animal carrying the vector according to any one of 1 to 8 above.

18. A pharmaceutical composition comprising the vector according to any one of 1 to 8 as a component. The invention's effect

 [0011] According to the present invention, it is possible to express a plurality of shRNAs with a single vector force.

 Therefore, it is also possible to increase the number of copies expressed in a cell by making multiple copies of a single shRNA on the vector. In addition, it is expected that gene expression can be more strongly suppressed by mounting DNA encoding different shRNAs targeting different parts of one gene in one vector. Furthermore, as the most effective use in the present invention, multiple genes can be simultaneously expressed by RNAi effect by introducing a single vector by mounting multiple shRNAs targeting different genes on a single vector. It can be suppressed. For example, in the case of a multiple shRNA vector that targets multiple genes involved in a single signal transduction pathway, multiple points in the signal transduction pathway are suppressed, so it is possible to strongly suppress the pathway transmission. it can. This can be expected to elucidate functions that cannot be detected by suppression of a single gene involved in the signal transduction pathway. In particular, in the case of a signal transduction system in which bypass pathways exist separately, if the multiple shRNA expression vector of the present invention is used, genes involved in both the mainstream and the nopass pathway are expressed simultaneously as targets. It is also possible to suppress it.

 In addition, according to the multiplex shRNA expression vector of the present invention, it is possible to obtain an animal in which a large number of genes are suppressed at the same time without creating a double Z triple knockout mouse, which has conventionally required time and labor to produce it. It becomes possible.

 As described above, according to the present invention, it can be expected to be an important tool for accelerating the elucidation of the function of gene Z protein and to contribute to the elucidation of the mechanism of disease occurrence. Brief Description of Drawings

[0012] [Fig. La] A photograph showing the results of examining the RNAi effect using a single knockdown construct. Four target sites were designed for each of the Smad2, Smad3, or TGFRB2 genes and subcloned into the pcPUR + U6i cassette to construct each siRNA expression vector. Each siRNA expression vector was transiently transfected into HeLa cells simultaneously with Smad2-, Smad3_, or TGFRB2-expression vectors, and the silencing efficiency was examined by Western blot analysis. Two identical samples were loaded per sample. [Fig. Lb] A photograph showing specific knockdown of Smad-2 or -3 by siSmad2 or siSmad3. For HaCaT cells, pcDEF3—Flag (N) —Smad2, pcDEF3—Flag (N) —Smad3, and pcP UR + U6- Smad2i (left lane), or pcPUR + U6- Smad3i (center lane), or pcP UR + U6-GFPi (right lane) was transiently transferred. After lysing the cells, the inventors performed a Western plot analysis.

 [FIG. Lc] FIG. Lc to FIG. Le are diagrams schematically showing the construction and construction method of RNAi vectors. Figure lc schematically shows the strategy for generating multitarget siRNA expression vectors. The pc PUR + U6-i cassette has a Bglll restriction enzyme site upstream of the U6 promoter, BamHI downstream of the RNAi cloning site, and a seal site for the ampicillin resistance gene. These sites are all single sites in this plasmid. “N; D5” in the figure means an aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctc (Table ¾ · No .: 162) and a base sequence of 55 bases in length.

 [Fig.ld] PCPUR + U6- Smad2i was digested with BamHI and Seal and pcPUR + U6- Smad3i was digested with Bglll and Seal to create a double knockdown vector targeting both Smad2 and -3 FIG. “N55” in the figure means the nucleotide sequence set forth in SEQ ID NO: 162.

FIG. 2 shows that a double knockdown vector was prepared by ligation of two fragments linearized with a restriction enzyme. “N55” in the figure means the nucleotide sequence set forth in SEQ ID NO: 162.

 FIG. If is a schematic diagram of a single, double, and triple siRNA expression vector prepared in Examples. Of these, pcPUR + U6-Smad4i has already been reported (Non-Patent Documents 1 and 13) o

[FIG. 2a] FIGS. 2a to 2g are diagrams and photographs showing the effectiveness and specificity of stable knockdown cells prepared in Examples. Figure 2a shows endogenous Smad knockdown: HaCaT cells were treated with pcPUR + U6— GFPi ゝ pcPUR + U6— Smad2i, pcPUR + U6— Smad3i, pcPUR + U6— Smad4i, pc PUR + U6— Smad23i, pcPUR + U6— Smad24i FIG. 3 shows that cells transformed stably by purifying pcPUR + U6-Smad34i or pcPUR + U6-Smad234i and incubating with puromycin (1 μg / ml) were selected. Total cell lysate Western blot analysis was performed.

 FIG. 2b is a photograph showing the results of detection of siRNA expression against Smad2, 3 and 4 by Northern analysis in each cell line.

 FIG. 2c shows that the relative expression of BTBD1 in the cell lines shown was evaluated by quantitative reverse transcriptase PCR. A value of 1.0 was assigned to the gene expression level in parental HaCaT cells. The knockdown vector did not induce silencing of the non-specific target gene, BTBD1.

圆 2d] shows that the relative expression of prominin 2 in the cell lines shown was evaluated by quantitative reverse transcriptase PCR. A value of 1.0 was assigned to the gene expression level in parental HaCaT cells. The knockdown vector did not induce silencing of the nonspecific target gene, prominin 2.

 FIG. 2e shows that the relative expression of OAS1 in the cell lines shown was evaluated by quantitative reverse transcriptase PCR. A value of 1.0 was assigned to the gene expression level in parental HaCaT cells. The knockdown vector did not induce interferon induction.

[FIG. 2f] Inhibition of TGF-β-Smad signaling in knockdown cells. Knockdown cells and control cells were transfected with (CAGA) 9-luc and pRL-SV40. After 24 hours, cells were incubated for an additional 24 hours in the presence or absence of TGF- | 8 1 2.5 ng / ml for double luciferase assay. (CAGA) 9-luc firefly luciferase activity was normalized to pRL-SV40 renilla luciferase activity. The value of 1.0 was assigned to the level of luciferase in cells in the absence of TGF-jS 1 (white bar) and the relative activity was calculated. Experiments are performed 3 times, 2 per sample, and mean and standard error (S.E.) are shown.

 [Fig. 2g] A photograph showing the induction of ΡΑΙ-1 by TGF-β1 by Western blotting. Cells were incubated for 10 hours in the presence or absence of 5 ng / ml TGF-jS to prepare total cell lysates.

 [FIG. 3a] FIGS. 3a to 3e are diagrams and photographs showing analysis of TGF-β-dependent cell phenotypes. Figure 3a is a photograph showing a representative image of infiltration.

FIG. 3b is a diagram showing the number of infiltrating cells. In the absence (white bar) and presence of TGF-β1 (white bar) The number of cells infiltrated in Matrigel with black bars was counted in 5 random fields. The experiment is repeated three times and shows the mean and standard error (SE).

 [Fig. 3c] A photograph showing a representative image of the running Atsey. Wound closure was assessed 24 hours later in the presence or absence of TGF-18 (15 ng / ml).

 [Fig. 3d] The distance from end to end of the wound after culturing in the absence (white bar) and presence (black! / ヽ bar) of TGF-β1 in the cell line shown It is a figure shown as a percentage based on distance. Repeat the experiment 3 times and show the mean and standard error (S.E.).

 [FIG. 3e] Percentage of apoptotic cells in 24 hours in the absence (white bars) or presence (black bars) of TGF- | 8 (15 ng). Three independent experiments were performed, but similar results were obtained and representative results are shown.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0013] [First embodiment]

 The present invention firstly relates to a multiple shRNA expression vector capable of generating a large number of shRNAs for knocking down a target gene.

 shRNA is an abbreviation for short hairpin RNA, and is a double-stranded RNA that can induce RNA interference. Double-stranded RNAs used for RNA interference generally include siRNA and shRNA. siRNA has a structure in which both ends are open, whereas shRNA has a structure in which one end is closed by a loop. In this invention, it is related with the system which expresses shRNA among these. Since shRNA has a loop structure at one end and can be synthesized as a single continuous RNA sequence, it is advantageous for production based on expression vectors. In this way, the present invention has a structure in which a plurality of shRNAs are expressed from a single vector because of the efficiency of production from the expression vector.

[0014] In order to generate a plurality of shRNAs from a single vector, the vector of the present invention is equipped with a plurality of shRNA units. The combination of a plurality of shRNA generation units mounted on one vector can be any of the following or a combination of two or more of these.

 (1) Multiple units that generate the same shRNA targeting the same part of the same gene

(2) Multiple units that generate different shRNAs targeting different parts of the same gene

(3) Multiple units that generate shRNA targeting two or more different genes As described above, the term “multiplex” in the present invention means that the shRNA sequence is the same or different, but V or not, but the number of DNA units encoding shRNA! Means.

 [0015] The number of shRNA generating units to be mounted on one vector can be determined by the length of the insert that can be inserted into a vector that is not particularly limited. For example, the number of shRNA generating units to be mounted can be generally 2 to 30, preferably 2 to 10, and more preferably 2 to 5.

 [0016] Each shRNA generation unit is provided with an shRNA-encoding DN A and a promoter upstream thereof as essential elements for shRNA generation. The shRNA-encoding DNA consists of an antisense DNA having a sequence complementary to the target gene and a sense DNA consisting of a sequence that can be paired with the antisense DNA, linked by a linker DNA. . That is, the DNA encoding shRNA is composed of antisense DNA-linker-DNA-sense DNA. From this structure, RNA having the structure of antisense RNA—linker RNA—sense RNA is expressed, and among these, antisense RNA and sense RNA form a double strand to generate shRNA.

 [0017] Here, it is preferable that the antisense DNA has a sequence that is completely complementary to the target gene so that an off-target effect can be avoided, and a sequence that has a mismatch with a gene other than the target is selected. For example, the sequence design can be carried out using a program shown in Table 1 below.

 [0018] [Table 1]

[0019] For antisense DNA, it is preferable to select a sequence that completely complements the RNA of the target gene. On the other hand, the sense DNA may have a mismatch sequence with the antisense DNA as long as it can be paired with the antisense DNA. Rather, when the sense DNA is composed of a sequence that is completely complementary to the antisense DNA, the above-mentioned “antisense DNA-linker DNA-sense DNA” has an inverted repeat structure, and the vector may be rearranged. May become unstable. Therefore, the sense DNA preferably has a mismatch sequence that does not complement the antisense DNA. The ratio of mismatch sequences can be, for example, 2 to 55%, preferably 10 to 35%, more preferably about 15 to 25%. As an example, the number of mismatched bases on the sense strand in a shRNA having a length of 21 bases as the length of the duplex forming region is 1 to: L 1 base, preferably 2 to 7 bases. Or 3-5 bases. Note that the length of the duplex forming region has a wide range of selections such as 17 to 200 as will be described later, so the number of mismatch bases on the sense strand can be determined according to the selection length. .

 [0020] The mismatch is generally a force formed by substitution, that is, substitution to another base that does not complement the antisense DNA sequence in the sequence of the sense DNA, but the present invention is not limited to this. For example, a mismatch occurs when one or more bases are deleted from the sense DNA sequence and the base corresponding to the antisense DNA is removed, and a base that does not correspond to the antisense DNA is included in the sense DNA sequence. A mismatch formed by insertion and a mismatch formed by adding an arbitrary base to the end of the sense DNA sequence are also included in the mismatch sequence of the present invention. When forming a mismatch sequence by substitution, cytosine (C) complementary to the antisense DNA on the sense DNA is converted to thymine (T), or guanine (G) complementary to the antisense DNA is converted to adenine (A). Replacement is preferred. Compared to C and G, 結合 and Τ have a weaker coupling force, so the rear range of the vector due to the unstable inverted repeat structure can be suppressed.

[0021] The length of the antisense DNA and the sense DNA may be longer than the length capable of maintaining specific binding with the target gene. On the other hand, in mammalian cells, a long double-stranded RNA may be cytotoxic. Therefore, when a mammalian cell is used as a host, it is preferable that the length of the shRNA expressed in the cell is not cytotoxic. For example, when targeting genes in mammalian cells, antisense DNA and sense DN The length of A can be 17 to 200 bases, preferably 18 to 55 bases, more preferably 19 to 23 bases.

 [0022] The linker DNA that connects the antisense DNA and the sense DNA is a structure for forming a hairpin structure in shRNA. The sequence of the linker DNA can constitute any sequence ability other than the termination sequence that inhibits the generation of shRNA. The length may be any length that does not hinder pairing with antisense RNA or sense RNA. As an example, the sequence of a hairpin site of microRNA can be mentioned. Alternatively, the linker DNA may be composed of DNA encoding tRNA. In fact, in Examples described later, “gtgtgctgtcc” (SEQ ID NO: 1) and its complementary strand, “acgtgtgc tgtccgtj (SEQ ID NO: 2) and its complementary strand were used as the linker DNA sequence.

 [0023] In order to express shRNA from the DNA encoding the above shRNA, the shRNA generation unit is provided with a "promoter". This promoter may be either a polll system or a polIII system as long as it can express shRNA, but preferably a polIII system suitable for expression of short RNAs such as shRNA can be 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. it can.

 [0024] By using an inducible promoter as the promoter, siRNA can be expressed at a desired timing. These inducible promoters include tetracycline-inducible U6 promoter (Ohkawa, J. & Taira, K. Control of the functional activity of an antisense RNA by a tetracycline—responsive derivative of the human U6 snRNA promoter. Gene Ther. 11, 577-585 (2000)). You can also use a tissue-specific promoter or a DNA recombination system such as the Cre-LoxP system to control shRNA expression from the vector! /.

[0025] In addition, in order to express the shRNA and !! sequences from the promoter accurately, it is preferable to provide a terminator for terminating transcription at the 3 'end of the DNA encoding shRNA. If the terminator is a sequence that can terminate transcription of the promoter, For example, a sequence in which four or more T (thymine) or A (adenine) are continuous, or a sequence capable of forming a nodromedome structure can be used.

 [0026] The vector carrying the shRNA generating unit can be selected according to the introduced cell and purpose. In mammalian cells, for example, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, lentiwinores vectors, henolepusuinores vectors, ananolofinores vectors, EB winoles vectors, Examples include the papilloma winores vector and winomeless betaters such as the foamy winores betater. In addition, dumbbell-shaped DNA (Non-patent Document 14) or naked plasmid, which is not a viral vector, can also be suitably used (Non-patent Document 15). From the viewpoint of purpose, it is preferable to use a vector or plasmid existing outside the chromosome when performing transient gene suppression among the above-mentioned vectors. In order to stably suppress the expression of the target gene, it is preferable to select a vector having the ability to integrate into the chromosome.

 [0027] When a plurality of shRNA generation units are mounted on a vector, the expression directions of each shRNA generation unit may be mounted to be the same or may be mounted without setting the direction. In addition, the distances between the units may be connected with no gap between them or may be connected with a gap through an arbitrary arrangement.

 If necessary, 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. The marker etc. which can be selected as a parameter | index are mentioned. In addition, a selection marker or the like that can select a surface antigen such as EGF receptor, B7-2, or CD4 as an indicator may be used. Thus, by using a selectable marker, it can be useful for the production of a multiple shRNA expression vector as described later, which is not useful for introducing a shRNA expression vector into a host.

[0028] The multiple shRNA expression vector can suppress the expression of one or a plurality of target genes targeted by the mounted shRNA generation unit. Especially targeting multiple target genes This is an advantageous means. Cells or non-human animals into which this multi-shRNA expression vector has been introduced can be used as model cells or model animals in which a specific target gene is suppressed. In particular, when a vector having the ability to integrate a vector into a chromosome is used, one or more targeted genes can be stably suppressed. Therefore, it is possible to easily create a multiple knockout animal, which has been conventionally created by spending a lot of time, by introducing this vector. Thus, the knockdown animal produced using the vector of the present invention can be used for functional analysis of a target gene or the like, or used for drug discovery screening as a disease model animal. The cells that can be used here include cells derived from prokaryotes, cells derived from eukaryotes, and the like, which are not particularly limited. Non-human animals can also include mammals such as mice, rats, rabbits, goats, pigs, monkeys and the like.

 [0029] The target gene is not particularly limited, but a disease-related gene or a gene of a foreign substance (bacteria or virus) that induces a disease can also be targeted. As the virus, for example, HIV, RSV, HCV, HBV and the like can be targeted. When targeting these viruses, DNA encoding shRNA targeting multiple genes on the viral chromosome, or shRNA targeting multiple sites within one gene on the viral chromosome was encoded. Can be equipped with DNA.

 It is also possible to mount a unit group that generates multiple shRNAs that target genes on the viral chromosome and host endogenous genes. For example, in the case of HIV, it is known that receptors in the CCR5 family and surface antigens are involved as a route for HIV to infect cells in the host. The CCR5 family includes CCR5, CXCR4, and CD4. Therefore, multiple shRNA vectors for treating HIV infection include, for example, shRNA targeting any of the HIV genes, particularly those involved in proliferation and infection, and the host CCR5 family! / As an example, a configuration capable of expressing shRNA targeting the above gene can be mentioned.

 [0030] [Second Embodiment]

Secondly, the present invention relates to a method for efficiently producing the multiple shRNA expression vector. The production method of the present invention comprises one shRNA generation unit (for convenience, the first shRNA generation unit). A first shRNA expression vector having a circular structure provided with a “t”, and a second shRNA expression vector having a circular structure provided with a second shRNA generator are prepared. Since the configuration and design of the shRNA generation unit to be inserted into the vector are the same as those in the first embodiment, description thereof is omitted. The shRNA generation unit can be generated by a genetic engineering technique used by those skilled in the art, for example, by synthesis using a DNA synthesizer or PCR.

[0031] The vector skeleton portion of the first shRNA expression vector and the second shRNA expression vector may be heterogeneous, but preferably the same vector is used. Different restriction enzyme A and B recognition / deletion sites a and b (hereinafter referred to as `` site a '' and `` site b '', respectively) upstream and downstream of the site or region to which the shRNA generator is connected on the vector And a recognition site for restriction enzyme C that exists only in the a-b region on the side not containing the shRNA generation unit (hereinafter referred to as “site c”). Need to be. Sites a, b, and c may be either sequences provided in the vector itself or artificially inserted.

 First, restriction enzymes A and B can be arbitrarily selected from those satisfying the following conditions.

 (1) Sites & and b are different sequences

 (2) Sites a and b are single, including the vector and the shRNA generation unit (sites a and b exist only in one place throughout the vector and do not exist in the shRNA generation unit)

 (3) Restriction enzymes A and B generate ends that can be connected to each other (ends generated by restriction enzyme A deleting site a, and restriction enzyme B generated by digesting site b) Connectable to the end)

 Such restriction enzymes A and B may form blunt ends or sticky ends. For example, the combinations of restriction enzymes Α and Β that form sticky ends can be selected with the same number of protruding bases and the same number of protruding bases in Tables 2-1 and 2-2.

[0033] [Table 2-1] Protrusion tjm powder u meta code Proxy enzyme name Recognition sequence Base sequence Radix Type Enzyme

Eam1 105I GACNNNINNGTC N

(SEQ ID NO: 151) ¹ 3 'overhang 2

 Xcml CCANNNNNlNNNNTGG N

(SEQ ID NO: 152) ¹ 3 'overhang 2

 Tth1 1 1 I GACNINNGTC N 1 5 'protruding 3 PflFI

EcoNI CCTNNlNNNAGG N

 (SEQ ID NO: 153) 1 5 'overhang 3

 Fnu4HI GC | NGC N 1 5 'overhang 4

 ScrFI CC | NGG N 1 5 'protruding 4

 Pvul CGATlCG AT 2 3 'protruding 5

 Pad TTAATlTAA AT 2 3 'protruding 5

 Clal ATLCGAT CG 2 5 'protruding 6 8 BspDI

HapII C | CGG CG 2 5 'overhang 6 7 8 Mspi

Hinl l GRlCGYC CG 2 5 'protruding 6 7 8 BsaHI

Psp1406I AAlCGTT CG 2 5 'protruding 6 8

 TthHB8I T | CGA CG 2 5 'overhang 7 Taql

BstBI TT | CGAA CG 2 5 'protruding 8

 Ndel CA | TATG TA 2 5 'protruding 10 9

 PshBI AT | TAAT TA 2 5 'protruding 9

 Xspl C | TAG TA 2 5 'protruding 10 9 Bfal

Msel T | TAA TA 2 5 'Protrusion 10

 Bgll GCCNNNNlNGGC NNN 3 3 'protruding 12

 (SEQ ID NO: 154)

 Sfil GGCCNNNNlNGGCC NNN 3 3 'protruding 1 1

 (SEQ ID NO: 155)

 Van91 I CCANNNNINTGG NNN 3 3 'protruding 1 1 12

 (SEQ ID NO: 156)

 AlwNI CAGNNNlCTG NNN 3 3 'protrusion 13

 BstAPI GCANNNNINTGC NNN 3 3 'protrusion 12

 (SEQ ID NO: 157)

 DraHI CACNNNIGTG NNN 3 3 'protruding 1 3

 Cpol CGIGWCCG GWC 3 5 'protrusion 14

 PpuMI RGIGWCCY GWC 3 5 'protruding 14

 Bpu1 102I GClTNAGC TNA 3 5 'protruding 15 Blpl

Eco81 I CClTNAGG TNA 3 5 'overhang 15 Bsu36I

EcoT22I ATGCAlT TGCA 4 3 'protrusion 16 17 Nsil

Pstl CTGCAlG TGCA 4 3 'protrusion 16

 Sse8387I CCTGCAIGG TGCA 4 3 'protruding 17 Sbfl

EcoRI G | AATTC AATT 4 5 'Protrusion 18

Muni ClAATTG AATT 4 5 'protrusion 18 19 2- 2] Apol RlAATTY AATT 4 5 'Protrusion 19

 Ncol ClCATGG CATG 4 5 'protruding 20 23 Xmal,

 AccIII,

BspHI TlCATGA CATG 4 5 'protruding 20 23 BspEI

Pcil AlCATGT CATG 4 5 'protruding 20

 Cfrl OI RlCCGGY CCGG 4 5 'protruding 21 22 BsrFI

BsaWI WlCCGGW CCGG 4 5 'protruding 22

 NgoMIV GlCCGGC CCGG 4 5 'overhang 22

 SgrAI CRlCCGGYG CCGG 4 5 'protruding 23

 BssHII GlCGCGC CGCG 4 5 'overhang 24

 lul AlCGCGT CGCG 4 5 'protrusion 24 25

 Ascl GGlCGCGCC CGCG 4 5 'overhang 25

 AfllH AlCRYGT CRYG 4 5 'protrusion 26

 Btgl ClCRYGG CRYG 4 5 'protruding 26

 Blnl ClCTAGG CTAG 4 5 'protruding 27

 Nhel GlCTAGC CTAG 4 5 'protruding 27

 Spel AlCTAGT CTAG 4 5 'protruding 27

 Xbal T | CTAGA CTAG 4 5 'protruding 27

 BamHI GlGATCC GATC 4 5 'protruding 28

 BglH AlGATCT GATC 4 5 'protruding 28

 Fbal T | GATCA GATC 4 5 'protruding 28 29

 Mfll RlGATCY GATC 4 5 'protruding 29

 Kasl GIGCGCC GCGC 4 5 'protrusion 30 31 32 PspOMI

Eael YlGGCCR GGCC 4 5 'overhang 30

 Eco52I ClGGCCG GGCC 4 5 'protruding 31 Eagl

Notl GClGGCCGC GGCC 4 5 'overhang 32

 Bsp1407I TlGTACA GTAC 4 5 'overhang 33 BsrGI

Acc65I GlGTACC GTAC 4 5 'protrusion 33

 BsiWI ClGTACG GTAC 4 5 'protruding 33

 Sail GlTCGAC TCGA 4 5 'protruding 34

 Xhol ClTCGAG TCGA 4 5 ′ overhang 34 PaeR7I The combination of restriction enzymes A and B that form blunt ends can be selected from Table 3.

[Table 3] I y 丄 〇 | ov people IVesg

 I wv | 丄 丄 丄

 I vv 丄 | v 丄 丄 I! Sd

I VOOlDO 丄 'PS | A | V00 | 3CLL Ilea

I V03 | 33 丄 'l ^ds J II! ^A V

 I VE) 0 | SO 丄

 I V 丄 3 | 3V 丄

I OVid | AlE) H. ^U ! H

I 3VW | 丄 丄 丄 0 I ^SLU d

I OVV | 丄 丄 3 iedH

L OV 丄 | V1〇 'IZ1 ^S 8 OV 丄 | V 丄 3 thigh

 I 3V 0 m

L OONlNOD ΛΙ ^Β ΙΝ

I OOOlODO MS i 00 | OD III ^aB H

I OON | NOO

I ODDlOOD I ^9e N

Ϊ 0 丄 VNNINN 丄 VS

 I 0 丄 V | 丄

 I DDOlDVO

 (09 ^ 蓥

I 0 丄 3NN | NN3V3 IV4 ^s d

(69L ^:

 I μιιχ

 (3 丄 丄 NN | NNWE)

y 00 | DO im ^s ay DO | DO'imsa DO | 00 II. . V l DDDlOOO

(89L ^:

l 3 丄 NNINN people VO II ^S IAI

 S 丄 0 | 3V0

 l 0 丄 OlOVO'Ilwd S 丄 0 | 3V3

 l OLO | OVO

y 丄 VW | 丄 丄 丄 V IB S l 丄 OV | 丄 OV ie. S l 丄 30 | 03V I ⁿ lS l 丄 o loov'i ^ v 丄 30 | 00V ΙΗ1 ^ ° ν l Up 0 | OV in | V l 丄 丄 V | 丄 W | ds _S — c 峯 鋰 ½ mm Z0MC / 900Zdf / X3d 03 01780Ϊ0 / .00Ζ OAV In Tables 2 and 3, R is A or G, Y is C or T, M is A or C, K is G or T, S is C or G, W is A or T, H means A, C or T, B means C, G or T, V means A, C or G, D means A, G or T, N means A, C, G or T .

[0035] Restriction enzyme C can be selected arbitrarily as long as it satisfies the following conditions.

 (1) Site c is different from sites a and b

 (2) Site c is single, including the vector and shRNA generation unit (site c exists only in one place throughout the vector and does not exist in the shRNA generation unit)

 Restriction enzyme C is not particularly limited as long as restriction enzymes A and B have different blunt ends or cohesive ends.

 [0036] The vector is preferably provided with a selection marker such as drug resistance or auxotrophy. By providing a selection marker, the efficiency in recovering the product after genetic recombination described later from the transformant of the host can be improved. The selection marker can be selected depending on the host at the time of recovery, but in the case of prokaryotes such as E. coli, drug resistance markers such as kanamycin and ampicillin can be used. In addition, in order to facilitate recovery of the target multiplex shRNA expression vector, it is preferable to place the site c in a selection marker.

 [0037] After the first shRNA expression vector and the second shRNA expression vector are prepared, the following series of operations (i) to (2) are performed.

 (I) Digest the first shRNA expression vector with restriction enzymes A and C

 (Mouth) Digest the second shRNA expression vector with restriction enzymes B and C

 (C) Mix the digested fragments generated in (i) and (mouth) above and perform ligation.

 (2) Collect the circular construct in which the a-c fragment containing the first shRNA generating unit of the first shRNA expression vector and the b-c fragment containing the second shRNA generating unit of the second shRNA expression vector are connected.

[0038] In the steps (i) and (mouth), digestion is performed under conditions suitable for the selected restriction enzyme. After the steps (i) and (mouth), if necessary, the desired digested fragment, that is, the ac fragment containing the first shRNA generating unit derived from the first shRNA expression vector, derived from the second shRNA expression vector Each bc fragment containing the second shRNA generating unit may be isolated and purified. As an example of the isolation and purification method, fragments can be fractionated by electrophoresis, and fragments of the desired length can be recovered from the gel and purified. (C) The ligation reaction in the process can be carried out according to a conventional method.

[0039] Throughout the process (e), a general genetic engineering technique is not particularly limited as a technique for recovering the target circular construct, that is, the multiple shRNA expression vector. As an efficient technique, there is a method in which a host such as E. coli is transformed and recovered from the transformant. When the vector has a selection marker, efficiency can be increased by selecting a transformant using the selection marker as an index. Furthermore, by providing the restriction enzyme C site c in the selection marker, the target multiple shRNA expression vector can be recovered more efficiently. In other words, it is possible to selectively sort the products that have been ligated as intended using the selected marker as an indicator.

 [0040] As described above, the finally produced multiplex shRNA expression vector connects the ac fragment containing the first shRNA generation unit and the bc fragment containing the second shRNA generation unit derived from the second shRNA expression vector. Has been formed. In other words, a-c fragment a and b-c fragment b were connected, and c were connected. Here, when a and b are connected, a sequence is formed that is no longer digested by either restriction enzyme A or restriction enzyme B. Therefore, the finally produced multiplex shRNA expression vector has two or more shRNA generating units, and the site b derived from the first shRNA expression vector, the site a derived from the second shRNA expression vector, The site c derived from the first and second shRNA expression vectors connected to each other and formed again has a structure with a site c. Therefore, the multiple sh RNA expression vector generated here can be used as a starting material again as the first shRNA expression vector or the second shRNA expression vector which is the raw material of the production method. By using the obtained multiplex shRNA expression vector as a starting material, the number of shRNA generation units can be increased freely.

 [0041] [Third embodiment]

Thirdly, the present invention relates to a vector for producing a multiple shRNA expression construct. Hon Baek The vector is a vector for use in the production of the first or second shRNA expression vector that is the production raw material of the second embodiment. Specifically, it is as follows.

 The vector of the present invention comprises a promoter, a cloning region capable of cloning shRNA coding DNA provided downstream of the promoter, and one or more selectable markers. A recognition digestion site a of restriction enzyme A is provided upstream of the promoter, a recognition site b of restriction enzyme B is provided downstream of the cloning region, and a recognition digestion site c of restriction enzyme C is included in one selection marker. Is provided. A and B are restriction enzymes that can generate connectable ends, and restriction enzyme C is a restriction enzyme that can form a stump that cannot be connected to the digestive ends of A and B. The a and b sites recognized by the restriction enzymes Α and Β are provided as a single site in the vector.

 [0042] As described above, in order to carry out the production method of the second embodiment, it is necessary for the vector to have sites a, b, and c corresponding to the restriction enzymes A, B, and C. In order to facilitate the installation of such restriction enzyme sites, an empty vector in which restriction enzyme sites used for the production of the multiplex shRNA expression vector of the present invention are prepared in advance (an empty vector is a DNA encoding shRNA). Providing a pre-staged vector) can facilitate the production of multiple shRNA expression vectors. In addition, by providing one or more restriction enzyme sites with a single recognition site different from the recognition sites of restriction enzymes A, B, and C as the cloning region for inserting DNA encoding shRNA, the first or second shRNA It is possible to facilitate insertion of DNA encoding the expression vector.

 Since the types of vectors and promoters that can be used are as described in the first embodiment, the description thereof is omitted. Since the restriction enzymes A, B, C and the corresponding sites a, b, c are as described in the second embodiment, including preferred embodiments, the description thereof is omitted.

 [0043] [Fourth embodiment]

 The present invention relates to a pharmaceutical composition comprising the multiple shRNA expression vector of the first embodiment as a component.

By using the target shRNA expressed from the multiple shRNA expression vector shown in the first embodiment as a disease-related gene, this shRNA expression vector can be used as a pharmaceutical product. can do. For example, as shown in the Examples described later, a multiple shRNA expression vector targeting a plurality of SMAD families is useful for the prevention and treatment of diseases involving the TGF- | 8 pathway, such as cancer. In particular, knocking down Smad2, Smad3, and Smad4 simultaneously with multiple shRNA expression vectors strongly inhibits the TGF-β pathway and induces apoptosis more strongly than knocking down either gene alone. It was shown that it was possible. Therefore, multiple shRNA expression vectors targeting these Smad2, Smad3, and Smad4 are expected to be applied as anticancer agents.

[0044] Regarding other diseases, if the cause of the disease is caused by the expression of multiple genes, the multiple shRNA expression vectors of the present invention are used to simultaneously suppress the expression of these causative factors. This makes it possible to prevent and treat diseases. Therefore, the multiple shRNA expression vector of the present invention can be applied to the development of therapeutic agents for diseases caused by gene expression, particularly, expression of two or more, preferably three or more genes. All prior art documents cited in this specification are incorporated herein by reference.

 Example

 [0045] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples. First, in describing the example, the method and materials used in this example will be described.

 [Methods and Materials]

 1) siRNA design and construction

Four different sequences targeting the Smad2, Smad3, or TGFRB2 gene were selected using a unique algorithm (9). In order to improve silencing activity and to overcome the technical obstacles described in the text, many C to T or A to G mutations were introduced only into the hairpin loop sense DNA, and the antisense DNA and sense DNA Multiple mismatches were formed. To construct a single hairpin RNAi vector, it was mixed with 1 M NaCl (11). Incubate the mixture at 95 ° C for 2 minutes, quickly cool to 72 ° C, and gradually cool to 4 ° C over 2 hours to allow sense and antisense Rigonucleotides were annealed. We diluted the annealed oligonucleotide 200-fold with TE buffer and used 1 μl for ligation with plasmid DNA. The pcPUR + U6i cassette plasmid (3-5 g) was digested with Bs pMI in the reaction solution (100 1). The digested reaction solution was electrophoresed. The gel piece containing the DNA fragment of the desired length was excised, and the DNA in the piece was purified using the MinElute gel purification kit (Qiagen). Using the DNA ligation kit version 2.1 (TAKARA), the linearized plasmid DNA and the annealed oligonucleotide were ligated. E. coli host cells were transformed with the ligation product.

Smad-2 and -3 double knockdown constructs were made as follows. pcPUR + U6-Smad2i was digested with BamHI and Seal, while pcPUR + U6-Smad3i was digested with Seal and Bglll (Step 1). A fragment containing the U6 promoter and hairpin loop unit was purified (step 2), followed by ligation to construct a double knockdown vector (step 3). The joint formed by filing the ends formed by Bglll and BamHI digestion can no longer be cleaved by either Bglll or BamHI. Thus, the same technique can be repeated when coding regions of multiple siRNAs are sequentially incorporated into the vector. The Seal site is present in the ampicillin resistance gene, which reduces the number of background bacterial colonies. The same method was applied to other double or triple knockdown constructs. The pcPUR + U6i cassette (Non-Patent Document 13), pcPUR + U6iGFP, and pcPUR + U6 + Smad4i (Non-Patent Documents 7 and 12) have already been reported. pCMV5- TGF jS RII / HA was provided by J 丄. Wrana, and pc DEF3-Flag (N) -Smad2 and pcDEF3-Flag (N) -Smad3 were donated by K. Miyazono. (CAGA) 9-Luc (Non-Patent Document 16) was donated by SE Kern. And p3TP-Lux was donated by J. Massague and has already been reported (Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang X—F, Massague J. TuF- β sign als through a heteromeric protein kinase receptor complex. Cell 1992; 71: 1003-1014). PRL-SV40 (Promega) containing the Simian virus (SV) 40 promoter upstream of the Renilla luciferase coding sequence was used as an internal standard in the luciferase assembly. [0047] 2) Cell culture and transfection

 The HeLa cell line (American Type Culture Collection, Rockville, MD) was passaged in Dulbecco's modified Eagle's medium containing 10% sushi fetal serum (Sigma, St. Louis, MO). Human keratinocyte cell line HaCaT (J Cell Biol. 1988 Mar; 106 (3): 761-71.Normal keratinization in a spontaneously immortalized aneuploid nu man keratinocyte cell line.Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE.) Were subcultured in Danolebecko modified ignore medium containing 10% urine fetal serum. Transfusion was performed using “Effectene” according to the manufacturer's instructions. To establish stable RNAi cells, the cells were selected by culturing for 2 weeks in the presence of 1 g of puromycin (Wako). To establish cells expressing stable shRNA, the puromycin concentration for selection in the HaCaT cell line is assayed and all cells that do not express puromycin die within 3 days. A concentration of 1 μg / ml was selected. Viable cells were observed in wells into which the siRNA expression vector was introduced. The first colony appeared at 2 weeks, and then cultured for another 2 weeks to create original stocks or grow to a density sufficient for the number of cells to perform expression analysis. Knockdown of target endogenous gene expression in stable cell lines was tested using the original stock and cells passaged 20 times from the original stock. The cells were further cultured in a medium containing puromycin (1 μg / ml) (Wako, Tokyo, Japan).

 [0048] 3) Western blot

 Western blotting was performed as described (Non-patent Document 18). Equal volume of protein is electrophoresed and the protein is diluted 250-fold by anti-Smad2 / 3 or anti-Smd4 antibody (BD Transduction Laboratories), 2500-fold diluted by anti-β-actin or anti-Flag antibody (Sigma), respectively. , Anti-antipox antibody (Roche), or anti-antipox AI-1 antibody (BD Transduction Laboratories) diluted 1000-fold.

 [0049] 4) Northern analysis

Total RNA was collected from stably knocked-down cells using Isogen Reagent (Wako, Tokyo, Japan) according to the attached document. Total RNA (5 g) is 18% (weight / volume ) Size fractionated with polyacrylamide urea gel and transferred to Hybond N + membrane (Amersham, Litt le Chalfont, UK). The membrane after transfer was dried at room temperature and fixed with ultraviolet light. The membrane is prehybridized with a solution (30% formamide, 10% dextran sulfate, 5x SSC, 0.5% SDS, 1 x Denhardt s solution, and 0.2 mg / ml salmon sperm DNA (Sigma Aidrich Co., Saint Louis, MO)). We did a dialysis. Hybridization was performed with a synthetic oligonucleotide probe at 36 ° C for 3 hours. This probe was composed of a sequence complementary to siRNA for Smad2, 3 and 4. As a loading control, a complementary probe to human tRNA palin was used. The probe sequence is as follows.

 • Probe for tRNA valin: (SEQ ID NO: 150)

 5,-GAA CGT GAT AAC CAC TAC ACT ACG GAA ACC CTA TAG TGA GT C GTA TTA GGC GGG AAC CGC CTA ATA CGA CTC ACT ATA GG-3 '

 • Probe for Smad2 siRNA: (SEQ ID NO: 98)

 5 '-GGATTGAACTTC ATCTGAA- 3',

 • Probe for Smad3 siRNA: (SEQ ID NO: 99)

 5 -GGATTGAGCTGCACCTGAA-3 '

 • Probe for Smad4 siRNA: (SEQ ID NO: 100)

 5 and GTACTTCATACCATGCCGA-3 '.

The synthetic probe was labeled with ³² P (Amasham, Little Chalfont, UK) using T4 polynucleotide kinase (Takara Shuzo Co., Kyoto, Japan). The membrane was washed twice at 36 ° C using 2x SSC and then analyzed using Fujix Bio-Image Analyzer BASIOOO (Fuji Photo Film Co. Ltd., Tokyo, Japan).

5) Luciferase Atsey

Luciferase activity has been described and carried out as described (Non-patent Document 18). Cells were transfected with (CAGA) 9-luc, or p3TP-Lux and pRL-SV40. After 24 hours, cells were incubated for an additional 24 hours in the presence or absence of 2.5 ng of TGF-811 (R & D Systems) for double luciferase assay. (CAGA) 9-luc or p 3TP-Lux firefly luciferase activity was normalized based on pRL-SV40 renilla luciferase activity. Cellular luciferase levels in the absence of TGF- | 8 1 The value 1.0 was assigned to calculate the relative activity. The experiment was performed 3 times, 2 pieces per sample.

[0051] 6) Quantitative reverse transcriptase PCR

 Quantitative RT-PCR analysis was performed using the ABI 7000 real-time PCR system (Applied Biosystems) as previously described (Non-Patent Document 13). The ratio of the mRNA level of each gene to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated and assigned a value of 1.0 to the parental cells. Each experiment was repeated twice, 3 per sample. The following primers were used.

 • BTBD1

 Forward (5'-CAAGGCGTCGCTGAAGGA-3 '; SEQ ID NO: 3)

 Reverse (5'- CCCAGTACGAAGCGCACAT-3 '; SEQ ID NO: 4)

 • Prominin 2

 Forward (5'-TGCCCTCTGTGGACCATGT-3 '; SEQ ID NO: 5)

 Reverse (5'-GGCGTTGAAGGTGCTGTTCT-3 '; SEQ ID NO: 6)

 • OAS1

 Forward (5'-AGGTGGTAAAGGGTGGCTCC-3 '; SEQ ID NO: 7)

 Reverse (5 ACAACCAGGTCAGCGTCAGAT-3 '; SEQ ID NO: 8)

 • GAPDH

 Forward (5'-CCACCCATGGCAAATTCC-3 '; SEQ ID NO: 9)

 Reverse (5'-TGGGATTTCCATTGATGACAAG-3 '; SEQ ID NO: 10)

 M13 (for sequence confirmation), reverse (5'-CAGGAAACAGCTATGAC-3 '; SEQ ID NO: 1

1)

 [0052] 7) Infiltration Atsey

Infiltration was measured with a 8 mm diameter Matrigel coated polycarbonate membrane (pore size 8 μm) using a BD Biocoat Matrigel Infiltration Chamber (BD Biosciences). Total were seeded 10 ⁵ knockdown or control HaCaT cells in the upper chamber of Ueru and cultured. After 24 hours, cells were incubated for an additional 24 hours in the presence or absence of TGF-8 | 8 (15 ng / ml). Next, wipe the cells on the upper surface of the filter paper with cotton and fix the cells that have infiltrated on the lower side of the filter paper, and use `` DiffQuick '' (Dade Behring) After staining, five random fields were selected with a bright-field microscope, and stained cells in the field were counted at 200x magnification. The experiment was repeated three times.

[0053] 8) Wound closure

 Wound closure assembly was performed as previously described (13). Confluent cell monolayers were damaged by exfoliation using a 1.2 mm wide pipette tip and incubated for 24 hours in the presence or absence of TGF-j8 15 ng / ml. Cell migration was carefully observed at various times. The experiment was repeated three times.

 7) Apoptosis

Inoculate 2 x 10 ⁶ cells in a 10 cm culture dish, incubate for 24 hours in the presence or absence of TGF- (15 ng), and use ApopTag fluorescein in situ apoptosis detection kit S71 11 (Serologicals Corporation) Apoptosis was analyzed. For the measurement, a Betaton Dickinson flow cytometer equipped with “CellQuest” software was used. The experiment was repeated three times, two per sample.

 [Example 1]

 (1) Preparation of single siRNA expression vector

 Using software based on various algorithms for effective siRNA design that have already been reported (Non-patent Document 9), Smad2 gene (SEQ ID NO: 12), Smad3 gene (SEQ ID NO: 13), and TGFRB2 gene ( For each of SEQ ID NO: 14), four target sites with a chain length of 21 nucleotides were designed (Table 4). In addition, Smad4 has already been reported and the target site was used.

[0055] [Table 4]

TGFBR2 site 1 5'-atgagcaactgcagcatcacc-3 '(SEQ ID NO: 1 0 1)

site 2 S'-aggccaagctgaagcagaaca-S * (SEQ ID NO: 102) site 3 5'-acggctccctaaacactacca-3 '(SEQ ID NO: 1 03) site 4 5'aaggacatcttctcagacatc ^_ 3 ^T (SEQ ID NO: 10 04)

Smad2 site 1 5'-ggattgaacttcatctgaatg-3 '(SEQ ID NO: 1 05)

site 2 5'-ggcctgatcttcacagtcatc_3 '(SEQ ID NO: 1 06) site 3 5 ^T -ggtttactctccaat ^ ttaac-3' (SEQ ID NO: 1 07) site 4 5 ^T -gccacctcctggatatatcag- 3, (SEQ ID NO: 1 08)

Smad3 site 1 5'-ggccagacctgcacagccacc-3 '(SEQ ID NO: 1 09)

site 2 5'-ggattgagctgcacctgaatg-3 '(SEQ ID NO: 1 1 0) site 3 5'-gagttcgccttcaatatgaag-3' (SEQ ID NO: 1 1 1) site 4 5'gggctgctctccaatgtcaac-3 ^T (SEQ ID NO: 1 1 2)

Smad4 site 1 5 'GTAATGATGCCTGTCTGAGCA-3' (SEQ ID NO: 1 1 3)

site 2 5'_GGATTTCCTCATGTGATCTAT-3 '(SEQ ID NO: 1 14) site 3 5' GTAGGACTGCACCATACACAC-3 '(SEQ ID NO: 1 1 5) site 4 5 ^f GAAACACCTTGCTGGATTGAA-3 ^f (SEQ ID NO: 1 1 6) site 5 5 ^† GTACTTCATACCATGCCGATT-3 '(SEQ ID NO: 1 1 7) To improve silencing activity and technical difficulties such as frequent sequencing of siRNA expression constructs or the difficulty of sequencing due to the nordromic sequence We designed an array that overcomes various obstacles. As shown in Table 5, the sense strand inserts two or more C, T, A or G point mutations into the target site sequence, and the antisense strand can induce RNA interference so that it can induce RNA interference. The sequence was kept complementary. Regarding Smad4, the sequence was designed by inserting a mismatch into the sense strand of the target sequence shown in Table 4 for which the inhibitory effect was already reported. For Smad4, it is possible to select other target regions and design other shRNA sequences based on the gene sequence (SEQ ID NO: 15).

[Table 5] Smad2 region 1 sense 5'-ggattga ^ cttiatftgaatg-3 '(SEQ ID NO: 1 6) antisense 5'-cattcagatgaagttcaatcc-3' (eyes L! Sequence r: 1 I) region 2 sense 5'-ggcftgatctt ne gt ate-3 '(Distribution' J 畨 号: 1 8)

antisense 5'-gatgactgtgaagatcaggcc-3 '(eyes 歹¹ J number-: 1 9) region 3 sense 5'-ggtttactrtciagtgttaac-3' (SEQ ID NO: 20) antisense 5'-gttaacattggagagtaaacc-3 '(eyes d 歹') ¾ ·: 2 1) Region 4 sense 5'-gccacftcftggatatatfag-3 '(SEQ ID NO: 22) antisense 5'-ctgatatatccaggaggtggc-3' (酉 己 歹 ϋΐ! · Τ7: 23

Smad3 region 1 sense 5'-ggccagacftgiaiagciacc-3 '(酉 ΰ row ¼: 24) antisense 5'-ggtggctgtgcaggtctggcc-3' (Bti 歹¹ J r ： 2 o) region 2 sense 5'-ggattgagftg fctgaatg.3 '(酉[! 歹 ϋ 番 ^ ": 26) antisense S'-cattcaggtgcagctcaatcc-d '(酉 row ¾":) Region 3 sense 5'-gagttigfcttiaatatgaag-3' (SJ row, number: 28) antisense 5'-cttcatattgaaggcgaactc-3 '(Eyes d 歹¹ J number: 29) domain 4 sense 5'-gggitgftctfcaatgt aac-3' (酉 己酉 'J number: 30) antisense 5'-gttgacattggagagcagccc-3' (酉 self 歹¹ J equipment: 3 1 )

Smad4 domain 5 sense 5'- gtacttcata ifetgfcgatt -3 '(B self' J number: 32) antisense 5'-aatcggcatggtatgaagtac -3 '(S self ¹ J number 3 3)

TGFBR2 region 1 sense 5'-atgag artg gfetcacc.3 ' ( Rooster ΰ歹^{1: 34) antisense 5'-ggtgatgctgcagttgctcat} -3' ( SEQ ID NO: 35) regions 2 sense 5'-aggciaagftgaagiagaaca-3 ' (a] evening ^{- 1} J number: 3 o) antisense 5'-tgttctgcttcagcttggcct-3 '(酉 己酉¹ J number: 3 7) Region 3 sense 5'-acggftcfctaaaiactacca-3' (酉 己 歹¹ ) ¾ ■: 3 8) antisense 5 '-tggtagtgtttagggagccgt-3' (酉 L! sequence number ^ ": 39) region 4 sense 5'-aagga¾tcttctfega ne tc-3 '(sequence number 40) antisense 5'-gatgtctgagaagatgtcctt-3' (distribution ¹ Jft: 4) ) Mismatch bases are shown in italics.

 [0056] A linker DNA is connected to the sense strand and the antisense strand to construct a DNA encoding shRNA, and a DNA strand in which a restriction enzyme site for cloning and a transcription termination codon are inserted is chemically synthesized. The complementary strand was also chemically synthesized (Table 6). These two DNA strands were annealed to form double stranded DNA fragments.

[0057] [Table 6]

 Four

 (Sequence number): 47 Arrangement ^ (column): 46th ^ (column) 5: 4 Number (column): 44

 site 2 5cacccrtatcttaiat tcttcttccat-- ''

Number) (column: number 34 ^ (column): 42 si 1 TGFBR2 site 1 S 'accatgag ^ a / te ^ g ^ tcacc ^^ gct ^ ccggtsatectecaett ^ ctcatttttt-S'

 (Sequence number ^: 5 8)

 S'-gcataaaaaatgagcaactgcaecatcaccsgacaecacacgstgat ^ ctaca ^ tt ^ ctcat-S '

(SEQ ID NO: 5 9)

 site 2 5'_ciicciiggc grtgaag aaca t gtcct tctscttcagcttracctttttt-3 '

(SEQ ID NO: 6 0)

 5'-ecataaaaaae2ccaaect £: aagcagaacaggacagcacactsttctacttcaactt ^ cct-3 '

(Array number ^: 6 1)

 site 3 S'-caccacgffftc ctaaaiactaccagtgtgctstcctggtastgtttagsgragccstttttt-S '

(Sequence number ¾: 6 2)

 D'-srcataaaaaacE ectccctaaacactaccaegacaecacactegtaetatttaaaga ^ ccet-S '

(SEQ ID NO: 6 3)

 site 4 S'-caccaagea i ^ cttct ^ ga ^ tcst ^ ectetccgatgtct ^ asaaeatetccttttttt-S

(Sequence number: 6 4)

 S'gcataaaaaaaggacatcttctcagacatcggacagcacaceratatct ^ aeaaea ^ tcctt-S '

(SEQ ID NO: 6 5) Note) Mismatch sequences are shown in italics. The linker (loop) sequence is shown underlined. The terminator (transcription termination codon) is indicated with a double underline. BspMI recognition sites are shown with broken lines.

 [0058] The DNA fragments generated by annealing the DNAs complementary to each other were subcloned into pcPUR + U6-i cassette (Non-patent Document 7) to construct siRNA expression vectors. To screen silencing efficiency with these siRNA expression vectors, these vectors were transiently co-transformed into HeLa cells along with Smad2-, Smad3- or TGFRB2-expression vectors. After 48 hours of transfection, cells were collected, a cell lysate was prepared, and Smad2, Smad3 or TGFRB2 protein was detected by Western blotting (Fig. La).

[0059] siRNA targeting one of the four sites in Smad2, three of the four sites in Smad3, and all four sites in TGFRB2 Suppresses the expression of the target gene by 0-10% and is effective (Figure la). Select the target site that was found to be most effective for knocking down the target gene, specifically site 1 for Smad2, site 2 for Smad3, and site 1 for TGFRB2, and the following examples Used for. Expression vectors containing these sites knock down the Smad2, Smad3, or TGFRB2 protein, respectively, so "pcPUR + U6- Smad2i", "pcPUR + U6- Sm ad3i ”and“ pcPUR + U6-TGFRB2i ”. A schematic diagram of pcPUR + U6-Smad2i is shown as a representative (Figure If).

 [0060] Since Smad2 and Smad3 are proteins with extremely high homology to each other, the above silencing activity is determined whether Smad3 can be knocked down by PCPUR + U6- Smad2i and Smad2 can be knocked down by pcPUR + U6-Smad3i. Similar to the measured experiments, co-transfection with Smad3— or Smad2—expression vectors in Hela cells was followed by Western blot analysis (Figure lb). PcPUR + U6-Smad2i and pcPUR + U6-Smad3i, which are highly homologous to each other and target the Smad2 and Smad3 genes encoding proteins, respectively, can specifically knock down the target gene without cross-over effect. confirmed. Thus, the present inventors succeeded in obtaining knockdown vectors capable of specifically suppressing a single gene for Smad2, Smad3, and TGFRB2, respectively.

 [0061] (2) Development of double or triple siRNA expression vectors

Next, the present inventors tried to produce a multiple knockdown vector capable of simultaneously suppressing a large number of genes by combining these constructs. The pcPUR + U6 vector has a Bglll recognition site upstream of the U6 promoter, a BamHI recognition site downstream of the RNAi insertion region, and a Seal recognition site in the ampicillin gene. All of these restriction enzyme recognition sites exist in the plasmid. To create a double knockdown vector targeting Smad2 and Smad3, we digested pcPUR + U6-Smad2i with BamHI and Seal and PCPUR + U6- Smad3i with Bglll and Seal (Figure 1). Step 1) in c, the fragment containing the expression cassette was ligated to create a single vector that could double knockdown both Smad2 and Smad3 (Step 2 in Figure Id). Double knockdown vectors for Smad2 and Smad3, Smad2 and Smad4, and Smad3 and Smad4 were named PCPUR + U6- Smad23i, pcPUR + U6- Smad 24i, and pcPUR + U6-Smad34i, respectively (Fig. Lf). A triple knockdown vector pcPUR + U6-S mad234i targeting Smads (S mad2, Smad3, and Smad4) associated with all pathways was also constructed in the same way (Figure If). The advantage of this system is that the ends formed by Bglll and BamHI deletions are sticky ends with protruding complementary single strands, but the binding sites after ligation are also cleaved by Bglll or BamHI. You I can't. In other words, even in a new vector formed by linking two plasmids, one BglII, BamHI, and Seal recognition site can be maintained. Therefore, multiple knockdown vectors targeting a large number of genes can be easily produced by repeating the same technique. It should be noted that the only seal site in the ampicillin resistance gene is involved in reducing the number of knock-down bacterial colonies.

[Example 2] Confirmation of Smads knockdown

 In order to examine the effectiveness of the single or multiple knockdown vectors, the present inventors analyzed using a human keratinocyte cell line HaCaT having a functional TGF-β pathway. This cell line is known to be suitable for analysis of TGF-8 signaling (Non-patent Document 19). Transfect the above vector into HaCaT, and after 5 weeks, select the stable knockdown cell lines S2KD, S3KD, S4KD, S23KD, S24KD, S34KD, S234KD, and iGFP selected with 1 μg / m1 puromycin Respectively. Western plot analysis was performed using the disrupted solution obtained by disrupting these cells (Fig. 2a). In addition, the present inventors confirmed that there was no cell change due to the genomic integration of the plasmid using a stable cell pool that was a mixture of puromycin-resistant polyclonal cells, but not an independent cell clone. .

 [0063] As shown in Figure 2a, most of the stable transformants (lanes 2-8) have a dramatically reduced expression of the endogenous Smad2, Smad3, and Smad4 proteins as intended. It has been shown. In particular, the ability to express three different siRNAs in S234KD cells. This technique knocked down three target genes. Northern analysis shows that the same amount of siRNA for Smad2, Smad3, and Smad4 is stably expressed in single, double, and triple knockdown cells, and is maintained even after prolonged incubation (31 hours of transfection) (Figure 2b).

On the other hand, with the knockdown vector targeting TGFRB2, a stable TGFRB2 knockdown cell line could not be obtained due to the massive cell death that occurred after transfection. However, this result suggests that TGFRB2 is important for cell survival. The silencing efficiency of each gene in multiple knockdowns was unaffected by the location or number of siRNA expression constructs inserted into the vector (data shown Not)

 [0064] To induce silencing of non-target messenger RNA (mRNA) transcripts, 11-15 contiguous nucleotide sequences identical to siRNA have been reported to be sufficient (Non-patent Document 21). The inventors carefully selected the target sequence by using a unique algorithm, but could not rule out the possibility of knocking down non-specific genes. To investigate silencing of non-target genes in our cells, we use the BLAST database (http: 〃 www.ncbi.nlm.nih.gov/BLAST) The human genomic force was also searched for the presence of any complementary sequence with at least 11 contiguous nucleotides matching the RNAi site used. Regarding the TGFRB2 site, no gene appeared to have sequence similarity. Regarding Smad2 site 1, one protein containing the BTB / POZ domain (hereinafter referred to as “BTBD1”!) Was matched with 17 adjacent nucleotides, and the number of others searched was low in similarity. . The 5-transmembrane glycoprotein (prominin 2) had 15 contiguous nucleotides similar to site 2 of Smad3.

 [0065] The present inventors examined the mRNA expression of non-target genes in knockdown cells using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was recovered from knockdown cell lines (S2KD, S3KD) and control parental HaCaT cells or iGFP control cell lines, and RT-PCR was performed (Fig. 2c, d).

The expression of non-target genes BTBD1, promin 2 and OAS1 is decreased in SK2D or SK3D cells compared to parental HaCaT cells (Figure 2b, c) or iGFP control cell line (data not shown). It ’s nasty. From this, we conclude that non-target gene silencing existed and was viable in established cell lines. Another report demonstrated that the interferon response was induced in mammalian cells by the U6 promoter Z lentiviral shRNA construct (21). Interferon induces OAS1, a double-stranded RNA (dsRNA) -linked enzyme, that is activated in a dsRNA-dependent manner. Our construct was checked for OAS-1 induction to rule out any non-specific RNA degradation due to the inability of lentivirus. Theoretically, triple knockdown cells that could produce more siRNAs could induce more OAS-1, but the knockdown cell line and the parent HaCaT cell line (Figure 2e) Or iGFP pair There was no statistical difference in OAS-1 mRNA between the tumor cell line (data not shown).

 [0066] [Example 3] Functional analysis of TGF-β pathway

 Since it was suggested that the pcPUR-U6 construct has high specificity for the target gene and is effective in gene silencing, the functional analysis of the TGF-β pathway is performed using these cells. It was decided.

 Functional analysis of the TGF-β pathway was performed by luciferase assay using Smad-dependent reporter (CAGA) 9-Luc. In luciferase atsey, signal transduction dependent on canonical TGF-β-Smad was markedly inhibited in all cell lines except S2KD (Fig. 2f). Conservation of luciferase induction by TGF-j8 in S2KD cells (Fig. 2f) suggests that Smad3 and Smad4 bind to "CAGA" Smad binding elements. Consistent with literature 16).

 On the other hand, luciferase assay was performed using a p3TP-Luc reporter plasmid containing an element different from the “CAGA” Smad binding element. The p3TP-Luc reporter plasmid is activated via three TRE elements derived from the human collagenase gene (Smad) in the -740 / -636 region of the PAI-1 promoter, which is selectively induced by TGF-jS. A luciferase gene is connected downstream of a sequence that binds an element specific to the activin pathway of the TGF-β family. When this p3TP-Luc was used as a reporter plasmid, the induction of luciferase activity in S2KD cells when TGF-8 was added was down-regulated to 20% of control cells (data not shown). ,).

[0068] The present inventors examined the induction of -1AI-1, a well-known TGF-β-responsive gene, in knockdown cell lines. In order to perform this analysis, the knockdown cell line was cultured in the presence or absence of TGF- | 8, and then a cell lysate was prepared and subjected to Western plot analysis (FIG. 2g). As a result of the analysis, it was shown that PAI-1 inducibility caused by TGF-8 | 8 was inhibited in all knockdown cell lines except S2KD cell line. The PAI-1 Western blot results were consistent with the reporter assembly results shown above (Figure 2f). This suggests that TGF-jS-mediated PAI-1 induction is highly dependent on the “CAGA” Smad binding element in its promoter region. [Example 4] Phenotype of Smads knockdown cell line

 Findings regarding functional differences among multiple Smad proteins can be found in Smad dominant-negative constructs (Non-patent Document 22), antisense RNA (Non-patent Document 23), and siRNA duplexes (Non-patent Document 24). Accumulated in the past through use. These studies have focused primarily on changes in molecular targets or signaling rather than phenotypic changes, as phenotypic observations have been limited due to the transient nature of previous methods. I was there. In contrast, by obtaining a stable RNAi construct as described above, we were able to achieve TGF-β such as infiltration, wound closure, and apoptosis when knocking down Smad. -Smad-dependent phenotype focused on changes

[0070] (1) Infiltration

 We prayed for TGF-dependent invasion by an in vitro system using a matrigel infiltration chamber (Fig. 3b). Although there is a report that loss of Smad4 in mice results in increased metastasis and tumor invasion (Non-patent Documents 25 and 26), our results indicate that invasion of S4KD cells in the absence of TGF- It indicates that the activity is lower than any other cell, and that TGF-β strongly stimulates invasion (41.3-fold increase). In contrast, S2KD and S3KD cells have invasive characteristics at the basal level, but a weak response to TGF- | 8 (8.3 and 3.5 fold increase, respectively). TGF-β-dependent invasion of S23KD, S24KD, and S34KD cells was not statistically significant compared to control iGFP cells (fold increase was 4.3, 4.3, 3.8, and 3.8 times, respectively). The fold increase in invasion by TGF- in S4KD cells was more than 10 times that of control cells with intact Smad4 (Fig. 3a, b). Invasion induced by TG F- | 8 is statistically significantly reduced in triple knockdown cell lines compared to Smad4 knockdown cells (magnification, 8.25 times), and cells lacking Smad4 function Suggests that Smad2 and Smad3 are important for TGF-8 | 8-mediated cell invasion.

[0071] (2) Wound closure

Since TGF-18 is known to promote wound closure by activating cell migration, we have established TGF- | 8 in an established knockdown cell line. Was examined to induce wound closure. The confluent cell monolayer was damaged by a 1.2 mm wide tip, and wound closure was observed 24 hours after administration of TGF-. In our previous report, Smad4 knockdown inhibited wound closure induced by TGF-jS in Pac-1 cells (Non-patent Document 13). We observed similar results in HaCaT cells. As shown in Figures 3c and 3d, wound closure was accelerated when lost alone with Smad3 or with Smad2 or Smad4. This finding is consistent with a report in Smad3 knockout mice (Non-patent Document 27). In contrast, S2KD and S23 4KD cells were weaker in wound closure compared to control cells (FIGS. 3c, d). All Smads are downstream mediators of the same TGF-jS pathway. Each Smad appears to have a different involvement in cell migration.

(3) Apoptosis

 The effect of TGF-β on cell apoptosis varies greatly depending on the cell type (Non-patent Document 25). In HaCaT cells, TGF-j8 has been reported to increase cell viability by suppressing apoptosis through Akt-dependent regulation of FKHRL1 (Non-patent Document 28). We examined whether TGF- ability was affected by the destruction of its downstream regulators (Fig. 3d). The percentage of apoptotic cells for different cell types in the presence of TGF- | 8 in the absence of Z was as follows: iGFP 0.21 / 0.15

 S2KD 0.61 / 1.01

 S3KD 1.64 / 1.78

 S4KD 0.29 / 0.31

 S23KD 1.41 / 2.50

 S24KD 1.47 / 2.5

 S34KD 1.48 / 1.31

 S234KD 1.42 / 12.32

Surprisingly, TGF-j8 still prevented apoptosis in single or double knockdown cells. When the inventors knocked down all Smads (Smad2, Smad3, and Smad4), TGF-18 lost its anti-apoptotic function and lost apoptosis. Obtained the guidance function. The underlying mechanism is unknown, but our data is

By knocking down all Smads in this cell line, we suggest that the Smad-independent TGF-β pathway transmits apoptotic signals.

[Example 5] Design of shRNA sequence for HIV knockdown

 Next, we designed an shRNA to knock down HIV. The next 16 target sites of shRNA were selected based on the HIV genomic DNA sequence.

 Sitel AGACCAGGATGAACAACAA (SEQ ID NO: 118)

 Site2 AGACAAAGATGACTTATAA (SEQ ID NO: 119)

 Site3 AGAGAAGAATTCACTTAGA (SEQ ID NO: 120)

 Site4 GAGAAAGCGCTTAAGTTTA (SEQ ID NO: 121)

 Site5 GGATGAACAACAATCAAAC (SEQ ID NO: 122)

 Site6 GCGACATAGTTTACAGAGA (SEQ ID NO: 123)

 Site7 GGTGAGATGCTAAACACAT (SEQ ID NO: 124)

 Site8 AGAACACCTTATATCCCAA (SEQ ID NO: 125)

 Site9 GGAAGAAGCTGATTGAGAG (SEQ ID NO: 126)

 Site 10 GATAGAGTGTACATAACAG (SEQ ID NO: 127)

 Sitel 1 ACCCAAAGATTATCCAGGA (SEQ ID NO: 128)

 Site 12 GGCAGGACATTGGTGACAT (SEQ ID NO: 129)

 Site 13 GGGACAACTCTATCAAGAT (SEQ ID NO: 130)

 Sitel4 GCTCTGACGTGCATAGGAA (SEQ ID NO: 131)

 Site 15 GGCTCAAATGTATAGAAAG (SEQ ID NO: 132)

 Site 16 AATGGAACGTCATCTGTGA (SEQ ID NO: 133)

[0074] In the same manner as the construction of the shRNA for knocking down the Smad gene targeting the above sequence, a DNA sequence encoding the shRNA by connecting the antisense strand and the sense strand containing the mismatch sequence with a linker DNA. (Table 7). Examples of shRNA-encoded DNA for each target site are shown. This sequence can be changed depending on the presence / absence of mismatch, position, etc. In Table 7, italics indicate mismatch sequences, a single underline indicates a linker DNA, and a double underline indicates a terminator sequence. [Table 7]

Site 1

 CTTTTTT (K column number: 1 34)

 Site 2: AGA 7AAAGATGA 7TT 6TAAACGTGTGCTGTCCGTTTATAAGTCATCTTTGT

CTTTTTT (SEQ ID NO: 1 35)

 Site 3: AG GGAAG GATTCA 7TTAGA. CGTGTGCTGTCCGTTCTAAGTGAATTCTTC

TCTTTTTT (SEQ ID NO: 1 36)

 Site4 ： GAG GAAG 7ϋ 7TTAAGTTT.4ACGTGTGCTGTCCGTTAAACTTAAGCGCTTT CTCTTTTT (SEQ ID NO: 1 37)

 Site 5:

 TCCTTTTT (SEQ ID NO: 1 38)

 SiteG:

 CGCTTTTT (SEQ ID NO: 1 39)

 Site ?: GGTGGGATG 7TAAA mCATACGTGTGCTGTCCGTATGTGTTTAGCATCTC ACCTTTTT (SEQ ID NO: 1 40)

 Site8: AGGACAC 7TTATATC 7 AAACGTGTGCTGTCCGTTTGGGATATAAGGTGT

TCTTTTTT (SEQ ID NO: 14 1)

 Site9: GG <7AGAAG7TGATTG6GAlACGTGTGCTGTCCGTTTCTCAATCAGCTTCT TCCTTTTT (SEQ ID NO: 1 42)

TATCTTTTT (SEQ ID NO: 5: 143)

 Site 11

 GGTTTTTT (SEQ ID NO: 1 44)

GCCTTTTT (SEQ ID NO: 145)

 CCCTTTTT (SEQ ID NO: 146)

 Site 14: GCT TTGA 7GTGCAT GGGAAACGTGTGCTGTCCGTTTCCTATGCACGTCAG AGCTTTTT (SEQ ID NO: 1 47)

GCCTTTTT (SEQ ID NO: 148)

 Site 16 AATGG Gk 7GTCAT 7 GTCx. ACGTGTCxCTGTCCGTTCACAGATGACGTTCCA TTTTTTT (SEQ ID NO: 1 49)

A multiple shRNA expression vector for suppressing and protecting against HIV infection is prepared by introducing two or more DNA sequences encoding each sh RNA in Table 7 above into a vector. By introducing this vector into cells infected with HIV, Activities such as v multiplication can be suppressed. That is,

 A method for treating HIV infection, comprising the step of administering a multiple shRNA expression vector having a plurality of shRNA generating units, wherein any one of the nucleotide sequences set forth in SEQ ID NOs: 134 to 149 is connected downstream of the promoter,

 Use of a multiple shRNA expression vector having a plurality of shRNA generating units each comprising one of the nucleotide sequences set forth in SEQ ID NOs: 134 to 149 connected downstream of the promoter, for the manufacture of a therapeutic agent for HIV infection,

 Is possible.

 Industrial applicability

[0076] In the above examples, it was shown that the multiple shRNA expression vector of the present invention is effective in silencing two or more targets simultaneously. Both the technology for creating knockout animal models of multiple genes and the stable RNAi technology targeting multiple genes according to the present invention have the common feature of stably suppressing the expression of a large number of genes, which is a different technology. Have. The former is a technology that has left enormous achievements in life science research so far, but the method of the present invention cited as the latter is expected to be a technology that can replace the former.

 The mechanism of RNAi is a cyclically acting enzyme complex, and once activated, a single RISC complex can possibly cleave multiple copies of multiple RNA targets (29). ), This feature could effectively silence many targets simultaneously. If effective shRNA does not work due to the low concentration of shRNAs produced, efficient knocking can be achieved by increasing the number of copies of the RNAi cassette carried on the plasmid by a factor of 2, 4 or more. The results shown in this example suggest that the construct can be downed.

[0077] The advantage of this system is that the number of shRN As can be increased without significantly changing the overall plasmid length. For example, if the upper limit of the length of plasmid DNA that can be introduced into cells by transfection is 20 kb, theoretically, one siRNA expression plasmid should carry 30 or more shRNA cassettes. It will be possible. Therefore, the best target that can be effectively silenced using this method. In order to determine the large number or copy number, those skilled in the art will be able to adjust appropriately depending on the cells used, the amount of target, and the regenerative capacity.

 As described above, the present invention will be a technique widely used in the field of life science such as functional analysis of various genes and identification of drug discovery targets as a technique for suppressing the expression of a large number of target genes. .

Claims

The scope of the claims

 [1] A multi-shRNA expression vector carrying a plurality of shRNA generation units in a vector capable of generating shRNA for suppressing the expression of a target gene,

 The shRNA generator is configured by connecting DNA encoding shRNA downstream of the promoter,

 The DNA encoding the shRNA is composed of an antisense DNA having a sequence complementary to the target gene and a sense DNA comprising a sequence capable of pairing with the antisense DNA.

Multiple shRNA expression vector constructed by ligation with DNA.

 [2] The multiplex shRNA expression vector according to claim 1, wherein the sense DNA contains a mismatch sequence that does not complement the antisense DNA.

 [3] The multiplex shRNA expression vector according to claim 2, wherein the mismatch sequence is contained in the sense DNA in an amount of 2 to 55%.

[4] The mismatch sequence power on the sense DNA is formed by substitution, deletion, insertion, addition, or a combination thereof in relation to the corresponding base on the antisense DNA. Multiple shRNA expression vector.

[5] The multiple shRNA expression vector according to claim 4, wherein the substitution is performed by changing C on the sense DNA to T or G to A.

[6] The multiple shRNA expression vector according to claim 1, wherein the vector is a vector that induces transient expression or a vector that induces stable expression.

[7] The multiple shRNA expression unit according to claim 1, wherein the plurality of shRNA generation units carried in the vector correspond to the displacement force or a combination of two or more of the following (1) to (3): Kuter.

 (1) The same shRNA generation unit targeting the same site of a single gene can be used multiple times

(2) Consists of multiple shRNA generation units targeting two or more different sites of a single gene

 (3) Consists of multiple shRNA generation units targeting two or more different genes

[8] The multiple shRNA expression vector according to claim 1, wherein the promoter is a polIII promoter.

[9] A method for producing a multiple sh RNA expression vector from a circular structure first shRNA expression vector having a first shRNA generation unit and a circular structure second shRNA expression vector having a second sh RNA generation unit as raw materials Because

 Similarly, the first and second shRNA expression vectors are provided with recognition digestion sites a and b for restriction enzymes A and B, respectively, upstream and downstream of the first and second shRNA generating units, respectively. And the second shRNA expression vector is provided with a recognition digestion site c of restriction enzyme C that exists only in the a-b region on the side not containing the shRNA generation unit, and the restriction enzymes A, B, and C are all different, A and B are restriction enzymes that generate an end that can be connected to A and B, and an end that cannot be connected to the digested end of C. The a and b sites are the first, In conditions that exist as a single location in the second shRNA expression vector:

 A method comprising the following steps (1) to (4):

 (1) Digestion of the first shRNA expression vector with restriction enzymes A and C

 (2) Digestion of the second shRNA expression vector with restriction enzymes B and C

 (3) Step of mixing the digested fragments of (1) and (2) above and performing ligation

 (4) A step of recovering a circular construct in which the a-c fragment containing the first shRNA generating unit of the first shRNA expression vector and the b-c fragment containing the second shRNA generating unit of the second shRNA expression vector are connected.

 [10] The method according to claim 9, wherein the restriction enzymes A, B, and C are restriction enzymes that generate sticky ends.

[11] The method according to claim 9, wherein the restriction enzymes A and B can form different sequences from a and b when the generated ends are connected to each other.

[12] The first and second shRNA expression vectors are provided with a selection marker, and a site c is provided in the selection marker,

 The method according to claim 9, further comprising a step of selecting with the selection marker after the step (3).

 [13] A vector for producing a multiple shRNA expression construct,

 A promoter,

A claw into which DNA encoding shRNA provided downstream of the promoter can be inserted. Jung area,

 With one or more selection markers,

 A recognition digestion site a of restriction enzyme A is provided upstream of the promoter, a recognition site b of restriction enzyme B is provided downstream of the cloning region, and a recognition digestion site c of restriction enzyme C is included in one selection marker. The A and B are restriction enzymes that can generate connectable ends, and the restriction enzyme C is a restriction enzyme that can form a stump that cannot be connected to the digestive ends of the A and B. The a and b sites are provided as a single site;

 vector.

 [14] The vector according to claim 13, wherein the restriction enzymes A, B, and C are restriction enzymes that generate sticky ends.

15. The vector according to claim 14, wherein the restriction enzymes A and B can form different sequences from a and b when the generated ends are connected to each other.

[16] A cell carrying the vector according to any one of claims 1 to 8.

[17] A non-human animal carrying the vector according to any one of claims 1 to 8.

[18] A pharmaceutical composition comprising the vector according to any one of claims 1 to 8 as a component.