JP5625143B2 - Cancer therapeutic agent containing mortalin siRNA - Google Patents

Description

  The present invention relates to a cancer therapeutic agent containing mortalin siRNA. As a cross-reference of related applications, this application claims the priority of Japanese Patent Application No. 2007-162029 filed on June 20, 2007, the entire description of which is specifically incorporated herein by reference.

Mortalin was originally present in the cytoplasmic fraction of normal fibroblasts derived from CD1 ICR mice and was cloned as one of the hsp70 family of immortalized fibroblasts (Non-Patent Documents 1 and 2). At present, mortalin has been found to be a dynamic protein, that is, its intracellular localization is different between normal cells and immortalized cells, distribution to various organelles, various proteins and It functions in binding ability, suppression of p53, which is a powerful tumor suppressor, proteolysis, intracellular molecular transport, mitochondrial membrane import, and energy production (Non-Patent Documents 3 to 7). Increased mitotic life of normal human fibroblasts (Non-patent Documents 5 and 8), extension of nematode life (Non-patent Document 9), mouse by mass expression of mortalin gene mot-2 (mouse) or hmot-2 (human) Malignant tumor formation of immortalized cells (Non-patent Document 10) is caused. The mouse mot-1 protein differs from mot-2 in the two amino acids on the C-terminal side, resulting in functional differences between lack of molecular chaperone ability and induction of cell senescence-like phenotypes on immortalized cells (Non-Patent Documents). 2 and 11). Human mortalin is only one, unlike mouse, and is called hmot-2 because its activity is close to mouse mot-2 (Non-patent Document 10). PBP74 (Non-patent Document 12), mtHSP70 (Non-patent Document) Due to the wide variety of functions such as stress response, intracellular molecular transport, antigen presentation, cell division control, nephrotoxicity, differentiation, and carcinogenesis Document 13) and GRP75 (Non-patent Document 14) have also been reported. Recent studies have shown that Mot-2 binds to the tumor suppressor protein p53 and suppresses its transcriptional activity (Non-Patent Documents 16 to 20). Inhibition of p53 by Mot-2 causes the phenomenon of malignant tumor formation of NIH3T3 (Non-patent Document 10) and prolongation of the mitotic life of normal human fibroblasts (Non-patent Document 5). (Non-Patent Document 10). Mot-2 acts in cooperation with telomerase to immortalize human foreskin fibroblasts (Non-patent Document 8). It has also been clarified that knocking down mortalin with antisense or ribozyme promptly inhibits proliferation of human cancer cells (Non-Patent Documents 21 to 24).
Wadhwa, R., Kaul, SC, Ikawa, Y., and Sugimoto, Y. (1993) J Biol Chem 268, 6615-6621 Wadhwa, R., Kaul, SC, Sugimoto, Y., and Mitsui, Y. (1993) J Biol Chem 268, 22239-22242 Wadhwa, R., Kaul, SC, Mitsui, Y., and Sugimoto, Y. (1993) Exp Cell Res 207, 442-448 Ran, Q., Wadhwa, R., Kawai, R., Kaul, SC, Sifers, RN, Bick, RJ, Smith, JR, and Pereira-Smith, OM (2000) Biochem Biophys Res Commun 275, 174-179 Kaul, SC, Reddelb, RR, Sugiharac, T., Mitsuia, Y., and Wadhwac, R. (2000) FEBS Lett 474, 159-164 Wadhwa, R., Taira, K., and Kaul, SC (2002) Cell Stress Chaperones 7, 309-316 Liu, Y., Liu, W., Song, XD, and Zuo, J. (2005) Mol Cell Biochem 268, 45-51 Kaul, SC, Yaguchi, T., Taira, K., Reddel, RR, and Wadhwa, R. (2003) Exp Cell Res 286, 96-101 Yokoyama, K., Fukumoto, K., Murakami, T., Harada, S., Hosono, R., Wadhwa, R., Mitsui, Y., and Ohkuma, S. (2002) FEBS Lett 516, 53-57 Kaul, SC, Duncan, EL, Englezou, A., Takano, S., Reddel, RR, Mitsui, Y., and Wadhwa, R. (1998) Oncogene 17, 907-911 Kaul, SC, Aida, S., Yaguchi, T., Kaur, K., and Wadhwa, R. (2005) J Biol Chem 280, 39373-39379 Domanico, SZ, DeNagel, DC, Dahlseid, JN, Green, JM, and Pierce, SK (1993) Mol Cell Biol 13, 3598-3610 Webster, TJ, Naylor, DJ, Hartman, DJ, Hoj, PB, and Hoogenraad, NJ (1994) DNA Cell Biol 13, 1213-1220 Merrick, BA, Walker, VR, He, C., Patterson, RM, and Selkirk, JK (1997) Cancer Lett 119, 185-190 Wadhwa, R., Taira, K., and Kaul, SC (2002) Histol Histopathol 17, 1173-1177 Wadhwa, R., Takano, S., Robert, M., Yoshida, A., Nomura, H., Reddel, RR, Mitsui, Y., and Kaul, SC (1998) J Biol Chem 273, 29586-29591 Kaul, SC, Reddel, RR, Mitsui, Y., and Wadhwa, R. (2001) Neoplasia 3, 110-114 Wadhwa, R., Yaguchi, T., Hasan, MK, Mitsui, Y., Reddel, RR, and Kaul, SC (2002) Exp Cell Res 274, 246-253 Ma, Z., Izumi, H., Kanai, M., Kabuyama, Y., Ahn, NG, and Fukasawa, K. (2006) Oncogene Walker, C., Bottger, S., and Low, B. (2006) Am J Pathol 168, 1526-1530 Wadhwa, R., Takano, S., Taira, K., and Kaul, SC (2004) J Gene Med 6, 439-444 Wadhwa, R., Ando, H., Kawasaki, H., Taira, K., and Kaul, SC (2003) EMBO Rep 4, 595-601 Pizzatti, L., Sa, LA, de Souza, JM, Bisch, PM, and Abdelhay, E. (2006) Biochim Biophys Acta Shin, BK, Wang, H., Yim, AM, Le Naour, F., Brichory, F., Jang, JH, Zhao, R., Puravs, E., Tra, J., Michael, CW, Misek, DE , and Hanash, SM (2003) J Biol Chem 278, 7607-7616

  This invention made it the problem which should be solved to provide the anticancer agent using mortalin shRNA.

  Mortalin, one of the heat shock protein 70 family, plays a number of functions such as intracellular molecular transport, mitochondrial membrane import, energy production, molecular chaperone activity, and suppression of p53 activity. The present inventors have focused on mortalin as a target for anticancer treatment, demonstrated that cancer cells can be killed by using mortalin-specific siRNA, and found that mortalin-specific siRNA is useful as an anticancer agent. It was. The present invention has been completed based on these findings.

That is, according to the present invention, siRNA or shRNA having the base sequence described in any one of SEQ ID NOs: 1 to 6 as a target sequence is provided.
The present invention further provides a DNA comprising a combination of the following sense oligonucleotide and antisense oligonucleotide.
(1) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 7 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 8;
(2) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 9 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 10;
(3) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 11 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 12;
(1) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 13 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 14;
(2) a sense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 15 and an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 16; or (3) a sense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 17 and the nucleotide sequence of SEQ ID NO: 18. An antisense oligonucleotide consisting of;

  According to the present invention, there is further provided siRNA or shRNA obtained by expressing the above DNA and having the base sequence described in any one of SEQ ID NOs: 1 to 6 as a target sequence.

  The present invention further provides an anticancer agent containing the above-described siRNA or shRNA or DNA as an active ingredient.

  The present invention further provides a method for treating or preventing cancer using an anticancer agent containing the above siRNA or shRNA or DNA as an active ingredient.

  The present invention further provides use of the above-described siRNA or shRNA or DNA for producing an anticancer agent.

According to the present invention, there is further provided a recombinant expression vector capable of expressing the siRNA or shRNA comprising the DNA.
The recombinant vector of the present invention is preferably obtained by incorporating the above DNA into an adenovirus vector.
The recombinant vector of the present invention is preferably obtained by incorporating the above DNA into a vector containing a U6 promoter.
The present invention further provides an anticancer agent containing the recombinant expression vector as an active ingredient.

  According to the present invention, mortalin shRNA has been confirmed to kill cancer cells. That is, the present invention provides a cancer therapeutic agent containing mortalin siRNA.

Hereinafter, embodiments of the present invention will be described in detail.
RNAi (RNA interference) is a phenomenon in which double-stranded RNA introduced into a cell suppresses the expression of a gene having the same sequence. Specific examples of substances that inhibit mortalin expression by RNAi include siRNA or shRNA as described below.

  siRNA is an abbreviation for short interfering RNA and refers to double-stranded RNA having a length of about 21 to 23 bases. The siRNA may be in any form as long as it can cause RNAi. For example, siRNA obtained by chemical synthesis or biochemical synthesis, or synthesis in an organism, or a double-stranded RNA having about 40 bases or more. May be any short double-stranded RNA of 10 base pairs or more produced by degradation in the body. The siRNA sequence and the partial sequence of mortalin mRNA preferably match 100%, but do not necessarily match 100%.

  The region having homology between the siRNA base sequence and the mortalin gene base sequence preferably does not include the translation start region of the mortalin gene. The sequence having homology is preferably 20 bases away from the translation initiation region of the mortalin gene, more preferably 70 bases away. The sequence having homology may be, for example, a sequence near the 3 ′ end of the mortalin gene.

  As a substance that inhibits mortalin expression by RNAi, dsRNA of about 40 bases or more that generates siRNA may be used. For example, about 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% with respect to a part of the nucleic acid sequence of the mortalin gene. As described above, RNA containing a double-stranded portion or a variant thereof containing a sequence having 100% homology can be used most preferably. The sequence portion having homology is usually at least 15 nucleotides or more, preferably about 19 nucleotides or more, more preferably at least 20 nucleotides or more, and further preferably 21 nucleotides or more.

  As a substance that inhibits the expression of mortalin by RNAi, shRNA (short hairpin RNA) having a short hairpin structure having a protruding portion at the 3 ′ end can also be used. shRNA is a molecule of about 20 base pairs or more that has a double-stranded structure in the molecule and a hairpin-like structure by including a partially palindromic base sequence in single-stranded RNA. is there. The shRNA preferably has a 3 ′ protruding end. The length of the double-stranded part is not particularly limited, but is preferably 10 nucleotides or more, more preferably 20 nucleotides or more. Here, the 3 ′ protruding end is preferably DNA, more preferably DNA of at least 2 nucleotides, and further preferably DNA of 2 to 4 nucleotides.

  A substance that inhibits mortalin expression by RNAi may be artificially chemically synthesized, or DNA in a hairpin structure in which the DNA sequences of the sense strand and antisense strand are ligated in the reverse direction is used to produce RNA in vitro using T7 RNA polymerase. It may be produced by synthesis. In the case of synthesis in vitro, antisense and sense RNAs can be synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is caused and mortalin expression is suppressed. Introduction into cells can be performed, for example, by the calcium phosphate method, or a method using various transfection reagents (for example, oligofectamine, Lipofectamine, lipofection, etc.).

  As a substance that inhibits mortalin expression by RNAi, an expression vector containing a nucleic acid sequence encoding the above-described siRNA or shRNA may be used. Furthermore, cells containing the expression vector may be used. The kind of the expression vector or cell described above is not particularly limited, but an expression vector or cell already used as a medicine is preferable.

  In the present invention, siRNA or shRNA having the base sequence described in any one of SEQ ID NOs: 1 to 6 as a target sequence can be used. Specifically, for example, an siRNA having a base sequence of any one of SEQ ID NOs: 1 to 6 obtained by expressing a DNA comprising a combination of the following sense oligonucleotide and antisense oligonucleotide as a target sequence: shRNA can be used.

  The administration route of the anticancer agent containing the mortalin siRNA of the present invention is not particularly limited, and is orally or parenterally administered (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, intrarectal administration, intravaginal administration) Administration, local administration to the affected area, skin administration, etc.) may be used. Formulation forms suitable for oral administration include solid or liquid forms, and preparation forms suitable for parenteral administration include injections, drops, suppositories, external preparations, eye drops, nasal drops and the like. Can be mentioned. The anticancer agent of the present invention may be added with a pharmaceutically acceptable additive as required depending on the preparation form. Specific examples of pharmaceutically acceptable additives include, for example, excipients, binders, disintegrants, lubricants, antioxidants, preservatives, stabilizers, tonicity agents, coloring agents, taste masking agents. Agents, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, carriers, excipients and / or pharmaceutical adjuvants and the like.

  The anticancer agent of the present invention in the form of an oral solid preparation is, for example, an excipient added to mortalin siRNA which is an active ingredient, and further, a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, etc. as necessary Can be prepared as tablets, granules, powders, and capsules by conventional methods. The anticancer agent of the present invention in the form of an oral liquid preparation is obtained by adding one or more additives for pharmaceutical preparation such as a corrigent, stabilizer or preservative to mortalin siRNA which is an active ingredient, Syrup, elixir and the like.

  The solvent used to formulate the anticancer agent of the present invention as a liquid preparation may be either aqueous or non-aqueous. Liquid preparations can be prepared by methods well known in the art. For example, an injection is prepared by dissolving in a saline solution, a buffer solution such as PBS, a solvent such as sterilized water, sterilizing by filtration with a filter or the like, and then filling an aseptic container (for example, an ampoule). be able to. This injection may contain a conventional pharmaceutical carrier, if necessary. Alternatively, an administration method using a non-invasive catheter may be used. Examples of the carrier that can be used in the present invention include neutral buffered physiological saline, physiological saline containing serum albumin, and the like.

  With regard to the type of gene delivery such as mortalin siRNA or siRNA expression vector, the method is not particularly limited as long as the expression of RNA or siRNA expression vector encoding mortalin siRNA in the applied cells is obtained. It is possible to use gene transfer using vectors and liposomes. Examples of viral vectors include animal viruses such as retroviruses, vaccinia viruses, adenoviruses, and synthin liquiviruses.

  A substance that inhibits mortalin expression by RNAi may be directly injected into cells.

  The dosage of the anticancer agent of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the severity of the disease, the patient's age, weight, sex, medical history, type of substance used as an active ingredient, etc. it can. The dose of the anticancer agent of the present invention is, for example, about 0.1 ng to about 100 mg / kg, preferably about 1 ng to about 10 mg per adult as an active ingredient, and is administered as a viral vector or a non-viral vector. When used, it is usually 0.0001 to 100 mg, preferably 0.001 to 10 mg, more preferably 0.01 to 1 mg. The administration frequency of the anticancer agent of the present invention may be, for example, once a day to once every several months.

  The present invention will be described in more detail in the following examples, but the present invention is not limited thereto. Various changes and modifications can be made by those skilled in the art, and these changes and modifications are also included in the present invention.

(1) Experimental materials and methods (a) Cell culture, transfection, viral infection Normal human epithelial cells (TIG-1, WI-38), osteosarcoma cells (U2OS, mortalin overexpression strain U2OS), fibrosarcoma cells (HT1080) 10% fetal bovine serum (FBS), penicillin, streptomycin, Fungizone (Life Technologies, Inc.) added Dulbecco's modified Eagle's minimal essential medium (DMEM) as a medium, carbon dioxide concentration 5%, humidity 95% The cells were cultured in an incubator at 37 degrees.

  Fugene (Roche Applied Scinece) was used for transfection of shRNA expression plasmid targeting mortalin. Usually, 3 μg of plasmid DNA was transfected into about 60% confluent cells of a 6 cm culture dish. Thereafter, selection was performed for 48 hours in a medium supplemented with puromycin (2 μg / ml). For immunostaining, the cells were cultured on a cover glass for 24 hours after transfection and stained with an anti-mortalin antibody. The cell growth rate was examined by microscopic observation of proliferation assay and colony formation experiment.

  A replicable carcinogenic adenovirus (Adonco viruses) was generated using a method already reported (Kim, E., Kim, JH, Shin, HY, Lee, H., Yang, JM, Kim, J., Sohn, JH, Kim, H., and Yun, CO (2003) Hum Gene Ther 14, 1415-1428). In order to make shRNA targeting mortalin, a DNA fragment for the expression of shRNA targeting human mortalin was generated by annealing sense and antisense oligonucleotides.

No. 1 (target sequence: gcgtctcatt ggccggcgat a (SEQ ID NO: 1))
Sense oligonucleotide:
caccgcgtcttattggtcggtgatacgtgtgctgtccgttcttctttcagcttgttgcttttt (SEQ ID NO: 7)
Antisense oligonucleotides:
gcataaaaagcgtctcattggccggcgatagaacggacagcacacgttcttctttcaacttatcgc (SEQ ID NO: 8)

No. 2 (target sequence: gatgctggcc agatatctgg a (SEQ ID NO: 2))
Sense oligonucleotide:
caccgatgctggtcagatatctggacgtgtgctgtccgttcttctttcagcttgttgcttttt (SEQ ID NO: 9)
Antisense oligonucleotides:
gcataaaaagatgctggccagatatctggagaacggacagcacacgttcttctttcaacttatcgc (SEQ ID NO: 10)

No. 3 (target sequence: gactttgacc aggccttgct a (SEQ ID NO: 3))
Sense oligonucleotide:
caccgactttgatcaggtcttgctacgtgtgctgtccgttcttctttcagcttgttgcttttt (SEQ ID NO: 11)
Antisense oligonucleotides:
gcataaaaagactttgaccaggccttgctagaacggacagcacacgttcttctttcaacttatcgc (SEQ ID NO: 12)

No. 4 (target sequence: gcaacaagct gaaagaaga (SEQ ID NO: 4))
Sense oligonucleotide:
caccgcgataagttgaaagaagaacgtgtgctgtccgttcttctttcagcttgttgcttttt (SEQ ID NO: 13)
Antisense oligonucleotides:
gcataaaaagcaacaagctgaaagaagaacggacagcacacgttcttctttcaacttatcgc (SEQ ID NO: 14)

No. 5 (target sequence: gccagaagga caacatatg (SEQ ID NO: 5))
Sense oligonucleotide:
caccgctagaaggataacgtataacgtgtgctgtccgttatatgttgtccttctggcttttt (SEQ ID NO: 15)
Antisense oligonucleotides:
gcataaaaagccagaaggacaacatataacggacagcacacgttatacgttatccttctagc (SEQ ID NO: 16)

No. 6 (target sequence: gaatgaggct agaccttta (SEQ ID NO: 6))
Sense oligonucleotide:
caccgagtgaggttagatctttaacgtgtgctgtccgttaaaggtctagcctcattcttttt (SEQ ID NO: 17)
Antisense oligonucleotides:
gcataaaaagaatgaggctagacctttaacggacagcacacgttaaagatctaacctcactc (SEQ ID NO: 18)

The fragment was incorporated into the BamH1 and HindIII sites of the adenovirus pSP72-E3 shuttle vector (Cancer Gene Therapy, 12, p61-71, 2005) to create pSP72-E3-shMot. In order to generate an oncogenic adenovirus expressing a shRNA specific for mortalin, the SP72ΔE3-shMot shuttle vector was linearized with XmnI. Subsequently, E. coli BJ5183 was simultaneously transformed with the SpeI-treated E1A- and E1B-double mutant whole adenovirus vector pdl-ΔB7, and homologous recombination was performed to prepare a pΔB7-shMo adenovirus vector. To confirm homologous recombination, plasmid DNA was purified from overnight E. coli cultures, cut with HindIII, and analyzed for cleavage patterns. Appropriate homologous recombination Ad plasmid DNA was cut with PacI and transfected into 293 cells to produce adenovirus Ad-ΔB7-shMot. Replication incompetent Ad lacking E1 (Ad-ΔE1, deletion of nucleotide sequence of Ad genome 1341-3535; Int. J. Cancer, 88 p454-463, 2000) and double mutation of E1A and E1B A replacement replicating adenovirus (Ad-ΔB7) was prepared. All viruses were grown in 293 cells, purified by CsCl density centrifugation, and stored at −80 ° C. in a stock lysate of 10 mM Tris, 4% sucrose, 2 mM MgCl 2. The number of virus particles was measured by the absorbance of OD260 at 260 nm. One absorbance unit corresponded to 10 ¹² / ml virus. The infection titer (plaque forming unit / ml) was determined by the limiting dilution method of 293 cells. MOI was calculated from the infection efficiency. For virus infection, cells were prepared in 48-well plates and infected with MOI of 10-300. Analysis of cell growth rate due to virus infection was performed by the method described above.

  FIG. 1 shows a diagram of the adenovirus vector used in this example. All the adenoviral vectors shown used full length adenoviral genes and were engineered into E. coli plasmids. Ad-Δ E1 deleted all E1 regions. Ad-ΔB7 has lost the E1A and E1B regions with mutations in the retinoblastoma (RB) binding site. Ad-Δ B7-shMot acts on a mortalin-specific shRNA controlled by the U6 promoter. ΔE1A, ΔE1B and ΔE3 indicate deletion of the E1A, E1B and E3 genes, respectively. ITR = inverted terminal repeat; Ψ = package signal; IX = protein IX. The asterisk indicates a mutation in the E1A Rb protein binding site.

(B) Construction of siRNA expression vector
For the construction of the siRNA expression vector, a vector having a U6 promoter capable of being induced by tetracycline was used (Miyagishi, M., and Taira, K. (2002) Nat Biotechnol 20, 497-500) (Miyagishi, M. Sumimoto, H., Miyoshi, H., Kawakami, Y., and Taira, K. (2004) J Gene Med 6, 715-723). The target site of siRNA was designed using an algorithm (http://www.igene-therapeutics.co.jp). Table 1 shows the sequence of mortalin targeted by siRNA.

In a typical siRNA construct, a sense sequence oligo (5′-cacc GttGCtCACTtCAAGAGAG gtgtgctgtcc CTCTCTTGGAGTGGGCGGC tttttt-3 ′) and an antisense sequence oligo (5′-GCATaaaaaa GCCGCCCACTCCAAGAGAG ggacagcacac CTCTCTTGaAGTGTGCAGCaaC- 3 ′) were prepared. The underlined portion indicates the sequence of the target site. Mutations from C to T and G to A were inserted only into the sense sequence. Mix 5 μl of 100 μM sense and antisense oligonucleotides and use a thermal cycler (99 ° C. 2 min, decrease from 72 ° C. to 4 ° C. over 2 hours) in 100-150 mM NaCl in a final volume (20 μl). And annealed. The annealed oligonucleotides were diluted (1: 200), 2 μl of which was ligated to the pciPur vector purified by digestion with BspM I using the High Ligation Kit (Takara). The sequence of the plasmid DNA prepared from the transformed bacteria was determined using an antisense vector primer (CAGGAAACAGCTATGAC) (SEQ ID NO: 19), and the cloned DNA fragment was confirmed.

(2) Results Mortalin has increased expression in carcinogenesis and is involved in malignant cancer cells (Wadhwa, R., Takano, S., Kaur, K., Deocaris, CC, Pereira-Smith, OM, Reddel, RR, and Kaul, SC (2006) Int J Cancer; and Dundas, SR, Lawrie, LC, Rooney, PH, and Murray, GI (2005) J Pathol 205, 74-81) (JP 2006-89471 A) ). The present inventors constructed a mortalin-expressing siRNA and examined its effect on human cancer cells. (i) U6 promoter (Miyagishi, M., and Taira, K. (2002) Nat Biotechnol 20, 497-500), (ii) Adeno-oncolytic virus (Adonoc-motshRNAs) (Kim, E., Kim, JH , Shin, HY, Lee, H., Yang, JM, Kim, J., Sohn, JH, Kim, H., and Yun, CO (2003) Hum Gene Ther 14, 1415-1428) siRNA was produced. Mortalin shRNA (596, 696, 906) and homeostatic Adonoc-motshRNAs (7, 8, 9) using the tetracycline-inducible U6 promoter were constructed (Table 1, FIG. 1). In the data shown below, it was demonstrated that mortalin shRNA has an effect of killing cancer cells and can be used for cancer treatment.

  In FIG. 2, the displayed mortalin shRNA was transfected into human osteosarcoma cells. Transfected cells were selected by culturing for 48 to 96 hours in a medium supplemented with puromycin (2 ug / ul). The tetracycline-inducible construct was induced by adding doxycycline (1 μg / ml) to the medium.

FIG. 2A shows mortalin immunostaining in mortalin shRNA transfected and control cells. About 60% of the mortalin shRNA transfected cells showed very weak staining.
In FIG. 2B, the vector transfected cells are healthy, but the mortalin shRNA transfected cells are killed.
In FIG. 2C, puromycin-selected cells were seeded at low density (2000 cells / 10 cm dish) and colony formation was observed for 15 days. A significant reduction in colony formation rate was shown.

  In FIG. 3, mortalin shRNA-expressing adeno-oncolytic virus was prepared as already shown (Kim, E., Kim, JH, Shin, HY, Lee, H., Yang, JM, Kim, J., Sohn, JH, Kim, H., and Yun, CO (2003) Hum Gene Ther 14, 1415-1428). These viruses show selective replication in human cancer cells. These were virus infected normal and cancer cells with MOI100.

  FIG. 3A shows crystal violet staining of Adonco-motshRNA virus infected cells. Normal cells (TIG-1 and WI38) showed no effect at 48-60 hours after infection, whereas cancer cells (U2OS and HT1080) showed significant killing in each of the 6 shRNAs.

  In FIG. 3B, cell viability was examined 36-48 hours after infection. Cancer cells showed a very low survival rate, but normal cells did not show significant effects on any of the six shRNAs.

  FIG. 4 shows the results of proliferation of U2OS cells infected with Adonco-motshRNA and controls with MOI 0-300. At MOI 25, # 7 and # 9 cells began to die and at MOI 300 100% of the cells died. # 8 and control virus cells caused killing in massive infections (MOI 200-500 infected area).

  In FIG. 5, human normal fibroblasts infected with Adonco-motshRNA were also examined for proliferation at 36-48 hours after infection in parallel with the cancer cells shown in FIG. Cell killing was almost selectively seen in each of the six infected cancer cells.

  In FIG. 6, mortalin was overexpressed in U2OS cells by retrovirus as already shown (Wadhwa, R., Takano, S., Kaur, K., Deocaris, CC, Pereira-Smith, OM, Reddel, RR, and Kaul, SC (2006) Int J Cancer). Control and mortalin overexpressing cells were infected with 6 types of Adonco-motshRNA in the same manner as the original U2OS cells. Although mortalin overexpressing U2OS cells remained, the original U2OS cells were effectively killed by each of six types of Adonco-motshRNA. This result indicates that mortalin knockdown by shRNA leads to cancer cell death.

FIG. 1 shows a diagram of the adenoviral vector used in this study. FIG. 2 shows the results of transfection of mortalin shRNA into human osteosarcoma cells. FIG. 3 shows the results of infecting normal cells and cancer cells with a mortalin shRNA-expressing adeno-oncolytic virus. FIG. 4 shows the proliferation of U2OS cells infected with Adonco-motshRNA and control. FIG. 5 shows the results of examining the proliferation of human normal fibroblasts infected with Adonco-motshRNA in 36-48 hours after infection in parallel with the cancer cells shown in FIG. FIG. 6 shows the results of infection of control and mortalin overexpressing cells with 6 types of Adonco-motshRNA in the same manner as the original U2OS cells.

Claims (5)

A siRNA or shRNA having a base sequence described in any one of SEQ ID NOs: 1 to 3 as a target sequence, obtained by expressing DNA comprising a combination of the following sense oligonucleotide and antisense oligonucleotide.
(1) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 7 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 8;
(2) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 9 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 10;
(3) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 11 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 12; An siRNA or shRNA having a base sequence of any one of SEQ ID NOs: 4 to 6 as a target sequence, obtained by expressing DNA comprising a combination of the following sense oligonucleotide and antisense oligonucleotide.
(1) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 13 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 14;
(2) a sense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 15 and an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 16; or (3) a sense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 17 and the nucleotide sequence of SEQ ID NO: 18. An antisense oligonucleotide consisting of; The recombinant expression vector which can express siRNA or shRNA of Claim 1 obtained by integrating the DNA which consists of a combination of the following sense oligonucleotide and antisense oligonucleotide into the vector containing a U6 promoter.
(1) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 7 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 8;
(2) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 9 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 10;
(3) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 11 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 12; The recombinant expression vector which can express siRNA or shRNA of Claim 2 obtained by integrating DNA which consists of a combination of the following sense oligonucleotide and antisense oligonucleotide in an adenovirus vector.
(1) a sense oligonucleotide consisting of the base sequence of SEQ ID NO: 13 and an antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 14;
(2) a sense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 15 and an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 16; or (3) a sense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 17 and the nucleotide sequence of SEQ ID NO: 18. An antisense oligonucleotide consisting of; An anticancer agent comprising the siRNA or shRNA according to claim 1 or 2 or the recombinant vector according to claim 3 or 4 as an active ingredient.