WO1996016976A1

WO1996016976A1 - Antisense oligonucleotide and carcinostatic agent containing the same

Info

Publication number: WO1996016976A1
Application number: PCT/JP1995/002452
Authority: WO
Inventors: Masahiko Tsuchiya; Timothy G. Geiser
Original assignee: Pola Chemical Industries Inc.
Priority date: 1994-12-02
Filing date: 1995-12-01
Publication date: 1996-06-06
Also published as: JPH1135595A

Abstract

An antisense oligonucleotide against protein kinase A-RIα improved in the carcinostatic effect and containing as the carcinostatic agent an oligonucleotide having the structure represented by general formula (I) and having a base sequence that is substantially complementary to part of the base sequence of the coding strand of the human protein kinase A-RIα genes represented by SEQ ID No:1 or a base sequence that is substantially complementary to part of the base sequence of the noncoding strand of the above genes wherein n represents an integer; R represents hydrogen or hydroxy; and B represents a nucleic acid base.

Description

The present invention relates to anticancer agents comprising antisense oligonucleotides against human protein kinases, particularly human protein kinases A-RI, anticancer agents comprising the antisense oligonucleotides, and anticancer agents comprising the antisense oligonucleotides. The present invention relates to a pharmaceutical composition containing an anticancer agent. Prior Art Cancer has long been known as an incurable disease, but it has not changed much to date. It is a so-called rebellious molecule, in which cancer cells are derived from the organism itself.The basic physiological mechanism is not much different from normal cells, so if you try to kill cancer cells with anticancer drugs or the like, normal cells It also causes side effects. In view of this situation, methods for selectively attacking and treating only cancer have been studied. For example, a method has been devised in which a carrier such as a monoclonal antibody or ribosome against an antigen specific to cancer cells is used and a drug is carried on these carriers. Alternatively, there was a problem with the binding strength between the carrier and the drug and a problem with the release, and it was not possible to achieve a sufficient effect.

Various genetic manipulation techniques have advanced since the early 1990s and have been applied to the field of cancer treatment. Of these, a gene specifically required for the physiology and proliferation of cancer is reacted with an oligonucleotide (antisense oligonucleotide) having a nucleotide sequence complementary to a part of the gene, so that it is called a cancer-related gene. Antisense technology for covering has been devised, and various studies have been made on this technology. Since this antisense oligonucleotide inhibits gene expression indiscriminately for both cancer cells and normal cells, it is important to know what kind of gene expression is to be suppressed. I have.

Among the anti-cancer effects of these antisense oligonucleotides, And colleagues noted that protein kinase A is required for the division of proliferating cancer cells, and the technology of antisense oligonucleotides against protein kinase A—RIa that we developed (Japanese Patent Application Laid-Open No. 6-218189). However, its anticancer effect in vivo was not very good, and its effectiveness was problematic. SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and an object of the present invention is to provide an antisense oligonucleotide for a protein kinase A gene having an enhanced anticancer effect.

Based on the above-mentioned situation, the present inventors have conducted intensive studies in order to increase the anti-cancer effect of the antisense oligonucleotide on the protein kinase Ait gene to a practically usable level. As a result, they found that the anticancer effect was significantly increased and completed the invention.

That is, the present invention relates to an oligonucleotide having at least a part of a nucleotide sequence complementary to all or a part of the sequence of the human protein kinase A gene and having a structure represented by the general formula (I). It is.

(In the general formula (I), n represents an integer, R represents a hydrogen atom or a hydroxyl group, and B represents a nucleic acid base.) As a part of the human protein kinase A gene, the human protein kinase A Sequences encoding tree portions or portions thereof are included. Further, the regulatory portion includes RI-α.

Further, the present invention provides an anticancer drug comprising the above-mentioned oligonucleotide, and a pharmaceutical composition for treating cancer, comprising at least one selected from the anticancer drugs.

As used herein, the term “antisense oligonucleotide” refers to an oligonucleotide that suppresses the expression of its gene by hybridizing to a gene or mRNA, and the coding strand of gene DNA (( +) Strand) or an oligonucleotide having a nucleotide sequence complementary to the mRNA, as well as an oligonucleotide having a nucleotide sequence complementary to the non-coding strand ((-) strand).

Hereinafter, the present invention will be described in detail.

(1) Antisense oligonucleotide of the present invention

The antisense oligonucleotide of the present invention has a nucleotide sequence at least partially complementary to all or a part of the sequence of human protein kinase A 遗 gene. Oligonucleotide. As a part of the sequence of the human protein kinase A gene, a sequence encoding the regulatory part of human protein kinase A or a part thereof can be mentioned. Further, the regulatory part includes RI-na. The antisense oligonucleotide of the present invention has a base sequence as described above, and the bonding mode between the nucleotides constituting the oligonucleotide is a 3′-amino group represented by the general formula (I). This is due to the phosphoramidate bond to which the 5 'phosphate group is bound (hereinafter simply referred to as "phosphoraamidate bond").

The length of the oligonucleotide chain is not particularly limited as long as the specificity for the protein kinase A gene is maintained. The preferred length is 9 to 40 in terms of the number of bases. This is because if it is less than 9, the specificity for the gene may be low and the anticancer effect may not be sufficiently exerted, and if it exceeds 40, it will be extremely difficult to synthesize by controlling the nucleotide sequence. A more preferable chain length is one having a base number of 15 to 24, which has the best balance between effects and economics.

The antisense oligonucleotide of the present invention is not particularly limited as long as it is a nucleic acid having a sequence complementary to a gene involved in the biosynthesis of protein kinase A-RIa. That is, as long as the bond between nucleotides is a phosphoramidate bond, even if the oligonucleotide is of the DNA type (R is a hydrogen atom in the general formula (I)), the RNA type (R in the general formula (I)) May be a hydroxyl group). The sequence of the antisense oligonucleotide to protein kinase A-RIα is a sequence substantially complementary to a part of the base sequence of the coding or non-coding chain of the protein kinase Α-RIα gene, in other words, non-coding. If the sequence is substantially identical to a part of the nucleotide sequence of the protein or the code chain, it is expected that the biosynthesis of protein kinase Α-RIα can be suppressed. As a part of the nucleotide sequence of the protein kinase II-RIa gene, the above effect can be expected at any site in the gene, but when the expression of protein kinase A-RIα activity is more completely suppressed, From this point of view, it is preferably a part of the sequence consisting of 300 nucleotides from the initiation codon to the 100th codon, at the 5 'end of the coding strand of the gene. 300 nucleotides in the coding region of this protein kinase Α—RI α 遗 gene The sequence of the reotide is shown in SEQ ID NO: 1. The antisense sequence corresponding to this sequence (same as the sequence of the non-coding strand) is shown in SEQ ID NO: 6. Here, the relationship between the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 6 is complementary, and anticancer activity can be expected with antisense oligonucleotides for either strand of the gene.

Further, as a part of the nucleotide sequence shown in SEQ ID NO: 1 or 6, it is preferable that the nucleotide sequence is 5 'end in SEQ ID NO: 1 and 3' end in SEQ ID NO: 6. Here, “substantially complementary” does not require that all the nucleotides in the oligonucleotide be complementary to the protein kinase A gene, and that the oligonucleotide is the gene DNA or m. It means that it only has to hybridize to the corresponding site of RNA and be complementary enough to inhibit transcription or translation. Therefore, some nucleotides in the nucleotide chain may be substituted, deleted or inserted as long as specific hybridization is not inhibited under physiological conditions.

The nucleotides that can be replaced in an antisense oligonucleotide vary depending on the length and sequence of the oligonucleotide, but generally the effects of substitution, deletion and insertion are less near the ends of the oligonucleotide than inside. , AT (or AU) base pairs are less affected by substitution than GC base pairs. Methods for calculating the hybridization intensity of nucleic acids are known, and those skilled in the art may modify some of the sequences based on the sequences shown in SEQ ID NOs: 1 and 6 so that hybridization under physiological conditions is not impaired. It is easy to do. Specifically, those having a homology of 90% or more to the protein kinase A gene can be expected to have some expected effect and can be defined as substantially complementary. . Further, even if an oligonucleotide having a non-specific sequence is added to the 5 'end or 3' end of such an oligonucleotide, it is not substantially affected.

The type of nucleotides constituting the antisense oligonucleotide of the present invention may be a DNA-comprising nucleotide analog in which the base is thymine, adenine, cytosine, or guanine and the sugar is deoxyribose. RNA-constituting nucleotide analogs containing ribonucleic acid, cytosine, and guanine and sugar as ribose may be used. Furthermore, non-specific hydrogen bonding with other bases such as inosine is possible. It may contain a functional base. Most preferred are oligonucleotides of nucleotide analogs that are complementary to the nucleotide sequence of SEQ ID NO: 1. The antisense oligonucleotide sequence to protein kinase A-RIα has a sequence complementary to a part of the base sequence shown in SEQ ID NO: 1, that is, a sequence identical to a part of the sequence shown in SEQ ID NO: 6. Specific examples include oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 2, 3, 4, and 5. In addition, a sequence complementary to a part of the base sequence shown in SEQ ID NO: 6, that is, a sequence having the same sequence as a part of the sequence shown in SEQ ID NO: 1 is specifically shown in SEQ ID NOs: 7, 8, and 9. And oligonucleotides. The positions of these oligonucleotides on the sequence shown in SEQ ID NO: 1 or 6 are shown below. SEQ ID NO: 2: Nucleotide number 280 to 300 of SEQ ID NO: 6

SEQ ID NO: 3: Nucleotide numbers 46 to 57 of SEQ ID NO: 6

SEQ ID NO: 4: Nucleotide number of SEQ ID NO: 6 1 4 8 to 16 5

SEQ ID NO: 5: Nucleotide number of SEQ ID NO: 6 from 250 to 2 79

SEQ ID NO: 7: Nucleotide numbers 1 to 21 of SEQ ID NO: 1

SEQ ID NO: 8: Nucleotide number 2 4 4 to 25 5 of SEQ ID NO: 1

SEQ ID NO: 9: Nucleotide number of 13 to 6 in SEQ ID NO: 1

SEQ ID NO: 10: base number 280 to 300 of SEQ ID NO: 6 The antisense oligonucleotide of the present invention is characterized in that nucleotides are connected to each other by a nitrogen-phosphorus bond as shown in the general formula (I). The antisense oligonucleotide represented by such a general formula has a more excellent tumor-suppressing effect than a conventional antisense oligonucleotide having a binding mode such as phosphorothioate, as described in Examples below.

(2) A method for producing the antisense oligonucleotide of the present invention

The antisense oligonucleotide of the present invention can be synthesized while controlling the nucleotide sequence by successively extending nucleotides according to the following reaction formula.

(Where PO represents a polymer residue, R represents a hydrogen atom or an acyloxy group, and B represents a nucleic acid base.) That is, a dimethyltritylated nucleic acid fixed to a solid phase of a polymer or the like by an ester bond. The nucleoside is detritylated with dichloroacetic acid, and is reacted with (2-cyanoethoxy)-(NNN-diisopropylamino) phosphine and N, N-diisopropylethylamine to form a phosphityl, thereby obtaining a cyanoethoxy nucleoside. After reacting this with tetrazole, tetrazole is further substituted with 3'-amino group of 3'-amino-5'-dimethyltrityldeoxyribonucleotide in the presence of triethylamine, and this dimethyltrityl Condensation of 3'-amino5'-dimethyltrityldeoxyribonucleoside in the same manner If it returns Repetitive, ethoxy Xia Roh Foss phosphoroamidate on polymer attached by a bond of a fixed arbitrary nucleotide sequence in E ester bond O Rigo nucleotides are obtained. this Is subjected to dedimethyltritylation and deesterification using ammonium to obtain an oligonucleotide having a structure represented by the general formula (I).

In the above series of reactions, an oligonucleotide having an arbitrary nucleotide sequence can be automatically obtained by using a commercially available DNA synthesizer and the above reaction reagent. In the above reaction, if a nucleoside in which the sugar chain part is replaced with 2′-acyl-3′-amino-5′-dimethyltritylribose is used, an RNA-like antisense oligonucleotide can be obtained.

(3) The anticancer agent of the present invention

The anticancer agent of the present invention comprises at least one selected from the above-mentioned oligonucleotides. When an oligonucleotide is used as a mixture of two or more, a higher anticancer effect may be obtained.

As shown in the Examples below, the anticancer agent of the present invention exhibits an excellent anticancer effect on various cancer cells. The cancer for which the anticancer agent of the present invention is effective is not particularly limited as long as it is a human cancer. For animal cancer, the antisense oligonucleotide, which is the anticancer agent of the present invention, is expected to be ineffective in some cases because it is an antisense oligonucleotide to the human protein kinase A-RIα 遗 gene. You.

Specific examples of the cancer for which the anticancer agent of the present invention is effective include leukemia, colon cancer, rectal cancer, colon cancer, lung cancer, stomach cancer, liver cancer, kidney cancer, malignant lymphoma, tongue cancer, esophageal cancer, breast cancer, Includes pharyngeal cancer, brain tumor, malignant fibroids, melanoma, cervical cancer, etc. The preferred dosage of the anticancer drug of the present invention varies depending on the disease type, medical condition, age, weight, gender, physical condition, etc., but it is approximately 10 to 200,000 mg once or several times per adult per day. It is appropriate to administer in divided doses. Examples of the administration route include intravenous, intraarterial, intraportal, subcutaneous, intradermal, intramuscular, and intralesional injection, and oral administration.

(4) The anticancer pharmaceutical composition of the present invention

The pharmaceutical composition of the present invention comprises the above-mentioned anticancer agent and an optional component in the dosage form. The optional components in the dosage form can be used without any particular limitation as long as they are commonly used in pharmaceutical compositions. For example, oral preparations include bulking agents, binders, flavoring agents, disintegrating agents, lubricants, S-coated agents, sugar coatings, etc., and injections include pH regulators, isotonic agents , Stabilizers and the like can be exemplified. It may be formulated together with other drugs known to have anticancer effects. The method of forming these anticancer agents and optional components into dosage forms may be a conventional method. Detailed Description of Preferred Embodiments B Month The present invention will be described more specifically with reference to the following examples. It goes without saying that the present invention is not limited to these examples. Example 1 Production Example of Antisense Oligonucleotides (1) SEQ ID NOs: 2, 3, 4, 5, and 5 were prepared using the nucleic acid synthesizer ABI384 Synthesizer (manufactured by Applied Biosystems) by the method described above. DNA-type oligonucleotides having the nucleotide sequences shown in 7, 8, and 9 were synthesized. That is,

The dimethyltritylated nucleoside having the 3 'terminal base of each sequence fixed to the polymer with an ester bond was treated with a 3% methylene chloride solution of dichlorodic acid for 1.5 minutes to perform a dedimethyltritylation reaction. . Phosphitylation was carried out using 0.2 mol of (2-cyanoethoxy)-(N, N-diisopropylamino) clophosphine and 0.2 mol of N, N-diisopropylethylamine in methylene chloride. The reaction was carried out for 10 minutes to obtain cyanoethoxynucleotide. This was reacted with a solution of 0.4 mol of tetrazole in a 90% aqueous solution of acetonitrile for 5 minutes and tetrazolylated, and then 0.2 mol of 3 'having the second base from the 3' terminal. The coupling of these nucleosides was carried out for 20 minutes using a 50% solution of amino-5'-dimethyltrityl deoxyribonucleoside and 0.2 mol of triethylamine in 50% tetrachlorocarbon acetonitrile.

In the same manner as described above, nucleosides are sequentially linked in accordance with each base sequence. Nucleotide removal was performed. The base sequence was previously input to the automatic synthesizer. Example 2 Production Example of Antisense Oligonucleotide (2) A reaction was carried out in the same manner as in Example 1 except that the monomer nucleoside was changed to 2′-acetyl-13′-amino-1,5,1-dimethyltritylribonucleoside, An RNA-type oligonucleotide having the sequence shown in SEQ ID NO: 10 was produced. Example 3 Production Example of Antisense Oligonucleotide (3) Synthesizing a DNA type oligonucleotide having a sequence in which two bases GG at the 5 ′ end of the base sequence shown in SEQ ID NO: 2 were replaced with TT in the same manner as in Example 1. did. Example 4 Anticancer Action on Leukemia Cells (In Vitro) The oligonucleotides produced in Examples 1 to 3 were examined for their growth inhibitory action using HL-60 leukemia cells. That is, cells cultured in RPMI 1640 medium supplemented with 1096 FBS (fetal calf serum) and washed twice with PBS (phosphate buffered saline) were added to RPMI 1640 medium supplemented with 10% FBS. And adjusted to a concentration of 2 × 10 ⁶ Zm 1. To this was added a physiological saline solution of the oligonucleotide produced in Examples 1 to 3 at various concentrations adjusted to a predetermined concentration. After that, the cells were cultured for 1 week at 37 temperatures in a 5% carbon dioxide gas atmosphere, and the number of cells was counted every day. From the number of cells 7 days after the start of culture, the minimum concentration that inhibited cell growth by 100% was determined. As a positive control, the thiooligonucleotide of SEQ ID NO: 1 (21-mer) described in JP-A-6-211889, that is, the binding mode of the oligonucleotide of SEQ ID NO: 2 in Example 1 of the present invention was used as a positive control. What was converted from phosphoramidate to phosphorothioether was used. Table 1 shows the results.

From these results, it can be seen that the oligonucleotide of the present invention has an anticancer effect and that the oligonucleotide of the present invention has an excellent anticancer effect as compared with the conventional oligonucleotide. Oligonucleotide i * ¾r inhibition degree (uM) Example 1 SEQ ID NO: 202

Example 1 SEQ ID NO: 3 2

Example 1 SEQ ID NO: 40.8

Example 1 SEQ ID NO: 50.8

Example 1 SEQ ID NO: 7 4

Example 1 SEQ ID NO: 8 2

Example 1 SEQ ID NO: 94

Example 2 SEQ ID NO: 10

Example 3 1 6

Phosphorothioate SEQ ID NO: 2 3 2

Like the Antitumor effects (in vitro) Example 4 for Example 5 colon cancer cells, the _t complete growth inhibition minimum concentration viewed antiproliferative activity using colon cancer-derived cell LS 1 7 4 T in Table 2 Show. This indicates that the oligonucleotide of the present invention also has the same effect on colon cancer as leukemia.

Table 2 Oligonucleotide growth inhibitory concentrations Example 1 SEQ ID NO: 20.1

Example 1 SEQ ID NO: 3 4

Example 1 SEQ ID NO: 40.8

Example 1 SEQ ID NO: 50.4

Example 1 SEQ ID NO: 72

Example 1 SEQ ID NO: 8 4

Example 1 SEQ ID NO: 94

Example 2 SEQ ID NO: 1 0 1 6

Example 3 3 2

Phosphorothioate SEQ ID NO: 2 3 2 Example 6 Anticancer Action on Colon Cancer Cells (In Vivo) Colon cancer-derived cells LS174T were used to transplant them into nude mice (male, 5 mice per group), and the present invention was used to examine the proliferation. Was examined for its inhibitory effect. That is, the pre-cultured dispersion of cancer to adjust the concentration to 2 x 1 0 ⁷ or 1 injected into 0. 1 ml right dorsal skin transplanted cancer cells, was fed 7 days, were used in the experiment. That is, the oligonucleotide should be mixed and emulsified with 49.5% peanut oil, 49.5% saline and 196 Tween 80 so that the final concentration of the oligonucleotide would be 1 Omg / ml. The sample mixed with the vehicle was subcutaneously administered to a 0.0 lm 1 tumor and the left back skin, and the size of the tumor was measured after the administration. The average volume of tumor in the oligonucleotide-administered group was subtracted from the average volume of tumor in the non-administered group, and this value was divided by the average volume of tumor in the non-administered group, multiplied by 100. And

As the oligonucleotide of the present invention, an oligonucleotide having the sequence shown in SEQ ID NO: 2 was used, and as a control, the phosphorothioate-type oligonucleotide shown in Example 1 was used. The growth inhibitory rate of the oligonucleotide of the present invention was 71%, whereas the oligonucleotide of the control product was 35%. This indicates that the oligonucleotide of the present invention has an excellent anticancer effect even in vivo. In addition, no undesired toxicity was observed even after administration of the oligonucleotide, indicating that the therapeutic index indicated by the safety and the effective concentration for toxicity was high. Example 7 Effect of Combination of Antisense Oligonucleotides of Diplomacy Similar to Example 4, the oligonucleotide of the present invention having the sequence shown in SEQ ID NO: 2 and the oligonucleotide of the present invention having the sequence shown in SEQ ID NO: 3, etc. The molar mixture was tested for its anticancer effect on HL-60. The minimum cytostatic concentration was 0.4 μM (a mixture of 0.2 M of the oligonucleotide of the present invention of SEQ ID NO: 2 and 0.2% of the oligonucleotide of the present invention of SEQ ID NO: 3), which was additive. It can be seen that the effect is more than the effect. INDUSTRIAL APPLICABILITY The antisense oligonucleotide for the protein kinase A gene of the present invention has a high anticancer effect, and thus is very useful as an anticancer agent and a medical composition for treating cancer.

Sequence listing

(1) General information

(i) Applicant: Pola Chemical Industry Co., Ltd.

(ii) Title of the invention: Antisense oligonucleotide and anticancer agent using the same (iii) Number of sequences: 10

(iv) Contact:

(A) Address

(B) Address

(C) City

(D) State

(E) Country

(F) ZIP:

(V) Computer readable format

(A) Medium: Floppy disk

(B) Computer: IBM PC compatible

(C) Operation system: PC-DOS / MS-DOS

(D) Software: FastSEQ Version 1.5

(vi) Current application data

(A) Application number

(B) Filing date

(C) Classification

(viii) Agent Z office information

(A) Name:

(B) Registration number:

(C) Reference number:

(ix) Communication information

(A) Phone number:

(B) Facsimile number:

(2) Sequence information of SEQ ID NO: 1

(i) Properties of the array:

(A) Array length: 300 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand (D) Topology: linear

(ii) Sequence type: Genomic DNA

(iv) Antisense: NO

(xi) Sequence: SEQ ID N0: 1:

ATGGAGTCTG GCAGTACCGC CGCCAGTGAG GAGGCACGCA GCCTTCGAGA ATGTGAGCTC 60 TACGTCCAGA AGCATAACAT TCAAGCACTG CTCAAAGATT CTATTGTGCA GTTGTGCACT 120 GCTCGACCTG AGAGACCCAT GGCATTCCTC AGGGAATACT TTGAGAGGTT GGAGAAGGAG 180 GAGGCAAAAC AGATTCAGAA TCTGCAGAAA GCAGGCACTC GTACAGACTC AAGGGAGGAT 240 GAGATTTCTC CTCCTCCACC CAACCCAGTG GTTAAAGGTA GGAGGCGACG AGGTGCTATC 300

(2) Sequence information of SEQ ID NO: 2

(i) Properties of the array:

(A) Array length: 21 bases

(B) Sequence type: nucleic acid

(C) Number of 縝: single strand

(D) Topology: linear

(ii) Sequence type: other nucleic acid synthesis perico'5 [nucleotide

(iv) Antisense: YES

(xi) Sequence: SEQ ID N0: 2:

GGCGGTACTG CCAGACTCCA T 21

(2) Sequence information of SEQ ID NO: 3

(i) Properties of the array:

(A) Array length: 12 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand

(D) Topology: linear

(ii) Sequence type: other nucleic acid synthesis] 'cleotit'

(iv) Antisense: YES (xi) Sequence: SEQ ID N0: 3:

AT 12

(2) Sequence information of SEQ ID NO: 4

(i) Properties of the array:

(A) Array length: 18 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand

(D) Topology: linear

(ii) Sequence type: other nucleic acid synthesis; 51 nucleotides (iv) Antisense: YES

(xi) Sequence: SEQ ID N0: 4:

CCTGAGGAAT GCCATGGG 18

(2) Sequence information of SEQ ID NO: 5:

(i) Properties of array:

(A) Array length: 30 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand

(D) Topology: Direct letter

(ii) Sequence type: other nucleic acid synthesis ^] '? Cleochito '(iv) Antisense: YES

(xi) Sequence: SEQ ID N0: 5:

TTCTCGAAGG CTGCGTGCC TCCTCACTGGC 30

(2) Sequence information of SEQ ID NO: 6

(i) Properties of the array:

(A) Array length: 300 bases

(B) Sequence type: nucleic acid (C) Number of chains: single strand

(D) Topology: linear

(ii) Sequence type: Genomic DNA

(iv) Antisense: YES

(xi) Sequence: SEQ ID NO: 6:

GATAGCACCT CGTCGCCTCC TACCTTTAAC CACTGGGTTG GGTGGAGGAG GAGAAATCTC 60 ATCCTCCCTT GAGTCTGTAC GAGTGCCTGC TTTCTGCAGA TTCTGAATCT GTTTTGCCTC 120 CTCCTTCTCC AACCTCTCAA AGTATTCCCT GAGGAATGCC ATGGGTCTCT CAGGTCGAGC 180 AGTGCACAAC TGCACAATAG AATCHTGAG CAGTGCTTGA ATGTTATGCT TCTGGACGTA 240 GAGCTCACAT TCTCGAAGGC TGCGTGCCTC CTCACTGGCG GCGGTACTGC CAGACTCCAT 300

(2) Sequence information of SEQ ID NO: 7

(i) Properties of the array:

(A) Array length: 21 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand

(D) Topology: linear

(ii) Sequence type: synthesis of other nucleic acids;

(iv) Antisense: NO

(xi) Sequence: SEQ ID NO: 7:

ATGGAGTCTG GCAGTACCGC C 21

(2) Sequence information of SEQ ID NO: 8:

(i) Properties of the array:

(A) Array length: 12 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand

(D) Topology: linear

(ii) Sequence type: synthesis of other nucleic acids; H] '5t nucleotide (iv) Antisense: NO

(xi) Sequence: SEQ ID NO: 8:

ATHCTCCTC CT 12

(2) S £ Sequence number 9 sequence information:

(i) Properties of the array:

(A) Array length: 18 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand

(D) Topology: linear

(ii) Sequence type: other nucleic acid synthesis; H] 'nucleotide (iv) Antisense: NO

(xi) Sequence: SEQ ID NO: 9:

CCCATGGCAT TCCTCAGG 18

(2) Sequence information of SEQ ID NO: 10:

(i) Properties of the array:

(A) Array length: 21 bases

(B) Sequence type: nucleic acid

(C) Number of chains: single strand

(D) Topology: linear

(ii) Sequence type: Other nucleic acid synthesis; H] 'nucleotide (iv) Antisense: YES

(xi) Sequence: SEQ ID NO: 10:

GGCGGUACUG CCAGACUCCA ϋ 21

Claims

The scope of the claims

1. An oligonucleotide having a nucleotide sequence at least partially complementary to at least a part or all of the sequence of the human protein kinase A gene, and having a structure represented by the general formula (I).

(In the general formula (I), n represents an integer, R represents a hydrogen atom or a hydroxyl group, and B represents a nucleic acid base.)

2. The oligonucleotide according to claim 1, wherein a part of the human protein kinase A gene is a regulatory part of human protein kinase A or a sequence encoding a part thereof.

3. The oligonucleotide according to claim 1, wherein the regulatory part is RI-α.

4. The oligonucleotide according to claim 3, which has a nucleotide sequence at least partially complementary to the nucleotide sequence of the coding chain of the human protein kinase Α-RIα¾ gene shown in SEQ ID NO: 1.

5. The ovirus according to claim 3, which has a nucleotide sequence at least partially complementary to the nucleotide sequence of the non-coding strand of the human protein kinase Α-RIα gene shown in SEQ ID NO: 6. Rigo nucleotides.

6. The oligonucleotide according to any one of claims 1 to 5, wherein the size is 9 to 40 mer.

7. The oligonucleotide according to claim 4, which has a nucleotide sequence substantially identical to the nucleotide sequence shown in SEQ ID NOs: 2 to 5 or 10.

8. The oligonucleotide according to claim 7, wherein the nucleotide sequence is any of the nucleotide sequences shown in SEQ ID NOs: 2 to 5 or 10.

9. The oligonucleotide according to claim 5, which has a nucleotide sequence substantially identical to the nucleotide sequence shown in SEQ ID NOs: 7 to 9.

10. The oligonucleotide according to claim 9, wherein the nucleotide sequence is any of the nucleotide sequences shown in SEQ ID NOs: 7 to 9.

11. An anticancer agent _c comprising the oligonucleotide according to any one of claims 1 to 10.

12. A pharmaceutical composition for treating cancer, comprising at least one selected from the anticancer agents according to claim 11.