WO2012020839A1

WO2012020839A1 - Pharmaceutical composition for cancer therapy

Info

Publication number: WO2012020839A1
Application number: PCT/JP2011/068438
Authority: WO
Inventors: 義成我原; 六嶋　正知; 陽子山口; 和彦前川
Original assignee: 塩野義製薬株式会社
Priority date: 2010-08-12
Filing date: 2011-08-12
Publication date: 2012-02-16

Abstract

The present invention relates to: a human AQR protein; the use of the gene encoding the human NHP2L1 protein or the gene encoding the human NUP205 protein as a target for cancer therapy; an agent for cancer therapy, particularly a pharmaceutical composition for cancer therapy comprising siRNA, in which these genes serve as the target; and a method for screening for agents for cancer therapy in which these genes serve as the target.

Description

Pharmaceutical composition for cancer treatment

The present invention relates to a pharmaceutical composition for treating cancer comprising a substance that suppresses the expression of a specific gene. More specifically, the present invention relates to a pharmaceutical composition for cancer treatment containing a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene, and a method for screening a substance for cancer treatment based on the expression variation of any of the above genes.

The most common feature of cancer cells is their rapid and uncontrollable growth compared to normal cells. Taking advantage of this feature, chemotherapy using drugs that are toxic to proliferating cells has been performed in the oncology region. The types of drugs (anticancer agents) that suppress the growth of cancer cells include nucleic acid synthesis inhibitors such as cyclophosphamide, antibiotics such as actinomycin D and bleomycin, and antimetabolites such as 5-fluorouracil and methotrexate. In addition, microtubule depolymerization inhibitors such as paclitaxel and molecular target drugs such as imatinib and gefitinib are known. However, these drugs do not necessarily have a sufficient therapeutic effect on cancer, and have a problem that drug resistance is caused by use. In addition, since it acts on normal cells, it may have side effects. Therefore, it has been desired to develop an anticancer agent having a new mechanism of action, that is, an anticancer agent having a growth-inhibiting action against cancer cells and having little toxicity against normal cells.

An approach for treating cancer by suppressing the growth of cancer cells by finding a gene that is a target for cancer treatment and suppressing the expression or function of the target gene for cancer treatment has been extensively studied. In recent years, RNA oligonucleotide strands of about 21 bases complementary to the desired gene (mRNA) and double-stranded RNA (small RNA interfering RNA: siRNA) consisting of the antisense RNA oligonucleotide strand have been developed, and can be arbitrarily selected in mammalian cells. It has become possible to suppress the expression of these genes (Non-patent Document 1: Nature, Vol. 411, 494-498, 2001). Currently, siRNA technology is actively used in life science research and is expected to be applied to various diseases including cancer. However, there are no siRNAs for treating diseases that are sold as pharmaceuticals including oncology.

An object of the present invention is to find a novel cancer treatment target gene, that is, a gene capable of suppressing cancer cell growth by suppressing its expression and enabling the treatment of cancer. Is to provide.

The present inventors introduced three types of genes that remarkably suppress the growth of lung cancer-derived cell lines by suppressing gene expression by introducing a siRNA library covering all human genes into the lung cancer-derived cell lines. AQR, NHP2L1 and NUP205) were identified. By suppressing the expression of one or more of these three genes, it was confirmed that the growth of cancer cells could be suppressed, and the present invention was completed.

That is, the present invention provides the following [1] to [13].
[1] A pharmaceutical composition for treating cancer comprising a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene.
[2] The pharmaceutical composition according to [1], wherein the substance that suppresses gene expression is siRNA, an antisense oligonucleotide or a ribozyme, or an expression vector thereof.
[3] The pharmaceutical composition according to [1] or [2], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[4] The medicament according to [2] or [3], wherein the siRNA is one or more siRNA selected from the group consisting of siRNAs according to any one of (a) to (i) below: Composition:
(A) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 7 and the first to 19th nucleotide sequence in SEQ ID NO: 8;
(B) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 9 and the first to 19th nucleotide sequence in SEQ ID NO: 10;
(C) siRNA in which the double-stranded RNA portion comprises the first to the 19th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 11 and the first to the 19th nucleotide sequence in SEQ ID NO: 12;
(D) siRNA in which the double-stranded RNA portion consists of the first to the 19th nucleotide sequence in SEQ ID NO: 13 and the first to the 19th nucleotide sequence in SEQ ID NO: 14;
(E) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 15 and the first to 19th nucleotide sequence in SEQ ID NO: 16;
(F) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 17 and the first to 19th nucleotide sequence in SEQ ID NO: 18;
(G) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 19 and the first to 19th nucleotide sequence in SEQ ID NO: 20;
(H) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 21 and the first to 19th nucleotide sequence in SEQ ID NO: 22;
(I) The siRNA according to any one of (a) to (h), wherein 1 to several nucleotides are added, inserted, deleted or substituted in one or both nucleotide sequences in the double-stranded RNA portion.
[5] The pharmaceutical composition according to [4], wherein one or both strands of the siRNA contain a 3 ′ terminal overhang.
[6] The pharmaceutical composition according to any one of [1] to [5], wherein the cancer is any of lung cancer, colon cancer and pancreatic cancer.
[7] A method for treating cancer, comprising a step of administering a substance that suppresses expression of an AQR gene, NHP2L1 gene, or NUP205 gene.
[8] The method according to [7], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[9] A substance that suppresses the expression of an AQR gene, NHP2L1 gene, or NUP205 gene, which is used for cancer treatment.
[10] The substance according to [9], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[11] Use of a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene for the production of a therapeutic agent for cancer.
[12] The use according to [11], wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
[13] A method for screening a substance for treating cancer comprising the following steps (a) to (c):
(A) contacting a test substance with a cell capable of measuring the expression of AQR gene, NHP2L1 gene or NUP205 gene;
(B) measuring the expression level of the gene in cells contacted with the test substance, and comparing the expression level with the expression level of the gene in control cells not contacted with the test substance;
Next, (c) when the expression of the gene in a cell given the test substance is lower than the expression of the gene in a cell not given the test substance, the test substance is selected as a cancer treatment substance Process.

According to the present invention, it is possible to suppress the growth of cancer cells by suppressing the expression of AQR, NHP2L1 or NUP205 gene, and substances that inhibit the expression of these genes are effective for pharmaceutical compositions for cancer treatment. It can be used as a component. The pharmaceutical composition for cancer treatment according to the present invention provides an anticancer agent having a new mechanism of action that specifically suppresses the expression of a specific gene, and is characterized by being less toxic than conventional anticancer agents. To do. Furthermore, the present invention also provides a method for screening a cancer therapeutic substance using the above gene as a target.

FIG. 1 is a diagram showing the influence on cell proliferation by transfection of three types of siRNA (s18725, s18726, s18727) for the AQR gene. The results of each siRNA are shown for each of the six cell lines (A549, RERF-LC-AI, HCT116, BxPC-3, MRC-5, BEAS-2B). The vertical axis represents the number of viable cells (%). The number of viable cells when negative control siRNA (NC- # 1) was added was taken as 100%. FIG. 2 is a graph showing the effect on cell proliferation by transfection of two types of siRNA (s9548, s9549) for the NHP2L1 gene. The results of each siRNA are shown for each of the six cell lines (A549, RERF-LC-AI, HCT116, BxPC-3, MRC-5, BEAS-2B). The vertical axis represents the number of viable cells (%). The number of viable cells when negative control siRNA (NC- # 1) was added was taken as 100%. FIG. 3 is a diagram showing the effect on cell proliferation by transfection of three types of siRNA (s23175, s23176, s23177) for the NUP205 gene. The results of each siRNA are shown for each of the six cell lines (A549, RERF-LC-AI, HCT116, BxPC-3, MRC-5, BEAS-2B). The vertical axis represents the number of viable cells (%). The number of viable cells when negative control siRNA (NC- # 1) was added was taken as 100%. FIG. 4 is a diagram showing the influence of the transfection of three types of siRNA (s18725, RNAs18726, s18727) on the AQR gene on the mRNA expression of the target gene. The results of each siRNA are shown for each of six types of cell lines (A549, RERF-LC-AI, HCT116, BxPC-3, MRC-5, BEAS-2B). The vertical axis shows the expression level (%) of the target gene (mRNA). The expression level when adding negative control siRNA (NC- # 1) was 100%. FIG. 5 is a diagram showing the influence on mRNA expression of a target gene by transfection of two types of siRNA (s9548, s9549) for the NHP2L1 gene. The results of each siRNA are shown for each of six types of cell lines (A549, RERF-LC-AI, HCT116, BxPC-3, MRC-5, BEAS-2B). The vertical axis shows the expression level (%) of the target gene (mRNA). The expression level when adding negative control siRNA (NC- # 1) was 100%. FIG. 6 is a diagram showing the influence on mRNA expression of a target gene by transfection of three types of siRNA (s23175, s23176, s23177) for the NUP205 gene. The results of each siRNA are shown for each of six types of cell lines (A549, RERF-LC-AI, HCT116, BxPC-3, MRC-5, BEAS-2B). The vertical axis shows the expression level (%) of the target gene (mRNA). The expression level when adding negative control siRNA (NC- # 1) was 100%.

Unless otherwise stated, terms used in the present specification are used in the meaning normally used in this field.

In one aspect, the present invention provides a pharmaceutical composition comprising a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene. Such a pharmaceutical composition can be used, for example, for the treatment of cancer.

As shown in Example 3 of the present specification, the AQR gene, NHP2L1 gene or NUP205 gene was found as a novel target gene for cancer.

AQR is an RNA-binding protein (GeneBank Accession ID: NM_014691) and is presumed to be a component of the spliceosome complex (Gene Ontology database), but the details of the function are unknown. Inhibition of AQR expression with siRNA in human HEK293T cells has been reported to reduce the infection efficiency of human immunodeficiency virus HIV-1 (Cell, 135, 49-60, 2008). However, the relationship between AQR and cancer is not known.

NHP2L1 is a component of the spliceosome complex (GeneBank Accession ID: NM_001003796; Nature, 419, 182-185, 2002) and binds to U4 small nuclear RNA (snRNA), It plays an important role in the meeting (EMBO Journal, 18, 6119-6133, 1999). However, the relationship between NHP2L1 and cancer is not known.

NUP205 is one of the components of the nuclear pore complex called nucleoporin (GeneBank Accession ID: NM_015135; Molecular Biology of the cell, 15, 4261-4277, 2004) and is localized in the nuclear pore It is involved in protein transport from the cytoplasm to the nucleus (Molecular Biology of the cell, Vol. 8, 2017-2038, 1997). However, the effect of NUP205 expression on cancer cell proliferation is not known.

As used herein, “AQR gene” means a gene encoding a human AQR protein. The base sequence of the human AQR gene and the amino acid sequence of the human AQR protein are known. For example, the base sequence of the human AQR gene (SEQ ID NO: 1) and the human AQR protein amino acid sequence (SEQ ID NO: 2) are registered in GenBank (GenBank Accession No. NM_014691), published.
In this specification, the human AQR protein includes not only a protein consisting of the amino acid sequence of SEQ ID NO: 2 but also a variant that can occur in a human individual, and the protein consisting of the sequence of SEQ ID NO: 2 lacks one amino acid or several amino acids. Proteins that are lost, substituted, and / or added and that are caused by mutations based on polymorphisms or mutations are included. However, these mutant human AQR proteins have functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 2.
In the present specification, the “AQR gene” includes not only a gene consisting of the base sequence of SEQ ID NO: 1 but also mutants that can occur in human individuals. For example, in the gene consisting of the base sequence of SEQ ID NO: 1, Genes with bases deleted, substituted and / or added, which are caused by mutations based on polymorphisms or mutations are included. Further, the “AQR gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more with respect to the nucleotide sequence of SEQ ID NO: 1. Variants consisting of nucleotide sequences having 5% or more, 99.7% or more, or 99.9% or more identity are included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. Furthermore, the “AQR gene” includes a variant consisting of a base sequence that hybridizes to the gene consisting of the base sequence of SEQ ID NO: 1 under stringent conditions. Examples of stringent hybridization conditions include, for example, conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” as washing conditions after hybridization. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although there is no particular limitation, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.”. Can do. Hybridization: Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocol in Coil in Amplification (Clinic): Current Protocol in Coil, 1994 (W). It can be carried out according to the method described in Edition (1995) (Oxford University Press). However, these mutant genes encode proteins having functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 2.

In the present specification, “NHP2L1 gene” means a gene encoding human NHP2L1 protein. The base sequence of the human NHP2L1 gene and the amino acid sequence of the human NHP2L1 protein are known. For example, the base sequence of the human NHP2L1 gene (SEQ ID NO: 3) and the human NHP2L1 protein amino acid sequence (SEQ ID NO: 4) are registered in GenBank (GenBank Accession No. NM_001003796).
In this specification, human NHP2L1 protein includes not only a protein consisting of the amino acid sequence of SEQ ID NO: 4 but also a variant that can occur in a human individual, and the protein consisting of the sequence of SEQ ID NO: 4 lacks one amino acid or several amino acids. Proteins that are lost, substituted, and / or added and that are caused by mutations based on polymorphisms or mutations are included. However, these mutant human NHP2L1 proteins have functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 4.
In the present specification, the “NHP2L1 gene” includes not only a gene consisting of the base sequence of SEQ ID NO: 3 but also a variant that can occur in a human individual. For example, in the gene consisting of the base sequence of SEQ ID NO: 3, Genes with bases deleted, substituted and / or added, which are caused by mutations based on polymorphisms or mutations are included. Furthermore, the “NHP2L1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99. Variants consisting of nucleotide sequences having 5% or more, 99.7% or more, or 99.9% or more identity are included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. Furthermore, the “NHP2L1 gene” includes a variant consisting of a base sequence that hybridizes to the gene consisting of the base sequence of SEQ ID NO: 3 under stringent conditions. Examples of stringent hybridization conditions include those described above. However, these mutant genes encode proteins having functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 4.

In the present specification, “NUP205 gene” means a gene encoding human NUP205 protein. The base sequence of human NUP205 gene and the amino acid sequence of human NUP205 protein are known. For example, the base sequence of human NUP205 gene (SEQ ID NO: 5) and the amino acid sequence of human NUP205 protein (SEQ ID NO: 6) are registered in GenBank (GenBank Accession No. NM_015135).
In this specification, the human NUP205 protein includes not only a protein consisting of the amino acid sequence of SEQ ID NO: 6 but also a variant that can occur in a human individual, and the protein consisting of the sequence of SEQ ID NO: 6 lacks one amino acid or several amino acids. Proteins that are lost, substituted, and / or added and that are caused by mutations based on polymorphisms or mutations are included. However, these mutant human NUP205 proteins have functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 6.
In the present specification, the “NUP205 gene” includes not only a gene consisting of the base sequence of SEQ ID NO: 5 but also mutants that can occur in human individuals. For example, in the gene consisting of the base sequence of SEQ ID NO: 5, Genes with bases deleted, substituted and / or added, which are caused by mutations based on polymorphisms or mutations are included. Furthermore, the “NUP205 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more with respect to the nucleotide sequence of SEQ ID NO: 5. Variants consisting of nucleotide sequences having 5% or more, 99.7% or more, or 99.9% or more identity are included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. Furthermore, the “NUP205 gene” includes a variant consisting of a base sequence that hybridizes under stringent conditions to the gene consisting of the base sequence of SEQ ID NO: 5. Examples of stringent hybridization conditions include those described above. However, these mutant genes encode proteins having functions equivalent to those of the protein consisting of the amino acid sequence of SEQ ID NO: 6.

In the present specification, the “substance that suppresses gene expression” refers to a substance that suppresses the transcription of mRNA of the target gene, a substance that degrades the transcribed mRNA, or a substance that suppresses the translation of the protein from the mRNA. There is no particular limitation. Examples of such substances include siRNA, antisense oligonucleotides or ribozymes, or expression vectors thereof. Among these, siRNA and its expression vector are preferable, and siRNA is particularly preferable. In addition to the above, “substances that suppress gene expression” include proteins, peptides, and other small molecules. In the present invention, the target gene is AQR gene, NHP2L1 gene or NUP205 gene.

In the present specification, “siRNA” is an RNA molecule having a double-stranded RNA portion consisting of about 15 to about 40 bases, and cleaves the mRNA of the target gene having a sequence complementary to the antisense strand of the siRNA. And has a function of suppressing the expression of the target gene. Specifically, the siRNA in the present invention is an antisense comprising a sense RNA strand comprising a sequence homologous to a continuous RNA sequence in the mRNA of the AQR gene, NHP2L1 gene or NUP205 gene, and a sequence complementary to the sense RNA sequence. An RNA comprising a double-stranded RNA portion comprising an RNA strand. The design and production of such siRNAs and mutant siRNAs described below are within the skill of the artisan.

The length of the double-stranded RNA portion is about 15 to about 40 bases, preferably 15 to 30 bases, more preferably 15 to 25 bases, still more preferably 18 to 23 bases, and most preferably 19 to 21 bases as a base. It is. The end structure of the sense strand or antisense strand of siRNA is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may have a blunt end or a protruding end (overhang) It is preferable that the 3 ′ end protrudes. The siRNA having an overhang consisting of several bases, preferably 1 to 3 bases, more preferably 2 bases, at the 3 ′ end of the sense RNA strand and the antisense RNA strand suppresses the expression of the target gene. In many cases, the effect is large, which is preferable. The type of the overhanging base is not particularly limited, and may be either a base constituting RNA or a base constituting DNA.

Furthermore, siRNA in which 1 to several nucleotides are deleted, substituted, inserted and / or added in one or both of the sense strand or the antisense strand of the siRNA is also a pharmaceutical composition for treating cancer of the present invention. It can be used for things. Here, the 1 to several bases are not particularly limited, but are preferably 1 to 4 bases, more preferably 1 to 3 bases, and most preferably 1 to 2 bases. Specific examples of such mutations include those in which the number of bases in the overhang portion at the 3 ′ end is 0 to 3, or the base sequence in the overhang portion at the 3 ′ end is changed to another base sequence, or Those in which the length of the sense RNA strand differs from that of the antisense RNA strand by 1 to 3 bases due to insertion, addition or deletion, or in which the base is replaced with another base in the sense strand and / or antisense strand For example, but not limited to. However, it is necessary that the sense strand and the antisense strand can hybridize in these mutant siRNAs, and that these mutant siRNAs have the same ability to suppress gene expression as siRNA having no mutation.

Furthermore, the siRNA may be a molecule having a closed structure at one end, for example, an siRNA having a hairpin structure (Short hairpin RNA; shRNA). A shRNA is a RNA comprising a sense strand RNA of a specific sequence of a target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand RNA, and a linker sequence connecting both strands. The sense strand portion and the antisense strand portion Hybridize to form a double stranded RNA portion.

It is desirable that siRNA does not show a so-called off-target effect in clinical use. The off-target effect refers to the action of suppressing the expression of another gene that is partially homologous to the siRNA used in addition to the target gene. In order to avoid the off-target effect, it is possible to confirm in advance that there is no cross-reaction for the candidate siRNA using a DNA microarray or the like. In addition, by using a known database provided by NCBI (National Center for Biotechnology Information), etc., by confirming that there is no gene containing a portion having high homology with the candidate siRNA sequence other than the target gene, It is possible to avoid the off-target effect.

In order to produce the siRNA of the present invention, a known method such as a method using chemical synthesis or a method using a gene recombination technique can be appropriately used. In the synthesis method, double-stranded RNA can be synthesized by a conventional method based on sequence information. In the method using gene recombination technology, an expression vector incorporating a sense strand sequence or an antisense strand sequence is constructed, and the sense strand RNA or antisense strand RNA generated by transcription after introducing the vector into a host cell. It can also be produced by acquiring each of the above. In addition, by expressing a sense strand RNA of a specific sequence of the target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand RNA, and a linker sequence connecting both strands, and expressing a shRNA that forms a hairpin structure, Desired double-stranded RNA can also be prepared.

As long as the siRNA has an activity of suppressing the expression of the target gene, a part of the nucleic acid constituting the siRNA may be DNA. The siRNA may be a nucleic acid in which all or part of the nucleic acid constituting the siRNA is modified as long as it has the activity of suppressing the expression of the target gene.
The modified nucleic acid means a nucleic acid having a structure different from that of a natural nucleic acid, in which a nucleoside (base site, sugar site) and / or internucleoside binding site is modified. Examples of the “modified nucleoside” constituting the modified nucleic acid include an abasic nucleoside; an arabino nucleoside, 2′-deoxyuridine, α-deoxyribonucleoside, β-L-deoxyribonucleoside, and other sugars Examples include nucleosides having modifications; peptide nucleic acids (PNA), peptide nucleic acids to which phosphate groups are bound (PHONA), locked nucleic acids (LNA), morpholino nucleic acids and the like. Examples of the nucleoside having a sugar modification include substituted pentasaccharides such as 2′-O-methylribose, 2′-deoxy-2′-fluororibose, and 3′-O-methylribose; 1 ′, 2′-deoxyribose Arabinose; substituted arabinose sugars; nucleosides with hexose and alpha-anomeric sugar modifications. These nucleosides may be modified bases with modified base sites. Examples of such modified bases include pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases.
Examples of the “modified internucleoside linkage” constituting the modified nucleic acid include, for example, alkyl linker, glyceryl linker, amino linker, poly (ethylene glycol) linkage, methylphosphonate internucleoside linkage; methylphosphonothioate, phosphotriester , Phosphothiotriester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidate, etc. Non-natural internucleoside linkages.

As the nucleic acid sequence contained in the double-stranded siRNA of the present invention, the sequences described in SEQ ID NOs: 7 to 22 in the sequence listing can be preferably used. The nucleotide sequences of these siRNAs are shown in Table 3. In Table 3, uppercase letters indicate the sense RNA sequence and antisense RNA sequence, and lowercase letters indicate the 3 'terminal overhang sequence. For example, s18725 is a double-stranded siRNA composed of a sense strand shown in SEQ ID NO: 7 and an antisense strand shown in SEQ ID NO: 8, and tt at the 3 ′ end of SEQ ID NO: 7 and 3 of SEQ ID NO: 8 'The terminal ca is an overhang sequence. Of these siRNAs, s18725, s18726 and s18727 knock down the AQR gene, s9548 and s9549 knock down the NHP2L1 gene, and s23175, s23176 and s23177 knock down the NUP205 gene. is there. These siRNAs suppress the growth of a wide range of cancer cells including lung cancer, colon cancer, pancreatic cancer, etc., and the degree of inhibition is large, so that the effect of the pharmaceutical composition for treating cancer containing these siRNAs is great. It is.

Oligonucleotides complementary to the mRNA of the target gene are called “antisense oligonucleotides”, and the function of the mRNA is suppressed by forming a double strand with the gene (mRNA) targeted by the antisense oligonucleotide. “Antisense oligonucleotides” are not limited to those that are completely complementary to the target gene (mRNA), but may contain some mismatches as long as they can be stably hybridized with mRNA.

Antisense oligonucleotides may be modified. By applying an appropriate modification, the antisense oligonucleotide becomes difficult to be degraded in the living body, and the expression of the target gene can be inhibited more stably. Such modified oligonucleotides include S-oligo type (phosphorothioate type), C-5 thiazole type, D-oligo type (phosphodiester type), M-oligo type (methyl phosphonate type), peptide nucleic acid And modified antisense oligonucleotides such as phosphodiester bond type, C-5 propynyl pyrimidine type, 2-O-propyl ribose, 2′-methoxyethoxy ribose type. Furthermore, the antisense oligonucleotide may be one in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom. Such an antisense oligonucleotide is particularly excellent in nuclease resistance and affinity for RNA. Examples of the antisense oligonucleotide in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom include oligonucleotides such as S-oligo type.
An antisense oligonucleotide (or a derivative thereof) can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer (for example, Applied Biosystems). Examples of the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate.

The antisense oligonucleotide of the present invention targets AQR gene, NHP2L1 gene or NUP205 gene. Preferred antisense oligonucleotides of the present invention that target the AQR gene include, but are not limited to, those described in SEQ ID NOs: 23 to 62 (or derivatives thereof). Preferred antisense oligonucleotides of the present invention that target the NHP2L1 gene include, but are not limited to, those described in SEQ ID NOs: 63 to 78 (or derivatives thereof). Preferred antisense oligonucleotides of the present invention that target the NUP205 gene include those described in SEQ ID NOs: 79 to 107 (or derivatives thereof), but are not limited thereto. The antisense oligonucleotide of the present invention may be any one that suppresses the expression of the target gene. One to several, for example, 1, 2 in the nucleotide sequence of any of SEQ ID NOs: 23 to 107 Includes those in which 3 or 4 bases are substituted, added, or deleted.

“Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligoDNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. In the book, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. Specifically, the ribozyme can specifically cleave mRNA or an initial transcription product encoding a target gene within the coding region (including an intron portion in the case of the initial transcription product). The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends adjacent to the portion having the hammerhead structure (about 10 bases in total) are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, it should be a hybrid ribozyme further linked with a tRNA-modified sequence. (Nucleic Acids Res., 29 (13): 2780-2788 (2001)).

The substance that suppresses the expression of the target gene may be a nucleic acid molecule such as siRNA, antisense oligonucleotide or ribozyme, and an expression vector encoding the nucleic acid molecule. In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in a mammalian cell to be administered. The promoter to be used is not particularly limited as long as it can function in the mammal to be administered, but for example, polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter), mammalian promoter (eg, CMV promoter, CAG promoter, SV40 promoter) and the like.
The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. In addition, selectable marker genes for selection of transformed cells (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, etc.) You can also
The basic backbone vector used as the expression vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector. Suitable vectors for administration to mammals such as humans include viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, and the like. .

Examples of the expression vector of the present invention include siRNA, antisense oligonucleotide or ribozyme expression vectors. Among them, siRNA expression vectors are preferable.
As the nucleic acid sequence encoded by the siRNA expression vector of the present invention, the first to 19th nucleotide sequences in the respective base sequences described in SEQ ID NOs: 7 to 22 in the sequence listing can be preferably used. The nucleotide sequences of these siRNAs are shown in capital letters in Table 3. These siRNAs suppress the growth of a wide range of cancer cells including lung cancer, colon cancer, pancreatic cancer, etc., and the degree of inhibition is large, so that the effect of the pharmaceutical composition for treating cancer containing these siRNAs is great. It is.

In the present invention, a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene can be used as an active ingredient of a pharmaceutical composition. The pharmaceutical composition of the present invention can be used as a pharmaceutical composition for cancer treatment by administering the pharmaceutical composition in vivo.

In the pharmaceutical composition of the present invention, a single expression-suppressing substance may be used as an active ingredient, or a plurality of gene expression-suppressing substances may be used as active ingredients. The target genes of the plurality of gene expression inhibitors may be different genes. When the expression-suppressing substance of the pharmaceutical composition of the present invention is siRNA, one or more siRNA may be used as an active ingredient. For example, the pharmaceutical composition of the present invention may contain siRNA for AQR gene and siRNA for NHP2L1 gene.

The type of cancer to be treated by the pharmaceutical composition of the present invention is not particularly limited. For example, bladder cancer, breast cancer, colon cancer, colon cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer Esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, prostate cancer, squamous cell carcinoma, skin cancer, bone cancer, lymphoma, leukemia and brain tumor. Among these, the application of the pharmaceutical composition of the present invention is expected for lung cancer, colon cancer and pancreatic cancer.
The pharmaceutical composition for cancer treatment of the present invention can be used not only for cancer treatment but also for prevention of recurrence and metastasis after cancer treatment.

The pharmaceutical composition of the present invention can employ both oral and parenteral dosage forms. In the case of parenteral administration, it is also possible to administer directly to the tumor site.

The pharmaceutical composition of the present invention can be formulated according to a conventional method, and may contain a pharmaceutically acceptable carrier or additive. Such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl. Sodium starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, pharmaceutical Examples of acceptable surfactants include additives.
The additive is selected from the above alone or in appropriate combination depending on the dosage form of the pharmaceutical composition of the present invention. As for the dosage form, in the case of oral administration, it can be administered as a tablet, capsule, fine granule, powder, granule, liquid, syrup or the like, or in an appropriate dosage form. In the case of parenteral administration, pulmonary dosage forms (for example, those using a nephriser etc.), nasal dosage forms, transdermal dosage forms (for example, ointments, creams), injection dosage forms and the like can be mentioned. In the case of an injection dosage form, it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like.

When the substance that suppresses the expression of the target gene is a nucleic acid molecule such as siRNA, antisense oligonucleotide, or ribozyme, or an expression vector that encodes the nucleic acid molecule, the expression inhibitor is introduced into a phospholipid endoplasmic reticulum such as a liposome. In addition, the endoplasmic reticulum can be used as the pharmaceutical composition of the present invention.

The dosage of the pharmaceutical composition of the present invention varies depending on age, sex, symptoms, administration route, administration frequency, and dosage form. The administration method is appropriately selected depending on the age and symptoms of the patient. The effective dose is 0.01 μg to 1000 mg, preferably 0.1 μg to 100 μg, per kg body weight. However, the therapeutic agent is not limited to these doses.

The invention in a further aspect,
(I) a method for treating cancer, comprising a step of administering a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene;
(Ii) a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene used for cancer treatment, and (iii) expression of AQR gene, NHP2L1 gene or NUP205 gene for the production of a therapeutic agent for cancer. Provide the use of a suppressive substance.
The “substance that suppresses gene expression” is as described above. Further, when siRNA is used as the “substance that suppresses gene expression”, preferred siRNA is as described above.

In another aspect, the present invention provides a method for screening for a therapeutic agent for cancer using as an index suppression of expression of AQR gene, NHP2L1 gene or NUP205 gene.

The test substance to be subjected to the screening method may be any known compound and novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

In one embodiment, the screening method of the present invention comprises the following steps (a) to (c):
(A) contacting a test substance with a cell capable of measuring the expression of AQR gene, NHP2L1 gene or NUP205 gene;
(B) measuring the expression level of the gene in cells contacted with the test substance, and comparing the expression level with the expression level of the gene in control cells not contacted with the test substance;
Next, (c) when the expression of the gene in a cell given the test substance is lower than the expression of the gene in a cell not given the test substance, the test substance is selected as a cancer treatment substance Process.

In step (a) of the above method, cells capable of measuring the expression of the AQR gene, NHP2L1 gene or NUP205 gene and the test substance are placed under contact conditions. Contact of the test substance with cells capable of measuring the expression of these genes is performed in a culture medium.

A cell capable of measuring the expression of AQR gene, NHP2L1 gene or NUP205 gene is a cell capable of directly or indirectly evaluating the expression level of AQR gene, NHP2L1 gene or NUP205 gene product, for example, transcript or translation product. Say. A cell capable of directly evaluating the expression level of the gene product can be a cell capable of naturally expressing the gene, while a cell capable of indirectly evaluating the expression level of the gene product includes: Examples include cells that allow reporter assay for the gene transcription regulatory region. As the cells capable of measuring the expression of the gene, animal cells such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys or human mammalian cells can be used, and human-derived cells are particularly preferable.

A cell enabling a reporter assay for a gene transcription regulatory region is a cell containing a target gene transcription regulatory region and a reporter gene operably linked to the region. The target gene transcription regulatory region and the reporter gene can be inserted into an expression vector. The transcriptional regulatory region of the target gene is not particularly limited as long as it can control the expression of the target gene. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases in the base sequence of the region Examples include a region consisting of a base sequence deleted, substituted or added and having the ability to control the transcription of the target gene. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, the GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

A cell into which a gene transcription regulatory region and a reporter gene operably linked to the region are introduced can be used for quantitative analysis of the expression level of the reporter gene as long as the transcriptional regulatory function of the target gene can be evaluated. As long as it is, it is not particularly limited.

The culture medium in which the cell capable of measuring the expression of the AQR gene, NHP2L1 gene or NUP205 gene and the test substance is contacted is appropriately selected depending on the type of the cell used, for example, about 5 to 20%. Examples include minimal essential medium (MEM) containing fetal calf serum, Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, and 199 medium. The culture conditions are also appropriately determined according to the type of cells to be used. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 144 hours.

In step (b) of the above method, first, the expression level of the AQR gene, NHP2L1 gene or NUP205 gene in the cell contacted with the test substance is measured. The expression level can be measured by a method known per se in consideration of the type of cells used. For example, when a cell capable of naturally expressing any of the AQR gene, NHP2L1 gene or NUP205 gene is used as a cell capable of measuring the expression of the gene, the expression level is the gene product such as a transcription product or a translation product. The product can be measured by a method known per se. For example, the expression level of the transcription product can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological method, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. On the other hand, when a cell capable of performing a reporter assay for the gene transcription regulatory region is used as a cell capable of measuring the expression of the AQR gene, NHP2L1 gene or NUP205 gene, the expression level can be measured based on the signal intensity of the reporter.
Next, the expression level of the gene in the cell contacted with the test substance is compared with the expression level of the gene in the control cell not contacted with the test substance (step (c) of the above method). The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of the gene in the control cell not contacted with the test substance is the expression level measured at the same time, even if it is the expression level measured in advance compared to the measurement of the gene expression level in the cell contacted with the test substance. However, the expression level is preferably measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

The following experiment was conducted for the purpose of finding a gene capable of suppressing the growth of cancer cells by inhibiting the expression. Using human whole gene siRNA library (Human siGENOME siRNA Library-Genome; Thermo Scientific; 21,121 genes) distributed to 384-well plates for cell line RERF-LC-AI derived from human lung squamous cell carcinoma, A gene that suppresses the proliferation of the same cells by suppressing the expression was screened. As a screening protocol, first, 1.25 pmol of the above-mentioned library was dispensed into a 384 well plate (Corning; catalog number 3570). SiRNA against kinesin family member 11 (KIF11) as a positive control on each plate (Thermo Scientific; catalog number M-003317-01-0020), and Non-Targeting siRNA Pool # 2 (NT-2; Thermo Scientific) as a negative control Catalog number D-001206-14-20) was also dispensed in the same amount and used to calculate activity values and assay accuracy.
Meanwhile, 5 μl of Opti-MEM I (Gibco; catalog number 31985) and 0.025 μl of siRNA introduction reagent RNAiMAX (Invitrogen; catalog number 13778-150) are mixed and held at room temperature for 10 minutes. Was added to aliquoted 384 well plates. After incubation at room temperature for 20 minutes, D-MEM (Nacalai Tesque; catalog number 08456-65), 10% Fetal Bovine Serum (Sigma; catalog number 13778-150), 500 RERF-LC-AI cells 20 μl of the culture broth was added and cultured for 4 days in a 37 ° C., 5% CO 2 environment. 25 μl of CellTiter-Glo Luminescent Cell Viability Assay (Promega; Catalog No. G7571 / 2/3) was added to each well, and the amount of luminescence was measured using a Viewlux high-throughput microplate imager (PerkinElmer). Based on the luminescence value of each well, the growth inhibition activity of the positive control KIF11 siRNA is 100% and the growth inhibition activity of the negative control NT-2 is 0% for each plate. Inhibitory activity was calculated. A total of 341 genes that can be purchased from Ambion siRNA from 421 genes with growth inhibitory activity of 75% or more and that do not encode proteosome subunits were subjected to secondary evaluation. The reason for excluding genes encoding proteosome subunits is that anti-cancer drugs targeting proteosome are already on the market, but the gene group cannot be a new target for cancer, This is because a large number (26) were included in the 421 gene.

The 341 gene was subjected to secondary evaluation using RERF-LC-AI cells in the same manner as described above using 3 sequences of each gene of siRNA (product name Silencer Select) manufactured by Ambion. Ambion KIF11 siRNA (siRNA ID s7903; catalog number 4390822) was used for positive control, and Ambion Negative Control # 1 siRNA (catalog number 4390844, NC- # 1) was used for negative control. 0.625 pmol each was used. 42 genes were extracted, assuming that the average of the three sequences of siRNAs was 75% or higher than that of KIF11 siRNA and had reproducibility.
For these 42 genes, a human lung adenocarcinoma-derived cell line A549 was used as a third evaluation, and similarly, a gene showing an average of three sequences of siRNAs exhibiting growth inhibition of 75% or more of KIF11 siRNA was extracted. As a result, 38 genes were identified as important genes for proliferation and survival in both RERF-LC-AI cells and A549 cells.

Next, in order to extract genes having a more desirable profile from the 38 genes, the following high-order evaluation was performed. First, for the purpose of finding target molecules that have a difference in action between normal cells and cancer cells, Ambion of the above-mentioned three sequences was used for human lung normal fibroblast MRC-5 and human normal bronchial epithelial cell BEAS-2B. Similar experiments were performed using siRNA, and the growth inhibitory activity of each siRNA was calculated. Then, genes with an average growth inhibitory activity of 3 sequences of siRNA in each normal cell 30% or more lower than that of A549 cells were extracted, and 4 genes shown in Table 1 were identified. It was found that even if the expression of these genes was suppressed, the effect on the proliferation of normal cells was small. In addition, a gene having a divergence of 30% or more between RERF-LC-AI cells and two normal cells was not found.
On the other hand, RERF-LC-AI cells and A549 cells were selected from D-MEM (Nacalai Tesque; catalog number 08456-65), 10% Fetal Bovine Serum (MP Biomedicals; catalog number 2916754), penicillin: 5,000 units / ml, streptomycin: 5,000 μg / ml (Invitrogen; catalog number 15070-063), cultured in a 37 ° C, 5% CO2 environment to extract total RNA did. In addition, 23 types of normal human tissues (fat, bone marrow, brain hippocampus, brain, colon, fetal brain, fetal liver, heart, kidney, liver, lung, peripheral leukocytes, placenta, prostate, skeletal muscle, small intestine, spinal cord from Clontech Spleen, stomach, testis, thymus, trachea, uterus) and 3 types of human normal tissues (arteries, veins, skin) from BioChain. Expression profile data of these total RNAs was obtained using an Agilent microarray (Whole Human Genome Microarray Kit; catalog number G4112F). Using the data, we extracted genes that are more than 2-fold upregulated in RERF-LC-AI cells and A549 cells compared to normal human tissues among the 38 genes above, and found the 5 genes shown in Table 2 It was. Since these genes have increased expression in cancer cells compared to normal tissues, even if the expression of these genes is suppressed, the effect on normal tissues is considered to be small.
Regarding the 7 genes shown in Table 1 or 2 (2 genes are duplicated in Table 1 and Table 2), a clear link to cancer or cell growth is reported in the literature registered in Pubmed of the National Center for Biological Information. As a result of investigating whether or not it has been reported, no association has been reported with respect to AQR, NHP2L1 or NUP205, and it has become clear that this is a novel target molecule for cancer.

The following experiments were conducted with the aim of confirming knockdown by siRNA, reproducibility of growth suppression, and examining the effects on cancer cells derived from organs other than the lung for the three new cancer target genes. In addition to the aforementioned A549, RERF-LC-AI, MRC-5 and BEAS-2B, human colon cancer cell line HCT116 and human pancreatic cancer cell line BxPC-3 were used, and Ambion's siRNA was sequenced in each gene 2 or 3 Each was used (catalog number AQR, s18725, s18726, s18727; NUP205, s23175, s23176, s23177; NHP2L1, s9548, s9549), and the viability of the cells when each gene was knocked down was evaluated as described above. The sequence of each siRNA is shown in Table 3.

Sequences are written in the 5 ′ to 3 ′ direction.
Lower case letters are 3 ′ overhang nucleotides and DNA.

For the knockdown evaluation, 48 hours after siRNA introduction, template samples were prepared using Cell Amp Direct RNA Prep Kit for RT-PCR (Takara Bio Inc .; catalog number 3732), and One Step SYBR PrimeScript RT- PCR was performed by quantitative PCR using Kit II (Takara Bio Inc .; catalog number RR086A). As primers for quantitative PCR of each gene, products of Takara Bio Inc. (catalog number AQR, HA080236; NUP205, HA078518) or Qiagen (catalog number NHP2L1, QT00027867) were used. In the expression level measurement, β-actin was simultaneously measured as a correction gene and corrected. As a result, by introducing siRNA for the three genes, the proliferation of A549 cells and RERF-LC-AI cells was strongly suppressed, and the screening results of Example 1 were reproduced (FIGS. 1 to 3). Regarding NHP2L1 and NUP205, a phenomenon in which there is a difference in action between lung cancer cell A549 and normal lung cell MRC-5 was also reproduced (FIGS. 1 to 3). Moreover, by introducing siRNA for the three genes, not only lung cancer-derived cells but also colon cancer-derived cells HCT116 and pancreatic cancer-derived cells BxPC-3 were proliferated. In addition, knockdown of each gene by siRNA could be confirmed 48 hours after siRNA introduction (FIGS. 4 to 6).

Antisense oligonucleotide sequence design Design the base sequence of antisense oligonucleotides for 3 novel cancer target genes. As the nucleotide sequence information of each gene, for example, information registered in GenBank of the National Center for Biological Information (human AQR, NM_014691; human NHP2L1, NM_001003796; human NUP205, NM_015135) can be used. Based on each base sequence, for example, 1) the chain length is 13 bases, 2) does not contain a CpG sequence that can be a ligand for TLR9 so as not to show immunostimulatory action, and 3) does not have a higher order structure in the molecule. 4) It is possible to select an effective sequence on the basis that it does not show high homology to other genes. However, the selection criteria are not limited to the above as long as a sequence having an expression suppressing effect on the target nucleic acid can be selected. Examples of antisense oligonucleotides for each new target designed according to the above criteria are shown in Table 4. However, the antisense oligo is not limited to these as long as it has an expression suppressing effect on the target nucleic acid. These antisense oligonucleotides may contain a modified base, and the base may be bound by a bond other than a phosphodiester bond. For example, at least one of the bases indicated in lowercase letters in Table 4 may be LNA (Locked Nucleic Acid), and at least one of the bonds between the bases indicated in uppercase letters may be a phosphorothioate bond. Specific examples of such modified antisense oligonucleotides include those in which all the bases shown in lower case letters in Table 4 are LNAs, and the bonds between the bases shown in upper case letters are phosphorothioate bonds. Not.

Sequences are written in the 5 ′ to 3 ′ direction.

Antisense oligonucleotide synthesis Using standard phosphoramidite chemistry with oxidation by iodine, using an automated DNA synthesizer (NTS H-8-SE; Nihon Techno Service Co. Ltd.) with unsubstituted and substituted phosphodiesters (P = O) Synthesize oligonucleotides.

Standard bin oxide was replaced with a 0.2M solution of 3H-1,2-benzodithiol-3-one 1,1-dioxide in acetonitrile for phosphite-bonded thiation. Except for this, phosphorothioate (P = S) is synthesized in the same manner as the phosphorodiester oligonucleotide. The sulfurization standby process is extended to 70 seconds, and then the cap addition process is continued. After cleavage from the CPG column and deprotection in concentrated ammonium hydroxide at 55 ° C. (18 hours), the oligonucleotide is purified by known gel filtration or reverse phase HPLC methods.

Phosphinate oligonucleotides are prepared using the method described in US Pat. No. 5,508,270.
Alkyl phosphonate oligonucleotides are prepared using the method described in US Pat. No. 4,469,863.
3′-deoxy-3′-methylene phosphonate oligonucleotides are prepared using the methods described in US Pat. No. 5,610,289 or US Pat. No. 5,562,050.
Phosphoramidite oligonucleotides are prepared using the methods described in US Pat. No. 5,256,775 or US Pat. No. 5,366,878.
Alkylphosphonothioate oligonucleotides are prepared using the methods described in WO94 / 17093 or WO94 / 02499.
A 3′-deoxy-3′-aminophosphoramidate oligonucleotide is prepared using the method described in US Pat. No. 5,476,925.
Phosphotriester oligonucleotides are prepared using the methods described in US Pat. No. 5,032,243.
Borano phosphate oligonucleotides are prepared using the methods described in US Pat. No. 5,130,302 or US Pat. No. 5,177,198.

Using the method described in International Publication No. 2003/010284, an oligonucleotide (for example, LNA) having a modified nucleoside is prepared.

Evaluation of Antisense Oligonucleotide Expression Suppression Effect The target nucleic acid expression suppression effect of an antisense oligonucleotide can be evaluated in various cell types if the target nucleic acid is present at a measurable level. The expression suppression effect can be determined by a conventional method using, for example, a quantitative PCR method or Northern blot analysis. Hereinafter, examples using A549, which is a human lung adenocarcinoma-derived cell line, will be described, but the cell type is not particularly limited as long as the target nucleic acid is expressed in the cell type to be selected.
Dispense 1.0 pmol each of the antisense oligonucleotides designed and synthesized above into a 96-well plate (Costar; catalog number 3628). 20 μl of Opti-MEM I (Gibco; catalog number 31985) and 0.1 μl of RNAiMAX (Invitrogen; catalog number 13778-150) were mixed and held at room temperature for 10 minutes, and then the above-mentioned antisense oligonucleotide was dispensed in advance. Add to 96 well plate. After incubation at room temperature for 20 minutes, a culture solution consisting of D-MEM (Nacalai Tesque; catalog number 08456-65) and 10% Fetal Bovine Serum (Sigma; catalog number 13778-150) containing 2000 A549 cells was prepared. Add 80 μl and incubate for 48 hours in a 37 ° C., 5% CO 2 environment. For evaluation of knockdown, template samples were prepared using Cell Amp Direct RNA Prep Kit for RT-PCR (Takara Bio Inc .; catalog number 3732), and One Step SYBR PrimeScript RT-PCR Kit II (Takara Bio Inc .; catalog) It is possible to carry out by the quantitative PCR method using the number RR086A). In the expression level measurement, β-actin is simultaneously measured as a correction gene and corrected. As primers for quantitative PCR of each gene, products of Takara Bio Inc. (catalog numbers AQR, HA080236; NUP205, HA078518; β-actin, HA067803) or Qiagen (catalog numbers NHP2L1, QT00027867) can be used.

Evaluation of Antiproliferation Action of Antisense Oligonucleotide Antisense oligonucleotides that have been confirmed to have an expression inhibition effect on the target nucleic acid are evaluated for an antiproliferation action on cancer cells. The growth inhibitory action can be routinely determined by methods such as actually counting the number of cells, measuring the amount of ATP in the cells, and dehydrogenase activity. Hereinafter, examples using A549 cells are provided, but other cell types can be used as long as antisense oligonucleotides can be introduced into the cell types to be selected.
Dispense 0.25 pmol of the oligonucleotide into a 384 well plate. 5 μl of Opti-MEM I (Gibco; catalog number 31985) and 0.025 μl of RNAiMAX (Invitrogen; catalog number 13778-150) were mixed and held at room temperature for 10 minutes, and then the above-mentioned antisense oligonucleotide was dispensed in advance. Add to 384 well plate. After incubation at room temperature for 20 minutes, a culture solution consisting of D-MEM (Nacalai Tesque; catalog number 08456-65) and 10% Fetal Bovine Serum (Sigma; catalog number 13778-150) containing 500 A549 cells was prepared. Add 20 μl and incubate at 37 ° C in 5% CO2 for 4 days. To each well, 25 μl of CellTiter-Glo Luminescent Cell Viability Assay (Promega; Catalog No. G7571 / 2/3), a reagent for measuring ATP content, was added, and the amount of luminescence was measured by Viewlux high-throughput microplate imager. (PerkinElmer) is used for measurement, and this is a value reflecting the number of cells. Based on the luminescence value of each well, the growth inhibitory action of each antisense oligonucleotide is determined.

The present invention can be used in the field of pharmaceuticals, particularly in the field of development and production of anticancer agents.

Claims

A pharmaceutical composition for the treatment of cancer comprising a substance that suppresses the expression of AQR gene, NHP2L1 gene or NUP205 gene.
The pharmaceutical composition according to claim 1, wherein the substance that suppresses gene expression is siRNA, antisense oligonucleotide or ribozyme, or an expression vector thereof.
The pharmaceutical composition according to claim 1 or 2, wherein the substance that suppresses gene expression is siRNA or an siRNA expression vector.
The pharmaceutical composition according to claim 2 or 3, wherein the siRNA is one or more siRNA selected from the group consisting of siRNAs according to any one of the following (a) to (i):
(A) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 7 and the first to 19th nucleotide sequence in SEQ ID NO: 8;
(B) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 9 and the first to 19th nucleotide sequence in SEQ ID NO: 10;
(C) siRNA in which the double-stranded RNA portion comprises the first to the 19th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 11 and the first to the 19th nucleotide sequence in SEQ ID NO: 12;
(D) siRNA in which the double-stranded RNA portion consists of the first to the 19th nucleotide sequence in SEQ ID NO: 13 and the first to the 19th nucleotide sequence in SEQ ID NO: 14;
(E) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 15 and the first to 19th nucleotide sequence in SEQ ID NO: 16;
(F) siRNA in which the double-stranded RNA portion comprises a double strand consisting of the first to 19th nucleotide sequence in SEQ ID NO: 17 and the first to 19th nucleotide sequence in SEQ ID NO: 18;
(G) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 19 and the first to 19th nucleotide sequence in SEQ ID NO: 20;
(H) siRNA in which the double-stranded RNA portion consists of the first to 19th nucleotide sequence in SEQ ID NO: 21 and the first to 19th nucleotide sequence in SEQ ID NO: 22;
(I) The siRNA according to any one of (a) to (h), wherein 1 to several nucleotides are added, inserted, deleted or substituted in one or both nucleotide sequences in the double-stranded RNA portion.
The pharmaceutical composition according to claim 4, comprising a 3'-terminal overhang in one or both strands of the siRNA.
The pharmaceutical composition according to any one of claims 1 to 5, wherein the cancer is lung cancer, colon cancer or pancreatic cancer.
A method for screening a substance for treating cancer comprising the following steps (a) to (c):
(A) contacting a test substance with a cell capable of measuring the expression of AQR gene, NHP2L1 gene or NUP205 gene;
(B) measuring the expression level of the gene in cells contacted with the test substance, and comparing the expression level with the expression level of the gene in control cells not contacted with the test substance;
Next, (c) when the expression of the gene in a cell given the test substance is lower than the expression of the gene in a cell not given the test substance, the test substance is selected as a cancer treatment substance Process.