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WO2007010840A1 - VECTEUR D'EXPRESSION D'ARNsh MULTIPLES - Google Patents

VECTEUR D'EXPRESSION D'ARNsh MULTIPLES Download PDF

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WO2007010840A1
WO2007010840A1 PCT/JP2006/314028 JP2006314028W WO2007010840A1 WO 2007010840 A1 WO2007010840 A1 WO 2007010840A1 JP 2006314028 W JP2006314028 W JP 2006314028W WO 2007010840 A1 WO2007010840 A1 WO 2007010840A1
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shrna
vector
expression vector
dna
site
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PCT/JP2006/314028
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Japanese (ja)
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Fumihiko Kanai
Masao Omata
Amarsanaa Jazag
Hideaki Ijichi
Makoto Miyagishi
Kazunari Taira
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The University Of Tokyo
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Publication of WO2007010840A1 publication Critical patent/WO2007010840A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • 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.
  • 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).
  • siRNA effective short interfering RNA
  • 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:
  • Patent Document 1 Non-Patent Documents 7 and 8
  • 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.
  • 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 ⁇ .
  • sh short hairpin 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.
  • 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).
  • Non-Patent Document 11 Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nat Biot echnol 23, 227-231 (2005)
  • 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).
  • 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
  • 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)
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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):
  • restriction enzymes A, B, and C are restriction enzymes that generate sticky ends.
  • the first and second shRNA expression vectors are provided with a selection marker, and a site c is provided in the selection marker.
  • a cloning region into which DNA encoding shRNA provided downstream of the promoter can be inserted
  • 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
  • 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
  • the a and b parts are provided as a single part.
  • restriction enzymes A, B, and C are restriction enzymes that generate sticky ends.
  • a pharmaceutical composition comprising the vector according to any one of 1 to 8 as a component.
  • 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.
  • 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.
  • 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 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 3 ⁇ 4 ⁇ No .: 162) and a base sequence of 55 bases in length.
  • 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
  • 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 ⁇ pcPUR + U6— Smad2i, pcPUR + U6— Smad3i, pcPUR + U6— Smad4i, pc PUR + U6— Smad23i, pcPUR + U6— Smad24i
  • FIG. 1 shows endogenous Smad knockdown: HaCaT cells were treated with pcPUR + U6— GFPi ⁇ pcPUR + U6— Smad2i, pcPUR + U6— Smad3i, pcPUR + U6— Smad4i, pc PUR + U6— Smad23i, pcPUR + U6— Smad24i
  • 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.
  • ⁇ 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- ⁇ -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-
  • FIG. 2g A photograph showing the induction of ⁇ -1 by TGF- ⁇ 1 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.
  • FIGS. 3a to 3e are diagrams and photographs showing analysis of TGF- ⁇ -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- ⁇ 1 (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).
  • SE standard error
  • 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! / ⁇ bar) of TGF- ⁇ 1 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-
  • 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.
  • shRNA has a structure in which one end is closed by a loop.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sequence design can be carried out using a program shown in Table 1 below.
  • 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%.
  • 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.
  • 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. .
  • 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.
  • 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.
  • C cytosine
  • G guanine
  • A adenine
  • 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.
  • 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.
  • 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.
  • 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.
  • the sequence of a hairpin site of microRNA can be mentioned.
  • the linker DNA may be composed of DNA encoding tRNA.
  • 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.
  • 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.
  • siRNA 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! /.
  • 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.
  • the vector carrying the shRNA generating unit can be selected according to the introduced cell and purpose.
  • retrovirus vectors 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.
  • dumbbell-shaped DNA Non-patent Document 14
  • naked plasmid which is not a viral vector, can also be suitably used (Non-patent Document 15).
  • a vector or plasmid existing outside the chromosome when performing transient gene suppression among the above-mentioned vectors.
  • each shRNA generation unit 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • virus for example, HIV, RSV, HCV, HBV and the like can be targeted.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • restriction enzymes A and B can be arbitrarily selected from those satisfying the following conditions.
  • 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)
  • 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)
  • restriction enzymes A and B may form blunt ends or sticky ends.
  • the combinations of restriction enzymes ⁇ and ⁇ 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.
  • Restriction enzyme C can be selected arbitrarily as long as it satisfies the following conditions.
  • Site c is different from sites a and b
  • 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.
  • the vector is preferably provided with a selection marker such as drug resistance or auxotrophy.
  • a selection marker such as drug resistance or auxotrophy.
  • 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.
  • the steps (i) and (mouth) digestion is performed under conditions suitable for the selected restriction enzyme.
  • 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.
  • fragments can be fractionated by electrophoresis, and fragments of the desired length can be recovered from the gel and purified.
  • the ligation reaction in the process can be carried out according to a conventional method.
  • 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.
  • 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.
  • an efficient technique there is a method in which a host such as E. coli is transformed and recovered from the transformant.
  • efficiency can be increased by selecting a transformant using the selection marker as an index.
  • 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.
  • 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.
  • a-c fragment a and b-c fragment b were connected, and c were connected.
  • a sequence is formed that is no longer digested by either restriction enzyme A or restriction enzyme B.
  • 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.
  • 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
  • a recognition digestion site c of restriction enzyme C is included in one selection marker.
  • a and B are restriction enzymes that can generate connectable ends
  • 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 ⁇ and ⁇ are provided as a single site in the vector.
  • 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.
  • the first or second shRNA It is possible to facilitate insertion of DNA encoding the expression vector.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the multiple shRNA expression vector of the first embodiment as a component.
  • this shRNA expression vector can be used as a pharmaceutical product. can do.
  • a multiple shRNA expression vector targeting a plurality of SMAD families is useful for the prevention and treatment of diseases involving the TGF-
  • knocking down Smad2, Smad3, and Smad4 simultaneously with multiple shRNA expression vectors strongly inhibits the TGF- ⁇ 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.
  • 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.
  • 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.
  • TAKARA DNA ligation kit version 2.1
  • 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 ⁇ . Wrana, and pc DEF3-Flag (N) -Smad2 and pcDEF3-Flag (N) -Smad3 were donated by K. Miyazono.
  • 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.)
  • RNAi cells 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 ⁇ g / 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 ⁇ g / ml) (Wako, Tokyo, Japan).
  • 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.
  • 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).
  • 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.
  • 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.
  • Infiltration was measured with a 8 mm diameter Matrigel coated polycarbonate membrane (pore size 8 ⁇ m) 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
  • 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.
  • 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)
  • 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)
  • 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)
  • 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.
  • 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).
  • 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 ⁇ ⁇ : 1 8)
  • Smad3 region 1 sense 5'-ggccagacftgiaiagciacc-3 '( ⁇ ⁇ row 1 ⁇ 4: 24) antisense 5'-ggtggctgtgcaggtctggcc-3' (Bti ⁇ 1 J r : 2 o) region 2 sense 5'-ggattgagftg fctgaatg.3 '( ⁇ [!
  • 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 ⁇ 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 '( ⁇ ⁇ 1 J number: 3 7) Region 3 sense 5'-acggftcfctaaaiactacca-3' ( ⁇ ⁇ ⁇ 1 ) 3 ⁇ 4 ⁇ : 3 8) antisense 5 '-tggtagtgtttagggagccgt-3' ( ⁇ L!
  • 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.
  • Non-patent Document 7 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • the present inventors analyzed using a human keratinocyte cell line HaCaT having a functional TGF- ⁇ pathway.
  • This cell line is known to be suitable for analysis of TGF-8 signaling (Non-patent Document 19).
  • Western plot analysis was performed using the disrupted solution obtained by disrupting these cells (Fig. 2a).
  • 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. .
  • Non-patent Document 21 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.
  • BLAST database http: ⁇ www.ncbi.nlm.nih.gov/BLAST
  • 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.
  • RNA expression of non-target genes in knockdown cells was assessed using quantitative reverse transcriptase polymerase chain reaction (RT-PCR).
  • RT-PCR quantitative reverse transcriptase polymerase chain reaction
  • 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 wrong. From this, we conclude that non-target gene silencing existed and was viable in established cell lines.
  • dsRNA double-stranded RNA
  • the pcPUR-U6 construct 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- ⁇ pathway is performed using these cells. It was decided.
  • TGF- ⁇ pathway Functional analysis of the TGF- ⁇ pathway was performed by luciferase assay using Smad-dependent reporter (CAGA) 9-Luc.
  • Smad-dependent reporter CAGA 9-Luc.
  • signal transduction dependent on canonical TGF- ⁇ -Smad was markedly inhibited in all cell lines except S2KD (Fig. 2f).
  • 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).
  • 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- ⁇ family.
  • the present inventors examined the induction of -1AI-1, a well-known TGF- ⁇ -responsive gene, in knockdown cell lines.
  • the knockdown cell line was cultured in the presence or absence of TGF-
  • 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.
  • Non-patent Document 22 Smad dominant-negative constructs
  • Non-patent Document 23 antisense RNA
  • siRNA duplexes Non-patent Document 24.
  • TGF- ⁇ -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).
  • 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
  • TGF-18 Since TGF-18 is known to promote wound closure by activating cell migration, we have established TGF-
  • Smad4 knockdown inhibited wound closure induced by TGF-jS in Pac-1 cells (Non-patent Document 13).
  • 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).
  • 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.
  • Non-patent Document 25 The effect of TGF- ⁇ on cell apoptosis varies greatly depending on the cell type (Non-patent Document 25).
  • TGF-j8 has been reported to increase cell viability by suppressing apoptosis through Akt-dependent regulation of FKHRL1 (Non-patent Document 28).
  • Fig. 3d The percentage of apoptotic cells for different cell types in the presence of TGF-
  • TGF-j8 still prevented apoptosis in single or double knockdown cells.
  • Smad2 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
  • shRNA to knock down HIV.
  • the next 16 target sites of shRNA were selected based on the HIV genomic DNA sequence.
  • 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,
  • 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.
  • 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.
  • RNAi 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.
  • 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.
  • 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. .

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Abstract

Vecteur d'expression d'ARNsh multiples destiné à être utilisé pour l'inhibition de l'expression d'un gène cible, lequel porte de multiples unités produisant des ARNsh chacune pouvant produire un ARNsh. Dans le vecteur, chacune des unités produisant un ARNsh comprend un ADN codant pour un ARNsh ligaturé en aval à un promoteur, l'ADN codant pour l'ARNsh est composé d'un ADN antisens codant pour un ARN antisens qui a une séquence complémentaire à celle de l'ARNm du gène cible et un ADN sens codant pour un ARN sens ayant une séquence qui peut s'hybrider avec l'ADN antisens et à la fois l'ADN antisens et l'ADN sens sont ligaturés l'un à l'autre grâce à un ADN de liaison.
PCT/JP2006/314028 2005-07-15 2006-07-14 VECTEUR D'EXPRESSION D'ARNsh MULTIPLES WO2007010840A1 (fr)

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JP2009207417A (ja) * 2008-03-04 2009-09-17 Tottori Univ 線虫における簡便な多重遺伝子同時機能抑制法
RU2525935C2 (ru) * 2012-03-01 2014-08-20 Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) Способ получения кассетных генетических конструкций, экспрессирующих несколько рнк-шпилек
US10407677B2 (en) 2012-04-26 2019-09-10 Intana Bioscience Gmbh High complexity siRNA pools

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