WO2010021389A1 - Nucleic acid capable of inhibiting expression of bcl-2 protein - Google Patents

Abstract

Disclosed are: a nucleic acid which can inhibit the expression of Bcl-2 protein that acts as an inhibitory factor for apoptosis; a pharmaceutical composition comprising the nucleic acid; and a method for promoting apoptosis by using the nucleic acid.  Particularly disclosed are: a double-stranded nucleic acid composed of a nucleic acid comprising a nucleotide sequence for a part of an untranslated region of a gene encoding Bcl-2 protein and a nucleic acid comprising a nucleotide sequence complementary with the nucleotide sequence for the aforementioned nucleic acid; and a pharmaceutical composition comprising the double-stranded nucleic acid as an active ingredient.  The pharmaceutical composition can treat or prevent diseases which require the promotion of apoptosis for their healing such as cancer.

Description

Nucleic acids that suppress the expression of Bcl-2 protein

The present invention relates to a nucleic acid for use in suppressing the expression of Bcl-2 protein that acts as an inhibitor of apoptosis, and a pharmaceutical composition containing the nucleic acid.

Bcl-2 protein is a mitochondrial inner membrane protein showing inhibition of cell death by apoptosis in several cell types (see Non-Patent Document 1). Inhibition of apoptosis due to large-scale expression of Bcl-2 protein is thought to cause cancer and hematological malignancies. In fact, Bcl-2 protein is produced in large quantities in various solid cancers such as lymphosarcoma, prostate cancer, breast cancer, lung cancer, colon cancer and rectal cancer (see Non-Patent Documents 2 to 6). It has also been shown that Bcl-2 protein expression is involved in thymic apoptosis (see Non-Patent Document 7). Bcl-2 protein has also been shown to have some role in prostate cancer, and has been particularly associated with malignant tumors that are common in certain primates (see Non-Patent Documents 8 and 9).

In cells in which a large amount of Bcl-2 protein is produced, cell death is not induced due to its apoptosis-inhibiting action, and thus drug resistance to various anticancer agents is caused. On the other hand, it is known that suppression of Bcl-2 protein expression in prostate cancer cells results in suppression of cell proliferation and facilitates apoptosis (see Non-Patent Document 8). Therefore, in diseases that require promotion of apoptosis in their healing, such as solid cancers and hematological malignancies, the method of suppressing the expression of Bcl-2 protein can be an effective treatment or prevention method.

Suppressive oligonucleotide compounds configured to inhibit the expression by binding to a nucleic acid encoding a protein causing a disease such as cancer may be useful in the treatment of those diseases. For example, an antisense oligonucleotide for the bcl-2 gene can be considered.

As a method for suppressing the expression of a target gene, a method using RNA interference (hereinafter also referred to as RNAi) is known. For example, it is known that expression of a target gene is suppressed by introducing a double-stranded RNA having a length of 21 to 23 bases into a cell (see Patent Document 1). Such RNA is named small interfering RNA (siRNA).

SiRNA for the translation region of bcl-2 mRNA is known (see Patent Documents 2 and 3, Non-Patent Documents 10 to 12), but siRNA for the untranslated region of bcl-2 mRNA is not known.

Special Table 2003-529374 JP-T-2004-519457 International Publication No. 04/106511 Pamphlet

An object of the present invention is to provide a nucleic acid capable of suppressing the expression of Bcl-2 protein that acts as an inhibitor of apoptosis. Another object of the present invention is to provide a pharmaceutical composition for treating or preventing a disease such as cancer that requires promotion of apoptosis in its healing.

The present invention relates to the following (1) to (28).
(1) a nucleic acid comprising a partial base sequence of a non-translated region of a gene encoding a Bcl-2 protein, a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid, and Bcl- 2. A nucleic acid having protein expression-inhibiting activity.
(2) The nucleic acid according to (1), wherein a part of the base sequence of the untranslated region of the gene encoding Bcl-2 protein is a sequence consisting of 15 to 27 bases.
(3) A nucleic acid comprising a partial base sequence of the untranslated region of the gene encoding Bcl-2 protein is a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 54 to 106 or at least one of the nucleic acids The nucleic acid according to (1) or (2), which is a nucleic acid from which one to four bases have been deleted.
(4) a nucleic acid comprising a partial base sequence of the untranslated region of the gene encoding the Bcl-2 protein according to (1), and at least a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid One of the nucleic acids, wherein 1 to 3 bases are substituted, deleted or added, and has a Bcl-2 protein expression inhibitory activity.
(5) A double-stranded nucleic acid comprising a nucleic acid comprising a partial base sequence of the untranslated region of the gene encoding Bcl-2 protein, and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid.
(6) The double-stranded nucleic acid according to (5), which has a double-stranded forming part consisting of 15 to 27 base pairs.
(7) A nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 54 to 106 or a nucleic acid from which at least one end of the nucleic acid has been deleted by 1 to 4 bases, and complementary to the base sequence of the nucleic acid A double-stranded nucleic acid comprising a nucleic acid comprising a base sequence.
(8) In at least one strand of the double-stranded nucleic acid according to any one of (5) to (7), 1 to 3 bases are substituted, deleted or added, and the expression of Bcl-2 protein is suppressed. A double-stranded nucleic acid having activity.
(9) A double-stranded nucleic acid comprising the double-stranded nucleic acid according to any one of (5) to (8) and having a duplex forming part of 27 base pairs or less.
(10) A double-stranded nucleic acid in which 1 to 4 bases are added to the 3 ′ end or 5 ′ end of at least one strand of the double-stranded nucleic acid according to any one of (5) to (9).
(11) A nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 1-212, 216 and 217.
(12) The nucleic acid according to (11), wherein 1 to 3 bases are substituted, deleted or added, and has Bcl-2 protein expression inhibitory activity.
(13) A nucleic acid having 30 bases or less, including the nucleic acid according to (11) or (12).
(14) A nucleic acid in which part or all of the nucleotides constituting the nucleic acid according to any one of (1) to (13) are ribonucleotides and have Bcl-2 protein expression inhibitory activity.
(15) A nucleic acid in which a part or all of the nucleotides constituting the nucleic acid according to any one of (1) to (14) are deoxyribonucleotides or modified nucleotides and have Bcl-2 protein expression inhibitory activity .
(16) The nucleic acid according to (10) or (15), wherein at least one of the 1 to 4 bases added to the 3 ′ end or 5 ′ end is deoxyribonucleotide.
(17) A vector encoding the nucleic acid according to any one of (1) to (16).
(18) A pharmaceutical composition comprising the nucleic acid or vector according to any one of (1) to (17) as an active ingredient.
(19) The pharmaceutical composition according to (18), further comprising a carrier effective for transferring the nucleic acid into cells.
(20) The pharmaceutical composition according to (19), wherein the carrier effective for transferring the nucleic acid into the cell is a cationic carrier.
(21) The pharmaceutical composition according to (20), wherein the carrier effective for transferring nucleic acid into cells is a liposome.
(22) The pharmaceutical composition according to (21), wherein the liposome reaches the tissue or organ containing the expression site of the gene encoding Bcl-2 protein.
(23) A liposome composed of a composite particle comprising a nucleic acid or vector and a lead particle as constituent components and a lipid bilayer coating the composite particle, wherein the lipid bilayer component is soluble in ethanol The pharmaceutical composition according to (18), wherein the composite particles are dispersed in a 5 vol% ethanol aqueous solution, and the composite particles are dispersed in a 5 vol% ethanol aqueous solution.
(24) The pharmaceutical composition according to any one of (18) to (23), which suppresses the expression of Bcl-2 protein.
(25) The pharmaceutical composition according to (24), which promotes apoptosis.
(26) The pharmaceutical composition according to any one of (18) to (25), which is used for treatment or prevention of cancer.
(27) A method for suppressing the expression of Bcl-2 protein in a subject, which comprises administering the nucleic acid, vector or pharmaceutical composition according to any one of (1) to (26) to the subject.
(28) Use of the nucleic acid or vector according to any one of (1) to (17) for the production of a pharmaceutical composition.

FIG. 1 shows that the double-stranded nucleic acid of the present invention (SEQ ID NOs: 109 and 162) was transfected into PC-3 cells at a final concentration of 50 nM and cultured for 72 hours, and then the expression of Bcl-2 protein was evaluated by Western blotting. It is a figure which shows a result. FIG. 2 shows that the double-stranded nucleic acid of the present invention (SEQ ID NOs: 109 and 162) was transfected into PC-3 cells at a final concentration of 30 nM, cultured for 24 hours, and then bcl-2 mRNA was prepared by RT-PCR. It is a figure which shows the result of having quantified. When quasi-quantifying bcl-2 mRNA, the amount of GADPH mRNA in each sample was used as an internal control. FIG. 3 shows the inhibitory effect on cell proliferation of double-stranded nucleic acids (sense strand SEQ ID NOs: 107 to 119, antisense strand SEQ ID NOs: 160 to 172) targeting the untranslated region of the bcl-2 gene. It is a figure which shows the result evaluated by measurement. After transfection of double-stranded nucleic acid into PC-3 cells, the number of viable cells after 6 days of culture was measured. The ratio of the number of viable cells when each double-stranded nucleic acid is transfected when the number of viable PC-3 cells not transfected with the double-stranded nucleic acid is 1 is shown on the vertical axis. Each left bar graph is the result when double-stranded nucleic acid is added at a final concentration of 3 nM, and the right bar graph is the result when double-stranded nucleic acid is added at a final concentration of 30 nM. FIG. 4 shows the inhibitory effect on cell proliferation of double-stranded nucleic acids (sense strand SEQ ID NO: 120-159, antisense strand SEQ ID NO: 173-212) targeting the untranslated region of the bcl-2 gene. It is a figure which shows the result evaluated by measurement. After transfection of PC-3 cells with double-stranded nucleic acid, the number of viable cells after culturing for 5 days was measured. The ratio of the number of viable cells when each double-stranded nucleic acid is transfected when the number of viable PC-3 cells not transfected with the double-stranded nucleic acid is 1 is shown on the vertical axis. The results are obtained when double-stranded nucleic acid was added at a final concentration of 30 nM. FIG. 5 is a diagram showing the antitumor effect of each double-stranded nucleic acid-WL on PC-3 cells transplanted into nude mice. The vertical axis shows the tumor volume ratio. * And ** indicate a P value <0.05 and a P value <0.01, respectively, between the negative control group and the double-stranded nucleic acid (SEQ ID NO: 109, 162) administration group targeting the untranslated region of the bcl-2 gene. Student-t test) indicates a significant difference. ◆ (diamonds) indicates a negative control group, □ (squares) indicates a double-stranded nucleic acid (SEQ ID NO: 109, 162) -WL administration group of the present invention, and Δ (triangles) indicates a B717-WL administration group. FIG. 6 shows a double-stranded nucleic acid consisting of SEQ ID NOs: 216 and 217, a double-stranded nucleic acid consisting of SEQ ID NOs: 109 and 162, and two having a 2′-OMe modification in the ribose of a part of the nucleotides FIG. 2 shows the results of semi-quantification of bcl-2 mRNA by RT-PCR after strand nucleic acids were transfected into PC-3 cells at a final concentration of 30 nM and cultured for 24 hours. When quasi-quantifying bcl-2 mRNA, the amount of GADPH mRNA in each sample was used as an internal control.

The gene encoding the Bcl-2 protein targeted by the nucleic acid of the present invention (hereinafter also referred to as bcl-2 gene) is Genbank Accession No. A gene containing a DNA base sequence (SEQ ID NO: 215) corresponding to the full-length mRNA of bcl-2 registered as NM_000633. In the present invention, the nucleotide consisting of the base sequence represented by SEQ ID NO: 215 is also referred to as a target gene.

1. Nucleic acid of the present invention The nucleic acid of the present invention includes (a) a nucleic acid (single-stranded nucleic acid) containing a partial base sequence of the untranslated region of a gene encoding Bcl-2 protein, and (b) a Bcl-2 protein. A nucleic acid (single-stranded nucleic acid) containing a base sequence complementary to a nucleic acid consisting of a part of the base sequence of the untranslated region of the gene to be encoded, or (c) the nucleic acid of (b) and the base sequence of the nucleic acid Double-stranded nucleic acid composed of a nucleic acid containing a base sequence complementary to the above may be used, and any of them may be used. The nucleic acid of the present invention is preferably a nucleic acid that suppresses the expression of Bcl-2 protein.

In the present invention, a nucleic acid containing a base sequence complementary to a nucleic acid consisting of a partial base sequence of the untranslated region of the bcl-2 gene is referred to as an antisense strand nucleic acid, A nucleic acid containing a complementary base sequence is also referred to as a sense strand nucleic acid. The sense strand nucleic acid may be a nucleic acid itself consisting of a partial base sequence of the untranslated region of the bcl-2 gene.

The nucleic acid of the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides or molecules having functions equivalent to the nucleotides. For example, it is a polymer of RNA or deoxyribonucleotide that is a polymer of ribonucleotides. Examples thereof include DNA, chimeric nucleic acids composed of RNA and DNA, and nucleotide polymers in which at least one nucleotide of these nucleic acids is substituted with a molecule having a function equivalent to that of the nucleotide. The nucleic acid of the present invention includes siRNA, sh (short hairpin) RNA, and derivatives containing at least one molecule having a function equivalent to nucleotide in these nucleic acids. Uridine (U) in RNA can be uniquely read as thymine (T) in DNA.

Examples of molecules having a function equivalent to nucleotides include nucleotide derivatives.
The nucleotide derivative may be any molecule as long as it is a modified nucleotide. For example, compared to RNA or DNA, the affinity to complementary strand nucleic acid is increased in order to improve or stabilize the nuclease resistance of the nucleic acid. In order to increase cell permeability, to increase cell permeability, or to visualize nucleic acids, a molecule in which ribonucleotides or deoxyribonucleotides are modified is preferably used.

Examples of nucleotide derivatives include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and nucleotides modified with at least one of the sugar moiety, phosphodiester bond, and base.

The sugar moiety-modified nucleotide may be any nucleotide as long as it is a part or all of the chemical structure of the sugar of the nucleotide, modified or substituted with any substituent, or substituted with any atom. '-Modified nucleotides are preferably used.
2′-modified nucleotides include, for example, those in which the 2′-OH group of ribose is H, OR, R, R′OR, SH, SR, NH ₂ , NHR, NR ₂ , N ₃ , CN, F, Cl, Br and Substituted with a substituent selected from the group consisting of I (R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms and R ′ is alkylene, preferably alkylene having 1 to 6 carbon atoms) A 2′-modified nucleotide, preferably a 2′-OH group is F or a methoxy group. In addition, 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- [2- (N, 2'-substituted with a substituent selected from the group consisting of N-Dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) etoxy group and 2-cyanoetoxy group Examples thereof include modified nucleotides.

Examples of the sugar-modified nucleotide include a crosslinked structure-type artificial nucleic acid (BNA) having two circular structures by introducing a crosslinked structure into the sugar moiety, specifically, the 2 ′ position. Locked 原子 Nucleic Acid (LNA), Ethylene Bridged nucleic acid (ENA) [Nucleic Acid Research, 32 , E175 (2004)], and peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123 , 4653 (2001)], peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like.

The phosphodiester bond-modified nucleotide is any nucleotide that has been modified or substituted with an arbitrary substituent for a part or all of the chemical structure of the phosphodiester bond of the nucleotide, or with any atom. For example, a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, a phosphate Examples thereof include nucleotides in which a diester bond is substituted with a phosphoramidate bond.

As the base-modified nucleotide, any or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent or substituted with an arbitrary atom may be used. In which oxygen atoms are substituted by sulfur atoms, hydrogen atoms are substituted by alkyl groups having 1 to 6 carbon atoms, methyl groups are substituted by hydrogen or alkyl groups having 2 to 6 carbon atoms, amino Examples thereof include those in which the group is protected with a protecting group such as an alkyl group having 1 to 6 carbon atoms or an alkanoyl group having 1 to 6 carbon atoms.

Furthermore, as a nucleotide derivative, a nucleotide, sugar moiety, phosphodiester bond or nucleotide derivative modified with at least one of a base, a lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, Examples include dyes and other chemical substances added. Specifically, 5′-polyamine addition nucleotide derivatives, cholesterol addition nucleotide derivatives, steroid addition nucleotide derivatives, bile acid addition nucleotide derivatives, vitamin addition nucleotide derivatives, Cy5 Additional nucleotide derivatives, Cy3-added nucleotide derivatives, 6-FAM-added nucleotide derivatives, biotin-added nucleotide derivatives and the like can be mentioned.

The nucleotide derivative may form a cross-linked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, or a structure combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid. Good.

The nucleic acid of the present invention is a nucleic acid having a function equivalent to that of a nucleic acid comprising a partial base sequence of the untranslated region of the bcl-2 gene or a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid. , May be composed of any nucleotide or derivative thereof. That is, in a nucleic acid consisting of a partial base sequence of the untranslated region of the bcl-2 gene or a nucleic acid consisting of a base sequence complementary to the base sequence of the nucleic acid, the nucleotides constituting the base sequence are It may be substituted with a ribonucleotide, deoxyribonucleotide or a derivative thereof having an equivalent function.

In the nucleic acid of the present invention, a nucleic acid comprising a part of the base sequence of the untranslated region of the bcl-2 gene and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid may form a double strand. Any length is possible, but the length of the sequence capable of forming a duplex is usually 15 to 27 bases, preferably 15 to 25 bases, more preferably 15 to 23 bases, and further 15 to 21 bases. Preferably, 15 to 19 bases are particularly preferable.

As the nucleic acid of the present invention, a nucleic acid consisting of a partial base sequence of the untranslated region of the bcl-2 gene is used. Among the nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base is used. May be deleted, substituted or added.

The nucleic acid that suppresses the expression of Bcl-2 protein includes a partial base sequence of the untranslated region of the bcl-2 gene and a base sequence complementary to the base sequence of the nucleic acid, and Bcl-2 Any nucleic acid such as a single-stranded nucleic acid and a double-stranded nucleic acid can be used as long as it suppresses protein expression, but a double-stranded nucleic acid is preferably used.

In the present invention, the double-stranded nucleic acid means a nucleic acid having two strands paired and having a double-stranded forming part. The double-stranded forming part refers to a part where nucleotides constituting the double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand. The duplex forming part is usually 15 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, further preferably 15 to 21 base pairs, and particularly preferably 15 to 19 base pairs. .

The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 15 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and more preferably 15 to 25 bases. More preferably, it consists of 17 to 23 bases, most preferably 19 to 21 bases.

When the double-stranded nucleic acid of the present invention has an additional nucleotide or nucleotide derivative that does not form a duplex on the 3 ′ side or the 5 ′ side following the duplex forming portion, this is referred to as an overhang. Call it. In the case of having an overhang, the nucleotide constituting the overhang may be ribonucleotide, deoxyribonucleotide or a derivative thereof.

As the double-stranded nucleic acid, one having a protruding portion consisting of 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used, but one having a protruding portion consisting of 2 bases is preferably used. What has the protrusion part which consists of dTdT or UU is used more preferable. Overhangs can be on the antisense strand only, sense strand only, and both antisense and sense strands, but double-stranded nucleic acids with overhangs on both the antisense and sense strands, or antisense A double-stranded nucleic acid having an overhang only at the 3 ′ end of the strand is preferably used. In addition, a sequence that matches the target sequence following the duplex forming portion, or a sequence that matches the base sequence of the complementary strand of the target sequence following the duplex forming portion can also be used. In the present invention, a double-stranded nucleic acid having a nucleotide derivative is preferably used as the double-stranded nucleic acid having no 3'-end or 5'-end overhang.

In the double-stranded nucleic acid of the present invention, it is possible to use a nucleic acid comprising the same sequence as the base sequence of the target gene or its complementary strand, but the 5 ′ end of at least one strand of the nucleic acid or It is also possible to use a double-stranded nucleic acid comprising a nucleic acid from which 1 to 4 bases have been deleted at the 3 ′ end and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid. Examples of such a double-stranded nucleic acid include a double-stranded nucleic acid having a double-stranded forming part consisting of 15 to 19 base pairs.
Moreover, a single-stranded nucleic acid can also be used as the nucleic acid of the present invention. Examples of such nucleic acids include nucleic acids having the base sequences represented by any of SEQ ID NOs: 1-212, 216 and 217, and in these nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably Examples also include a nucleic acid in which one base is substituted, deleted or added and has Bcl-2 protein expression inhibitory activity. Examples of the nucleic acid containing these nucleic acids include 30 bases or less, preferably 28 bases or less, more preferably 26 bases or less, still more preferably 24 bases or less, and particularly preferably 23 bases or less.

The nucleic acid of the present invention may be a single-stranded nucleic acid obtained by linking the sense strand and the antisense strand of the above-described double-stranded nucleic acid via a spacer sequence. The single-stranded nucleic acid is preferably a single-stranded nucleic acid such as shRNA having a double-strand formation portion with a stem-loop structure. A single-stranded nucleic acid having a stem-loop structure is usually 50 to 70 bases in length.

The method for producing the nucleic acid of the present invention is not particularly limited, and examples thereof include a method using known chemical synthesis or an enzymatic transcription method. Examples of methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method [Nucleic® Acid® Research, 35, 20073287 (2007)]. For example, ABI3900 high-throughput nucleic acid synthesis Can be synthesized by a machine (Applied Biosystems). After the synthesis is completed, elimination from the solid phase, deprotection of the protecting group, purification of the target product, and the like are performed. It is desirable to obtain a nucleic acid having a purity of 90% or more, preferably 95% or more by purification. In the case of a double-stranded nucleic acid, the sense and antisense strands synthesized and purified are in an appropriate ratio, for example, 0.1 to 10 equivalents, preferably 0.5 to 1 sense strand to 1 equivalent of the antisense strand. Two equivalents, more preferably 0.9 to 1.1 equivalents, and even more preferably equimolar amounts may be mixed and then annealed, or used directly without the step of annealing the mixture. May be. Annealing may be performed under any conditions as long as double-stranded nucleic acid can be formed. Usually, the sense strand and the antisense strand are mixed in approximately equimolar amounts, and then heated at about 94 ° C. for about 5 minutes. Then, it is performed by slowly cooling to room temperature. As an enzymatic transcription method for producing the nucleic acid of the present invention, a transcription method using a phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having a target base sequence as a template can be mentioned.

The nucleic acid of the present invention can be introduced into cells using a transfection carrier, preferably a cationic carrier such as a cationic liposome. It can also be directly introduced into cells by the calcium phosphate method, electroporation method or microinjection method.

In place of the nucleic acid of the present invention, a vector that can be introduced into cells and expressed can be used. Specifically, the nucleic acid or the like can be expressed by inserting the sequence encoding the nucleic acid of the present invention downstream of the promoter in the expression vector, constructing the expression vector, and introducing it into a cell.

Expression vectors include pCDNA6.2-GW / miR (Invitrogen), pSilencer® 4.1-CMV (Ambion), pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), pENTR / U6 (Invitrogen) etc. can be mentioned.

It is also possible to use a recombinant viral vector produced by inserting a sequence encoding the nucleic acid of the present invention downstream of a promoter in a viral vector and introducing the vector into a packaging cell. Examples of virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like.

2. Nucleic acid that suppresses expression of Bcl-2 protein A first selection criterion for a double-stranded nucleic acid that suppresses expression of Bcl-2 protein is one of untranslated regions on the 5 ′ side or 3 ′ side of the bcl-2 gene. A double-stranded nucleic acid comprising a nucleic acid comprising a partial base sequence and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid. A nucleic acid comprising a part of the base sequence of the untranslated region is selected as a second selection criterion that (a) no G or C sequence has 4 or more bases, and (b) the GC content is 20 to 80%. It is preferable to do.

Nucleic acid consisting of a part of the base sequence of the untranslated region of bcl-2 gene is Genbank Accession No. It can be designed based on the untranslated region of the cDNA base sequence (SEQ ID NO: 215) of the full-length mRNA of bcl-2 registered as NM_000633. Here, the untranslated region refers to the region of residues 1 to 493 and residues 1214 to 6492 where the Bcl-2 protein is not encoded in the base sequence represented by SEQ ID NO: 215 (each region). Are also referred to as 5 ′ untranslated region and 3 ′ untranslated region).

The partial base sequence of the untranslated region of the bcl-2 gene may be any base sequence as long as it is a partial base sequence of the non-translated region, but a nucleic acid that suppresses the expression of the Bcl-2 protein is designed. For this purpose, for example, a partial base sequence of an untranslated region such as the base sequence represented by any one of SEQ ID NOs: 1 to 53 is preferable, and the base represented by any one of SEQ ID NOs: 1 to 7 and 9 to 53 More preferred is a partial base sequence of a 3 ′ untranslated region such as a sequence, and any one of SEQ ID NOs: 3, 14, 16, 17, 19, 25 to 28, 30, 32 to 34, 36 to 40, and 42 to 48. Is more preferable.
The nucleic acid thus selected includes, for example, a nucleic acid comprising a partial base sequence of the untranslated region of the bcl-2 gene and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid. And a double-stranded nucleic acid having Bcl-2 protein expression-inhibiting activity. The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 15 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and more preferably 15 to 25 bases. More preferably, it consists of 17 to 23 bases, most preferably 19 to 21 bases. The double-stranded nucleic acid usually consists of 15 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, still more preferably 15 to 21 base pairs, and particularly preferably 15 to 19 base pairs. Has a heavy chain forming part.

Specific examples of the double-stranded nucleic acid include the following (a) to (f).
(A) an antisense strand nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 54 to 106 corresponding to a complementary sequence of bcl-2 mRNA, and a sense strand complementary to the base sequence of the nucleic acid A double-stranded nucleic acid comprising a nucleic acid containing a base sequence.
(B) a double-stranded nucleic acid comprising an antisense strand nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 54 to 106, and a sense strand nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid .
(C) an antisense strand nucleic acid from which at least one of the 5 ′ terminal or 3 ′ terminal of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 54 to 106 has been deleted by 1 to 4 bases; A double-stranded nucleic acid comprising a sense strand nucleic acid comprising a base sequence complementary to the nucleic acid base sequence.
(D) a sense strand nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 53 and an antisense strand nucleic acid comprising a base sequence selected from SEQ ID NOs: 54 to 106 (wherein the sense strand SEQ ID NO: N (N is an integer from 1 to 53) and the antisense strand has a sequence number N + 53), or at least one strand of the double-stranded nucleic acid A double-stranded nucleic acid having -3 bases, preferably 1 to 2 bases, more preferably 1 base substituted, deleted or added, and having Bcl-2 protein expression inhibitory activity. The base substitution, deletion or addition is preferably performed in the sequence on the sense strand side.
(E) In the double-stranded nucleic acid according to any one of (a) to (d), the duplex forming part has 27 base pairs or less, preferably 25 base pairs or less, more preferably 23 base pairs or less, even more preferably Is a double-stranded nucleic acid of 21 base pairs or less, particularly preferably 19 base pairs or less.
(F) The double-stranded nucleic acid according to any one of (a) to (e), wherein a nucleotide or nucleotide derivative is present at the 3 ′ end or 5 ′ end of at least one of the antisense strand nucleic acid and the sense strand nucleic acid. A double-stranded nucleic acid added with 1 to 8 bases, preferably 1 to 6 bases, more preferably 1 to 3 bases, still more preferably 1 to 2 bases, and particularly preferably 2 bases.

As a specific example of the double-stranded nucleic acid of (f) above, a nucleic acid comprising a base sequence represented by SEQ ID NO: N ′ (where N ′ represents any integer of 107 to 159), From a double-stranded nucleic acid consisting of a nucleic acid consisting of a base sequence represented by SEQ ID NO: N '+ 53 as a complementary strand, or a nucleic acid consisting of a base sequence represented by SEQ ID NO: 216, and a base sequence represented by SEQ ID NO: 217 A double-stranded nucleic acid consisting of

As the single-stranded nucleic acid that suppresses the expression of Bcl-2 protein, an antisense strand nucleic acid consisting of a partial base sequence of the untranslated region of the bcl-2 gene can also be used. Examples of such nucleic acids include nucleic acids having the base sequence represented by any of SEQ ID NOs: 54 to 106, and in these nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base Examples also include nucleic acids that are substituted, deleted or added and have Bcl-2 protein expression inhibitory activity. Examples of the nucleic acid containing these nucleic acids include 30 bases or less, preferably 28 bases or less, more preferably 26 bases or less, still more preferably 24 bases or less, and particularly preferably 23 bases or less.

The single-stranded nucleic acid that suppresses the expression of Bcl-2 protein includes a nucleic acid consisting of a partial base sequence of the untranslated region of the bcl-2 gene and a base sequence complementary to the base sequence of the nucleic acid. Examples of the single-stranded nucleic acids shown in the following (A) to (G) are also included.
(A) an antisense strand comprising the base sequence represented by any of SEQ ID NOs: 54 to 106 corresponding to a complementary sequence of bcl-2 mRNA and a sense strand complementary to the base sequence as a spacer A single-stranded nucleic acid linked through a sequence.
(B) An antisense strand consisting of the base sequence represented by any of SEQ ID NOs: 54 to 106 and a sense strand consisting of a base sequence complementary to the base sequence are linked via a spacer sequence Single-stranded nucleic acid.
(C) an antisense strand from which at least one of the 5 ′ end or 3 ′ end of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 54 to 106 has been deleted by 1 to 4 bases, and the base A single-stranded nucleic acid obtained by linking a sense strand nucleic acid comprising a base sequence complementary to a sequence via a spacer sequence.
(D) an antisense strand consisting of a base sequence selected from SEQ ID NOs: 54 to 106 and a sense strand consisting of a base sequence represented by any of SEQ ID NOs: 1 to 53 complementary to the base sequence (here A single-stranded nucleic acid obtained by linking a sense strand SEQ ID NO: N (N represents an integer of 1 to 53) and an antisense strand SEQ ID NO: N + 53 via a spacer sequence 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base is substituted, deleted or added in at least one of the sense strand and the antisense strand contained in and the expression of Bcl-2 protein Single-stranded nucleic acid having inhibitory activity. The base substitution, deletion or addition is preferably performed in the sequence on the sense strand side.
(E) The sense strand and the antisense strand contained in the single-stranded nucleic acid according to any one of (A) to (D) are 27 base pairs or less, preferably 25 base pairs or less, more preferably 23 base pairs or less. More preferably, it is a single-stranded nucleic acid that forms a double strand of 21 base pairs or less, particularly preferably 19 base pairs or less.
(F) The single-stranded nucleic acid according to any one of (A) to (E), which forms a double-stranded forming part by a stem-loop structure.
(G) The single-stranded nucleic acid according to any one of (A) to (F), which has a length of 50 to 70 bases.

By introducing these single-stranded nucleic acid or double-stranded nucleic acid into cells, the expression of Bcl-2 protein can be suppressed. In the suppression of Bcl-2 protein expression using antisense DNA, for example, compared with the case where antisense DNA is generally required at a concentration of several hundreds nM to several tens μM, for example, the double-stranded nucleic acid of the present invention Can suppress the expression of Bcl-2 protein even at a concentration of several nM to several hundreds of nM even after culturing for 24 hours or more, for example, 72 hours after introduction into cells.

The evaluation of the Bcl-2 protein expression inhibitory activity of the single-stranded nucleic acid or double-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid or the like into a cultured cancer cell using a cationic liposome or the like for a certain period of time. After culturing, the expression level of Bcl-2 protein in the cancer cells can be quantified by Western blotting. Further, bcl-2 mRNA can be quantified by RT-PCR. Furthermore, the effect of suppressing cell proliferation can be evaluated by calculating the number of living cells of cells into which the single-stranded nucleic acid or double-stranded nucleic acid of the present invention has been introduced.

3. Pharmaceutical Composition of the Present Invention The present invention also relates to a pharmaceutical composition comprising a nucleic acid such as a single-stranded nucleic acid or a double-stranded nucleic acid of the present invention or a vector as an active ingredient. The pharmaceutical composition can further comprise an effective carrier for transferring the nucleic acid into the cell. The pharmaceutical composition of the present invention is a disease caused by overexpression of Bcl-2 protein, a disease in which apoptosis is desirably induced, cancer, AIDS, ARC (AIDS-related disease) or collagen disease (rheumatic), etc. Can be used for treatment or prevention. Examples of cancer include hematological malignancies such as lymphoma and leukemia, and solid cancers such as liver cancer, skin cancer, breast cancer, lung cancer, digestive organ cancer, prostate cancer, uterine cancer, and bladder cancer.

Examples of carriers that are effective for transferring nucleic acids into cells include cationic carriers. Examples of the cationic carrier include cationic liposomes and cationic polymers. Further, a carrier utilizing a viral envelope may be used as an effective carrier for transferring nucleic acids into cells. Cationic liposomes include 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol-containing liposomes (hereinafter also referred to as liposome A), oligofectamine (Invitrogen), lipofectin ( Invitrogen), Lipofectamine (Invitrogen), Lipofectamine 2000 (Invitrogen), DMRIE-C (Invitrogen), GeneSilencer (Gene Therapy Systems), TransMessenger (QIAGEN M), TransMens (QIAGEN Mus), TransMensi (O Etc. are preferably used. As the cationic polymer, JetSI (Qbiogene), Jet-PEI (polyethyleneimine; Qbiogene) and the like are preferably used. As the carrier using the virus envelope, GenomeOne (HVJ-E liposome; Ishihara Sangyo Co., Ltd.) is preferably used.

A composition comprising a single-stranded nucleic acid, double-stranded nucleic acid or vector and a carrier of the present invention can be prepared by methods known to those skilled in the art. For example, it can be prepared by mixing a carrier dispersion of an appropriate concentration with a single-stranded nucleic acid, double-stranded nucleic acid or vector solution. When a cationic carrier is used, a single-stranded nucleic acid, a double-stranded nucleic acid or a vector is negatively charged in an aqueous solution and can be easily prepared by mixing in an aqueous solution by a conventional method. Examples of the aqueous solvent used for preparing the composition include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution. Moreover, those skilled in the art can appropriately select conditions such as pH and temperature when preparing the composition.

The composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersion device or a high-pressure emulsification device if necessary. The optimal method and conditions for preparing a composition comprising a single-stranded nucleic acid, a double-stranded nucleic acid or a vector and a carrier depend on the carrier to be used, and those skilled in the art can use it without being bound by the above method. The optimum method for the carrier can be selected.

As a composition comprising a single-stranded nucleic acid, a double-stranded nucleic acid or a vector and a carrier, a composite particle comprising a single-stranded nucleic acid, a double-stranded nucleic acid or vector and a lead particle as constituent components, and the composite particle are coated. Liposomes composed of lipid bilayer membranes are also used, of which the constituent components of the lipid bilayer membrane are soluble in ethanol and part or all of the constituent components are dispersed in a 5 vol% ethanol aqueous solution. That is, it is preferable to use an aggregate, micelle or the like that is emulsified or emulsified, and part or all of the composite particles are dispersed in a 5 vol% ethanol aqueous solution.

As the lead particles constituting the composite particles, for example, fine particles containing lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations, and the like, preferably fine particles containing liposomes, are used. .

Examples of the fine particles containing liposomes as lead particles as constituents include lipids, surfactants, etc., preferably lipids, or those containing lipids and surfactants as constituents.

The lipid may be any of simple lipids, complex lipids or derived lipids, such as phospholipids, glyceroglycolipids, sphingoglycolipids, sphingoids, sterols or cationic lipids, preferably phosphorous Lipids or cationic lipids are used.

Examples of phospholipids include phosphatidylcholine (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, etc.), phosphatidylethanolamine (specifically distearoyl). Phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, etc.), glycerophospholipid (specifically phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, etc.), sphingophospholipid ( Specifically, sphingomyelin and ceramide phosphoethanol , Ceramide phosphoglycerol, ceramide phosphoglycerophosphate, etc.), glycerophosphonolipid, sphingophosphonolipid, natural lecithin (specifically egg yolk lecithin, soybean lecithin, etc.) or hydrogenated phospholipid (specifically hydrogenated soybean) Natural or synthetic phospholipids such as phosphatidylcholine.

Examples of the cationic lipid include N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride (DOTAP), N- [1- (2,3-dioleoyl). Propyl)]-N, N-dimethylamine (DODAP), N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyl Oxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA), N- [1- (2,3-ditetradecyloxypropyl)] -N, N-Dimethyl-N-hydroxyethyl ammonium bromide (DMRIE) or N- [1- (2,3-dioleyloxypropyl)]-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DORIE ) Etc.

The lead particles can contain, for example, a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant. One or more lipid derivatives or fatty acid derivatives or surfactants selected from sugars, peptides, nucleic acids and water-soluble polymers may be contained as lead particles, or may be used in addition to lead particles.

The lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers is preferably a glycolipid or a lipid derivative or fatty acid derivative of a water-soluble polymer. More preferred are water-soluble polymer lipid derivatives or fatty acid derivatives. Lipid derivatives or fatty acid derivatives or surfactants of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers are those in which part of the molecule and other components of the lead particle, such as hydrophobic affinity, electrostatic It is a substance with a two-sided property that has the property of binding due to mechanical interaction, etc., and the other part has the property of binding to the solvent at the time of lead particle production, for example, hydrophilic affinity, electrostatic interaction, etc. Is preferred.

Examples of lipid derivatives or fatty acid derivatives of sugars, peptides, or nucleic acids include sugars such as sucrose, sorbitol, and lactose, such as casein-derived peptides, egg white-derived peptides, soybean-derived peptides, peptides such as glutathione, or DNA, RNA, etc. Examples include nucleic acids and lipids or fatty acids such as stearic acid, palmitic acid, myristic acid, lauric acid and the like. Examples of the sugar lipid derivative or fatty acid derivative include glyceroglycolipid and glycosphingolipid.

Examples of the water-soluble polymer lipid derivative or fatty acid derivative include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, Chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol, etc. or their derivatives and lipids or fatty acids such as stearic acid, palmitic acid, myristic acid or lauric acid are combined More preferred are lipid derivatives or fatty acid derivatives such as polyethylene glycol derivatives and polyglycerin derivatives, and more preferred are polyethylene glycol derivatives. Lipid derivative or fatty acid derivative of the call derivatives.

Examples of lipid derivatives or fatty acid derivatives of polyethylene glycol derivatives include polyethylene glycolated lipids (specifically, polyethylene glycol-phosphatidylethanolamine (more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine). -N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), etc.), polyoxyethylene hydrogenated castor oil 60, Cremophor EL, etc.), polyethylene glycol sorbitan fatty acid esters (specifically mono Oleic acid polyoxyethylene sorbitan, etc.) or polyethylene glycol fatty acid esters, and the like, more preferably polyethylene glycolated lipids.

Examples of lipid derivatives or fatty acid derivatives of polyglycerin derivatives include polyglycerinized lipids (specifically polyglycerin-phosphatidylethanolamine) and polyglycerin fatty acid esters, and more preferably polyglycerinized lipids. can give.

Examples of the surfactant include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

Nonionic surfactants include, for example, polyoxyethylene sorbitan monooleate (specifically polysorbate 80 etc.), polyoxyethylene polyoxypropylene glycol (specifically pluronic F68 etc.), sorbitan fatty acid ester (specifically Sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.), glycerin fatty acid esters and the like.

Examples of the anionic surfactant include acyl sarcosine, sodium alkyl sulfate, alkylbenzene sulfonate, sodium fatty acid having 7 to 22 carbon atoms, such as sodium dodecyl sulfate, sodium lauryl sulfate, sodium cholate, sodium deoxycholate. And sodium taurodeoxycholate.

Cationic surfactants include alkylamine salts, acylamine salts, quaternary ammonium salts, amine derivatives, etc., specifically benzalkonium chloride, acylaminoethyldiethylamine salts, N-alkylpolyalkylpolyamine salts, fatty acid polyethylene Examples thereof include polyamide, cetyltrimethylammonium bromide, dodecyltrimethylammonium bromide, alkylpolyoxyethyleneamine, N-alkylaminopropylamine, and fatty acid triethanolamine ester.

Examples of amphoteric surfactants include 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, and the like. It is done.

The lead particles preferably contain a cationic substance, and more preferably have a positive charge.
Examples of the cationic substance include a cationic substance in lipid, a cationic surfactant (as defined above), a cationic polymer, etc., such as a protein or peptide that exhibits a cationic property at a pH below the isoelectric point. N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)]-N, N -Dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl)]-N , N-dimethyl-N-hydroxyethylammonium bromide and 3β- [N- (N ′, N′dimethylaminoethyl) carbamoyl] cholesterol.

The lead particles may be composed of a complex comprising a combination of two or more lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle formulations, etc., and lipid aggregates, liposomes, emulsion particles, polymers In addition, a complex formed by combining a metal colloid, a fine particle preparation, and the like with another compound (eg, sugar, lipid, inorganic compound, etc.) may be used as a constituent component.

The size of the lead particle is preferably an average particle diameter of several nm to several μm, more preferably about 10 nm to 1000 nm, further preferably about 50 nm to 300 nm, and about 50 nm to 200 nm. It is particularly preferred.

Examples of the constituent components of the lipid bilayer membrane that covers the composite particles include lipids, surfactants, and the like, which are synonymous with the fine particles having the liposome as a constituent component.
As the lipid, phospholipid, glyceroglycolipid, sphingoglycolipid and the like are preferable, phospholipid is more preferable, and EPC is more preferable. As the surfactant, polyethylene glycol alkyl ether or the like may be used, and polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester, polyethylene glycol alkyl ether, or the like is preferably used.
These lipids or surfactants can be used alone or in combination of two or more.

The ratio of the lipid bilayer to the liposome composed of the composite particle and the lipid bilayer coating the composite particle is preferably about 1: 0.1 to 1: 1000 by weight, and about 1: 1 to 1:10. More preferred. The liposome preferably has an average particle diameter of several nm to several μm, more preferably about 10 nm to 1000 nm, further preferably about 50 nm to 300 nm, and about 50 nm to 200 nm. It is particularly preferred.

The liposome is obtained by dispersing the composite particles in a polar organic solvent in which the components of the lipid bilayer coating the composite particles are soluble or a mixture of the solvent and an aqueous medium. After dissolving or dispersing the components, an aqueous medium can be added to the liquid to form a liposome dispersion.
Examples of the polar organic solvent include alcohols such as methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, and tert-butanol, glycols such as glycerin, ethylene glycol, and propylene glycol, and polymers such as polyethylene glycol. Alkylene glycol and the like can be mentioned, but alcohol is usually used, and ethanol is preferably used. As the aqueous medium, water is usually used, but it may contain a salt, an acid, an organic solvent, etc. as long as it does not hinder the formation of a liposome dispersion.
The liposome can also be prepared according to the method described in, for example, WO2002 / 028367, WO2006 / 080118 and the like.

The compounding ratio of the single-stranded nucleic acid, double-stranded nucleic acid or vector and carrier contained in the pharmaceutical composition of the present invention is 1 to 200 carriers per 1 part by weight of the single-stranded nucleic acid, double-stranded nucleic acid or vector. Part by weight is appropriate. Preferably, the amount is 2.5 to 100 parts by weight, more preferably 10 to 20 parts by weight, based on 1 part by weight of the single-stranded nucleic acid, double-stranded nucleic acid or vector.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent in addition to the above carrier. Pharmaceutically acceptable carriers or diluents and the like are essentially chemically inert and harmless compositions that do not affect the biological activity of the pharmaceutical composition of the present invention at all. Examples of such carriers or diluents include but are not limited to salt solutions, sugar solutions, glycerol solutions, ethanol and the like.

The pharmaceutical composition of the present invention contains a single-stranded nucleic acid, double-stranded nucleic acid or vector effective in treating or preventing a disease and is provided in a form that can be appropriately administered to a patient. The preparation form of the pharmaceutical composition of the present invention may be, for example, injections, eye drops, liquids for inhalation, etc., for example, external preparations such as ointments, lotions and the like.

In the case of a liquid preparation, the concentration range of single-stranded nucleic acid, double-stranded nucleic acid or vector in the pharmaceutical composition of the present invention is usually 0.001 to 25% (w / v), preferably 0.01 to 5%. (W / v), more preferably 0.1 to 2% (w / v). The pharmaceutical composition of the present invention may contain an appropriate amount of any pharmaceutically acceptable additive, for example, an emulsification aid, a stabilizer, an isotonic agent, a pH adjuster and the like. Any pharmaceutically acceptable additive can be added in an appropriate step before or after dispersion of the complex.

The lyophilized preparation can be prepared by subjecting a single-stranded nucleic acid, a double-stranded nucleic acid, or a vector and a carrier to a dispersion treatment and then a freeze-drying treatment. The lyophilization treatment can be performed by a conventional method. For example, a predetermined amount of the complex solution after the dispersion treatment is aseptically dispensed into a vial and pre-dried for about 2 hours under the condition of about −40 to −20 ° C. and about 0 to 10 ° C. Primary drying under reduced pressure, followed by secondary drying under reduced pressure at about 15-25 ° C. and lyophilization. Then, for example, by replacing the inside of the vial with nitrogen gas and stoppering, a freeze-dried preparation of the pharmaceutical composition of the present invention can be obtained.

The pharmaceutical composition of the present invention can be redissolved and used by adding any appropriate solution. Examples of such a solution include electrolytes such as water for injection and physiological saline, glucose solution, and other general infusion solutions. The amount of this solution varies depending on the application and is not particularly limited, but is preferably 0.5 to 2 times the amount before lyophilization or 500 ml or less.

The pharmaceutical composition of the present invention can be administered to animals including humans, for example, intravenous administration, intraarterial administration, oral administration, tissue administration, transdermal administration, transmucosal administration, or rectal administration. It is preferable to administer by an appropriate method according to the symptoms. In particular, intravenous administration, transdermal administration, and transmucosal administration are preferably used. Moreover, local administration, such as local administration in cancer, can also be performed. Examples of dosage forms suitable for these administration methods include various injections, oral preparations, drops, absorbents, eye drops, ointments, lotions, suppositories and the like.

The dosage of the pharmaceutical composition of the present invention is preferably determined in consideration of the drug, dosage form, patient condition such as age and weight, administration route, nature and degree of disease, etc. The mass of the nucleic acid, double-stranded nucleic acid or vector is 0.1 mg to 10 g / day, preferably 1 mg to 500 mg / day, per day for an adult. In some cases, this may be sufficient, or vice versa. It can also be administered once to several times a day, and can be administered at intervals of one to several days.

Hereinafter, the present invention will be described with reference to examples. However, the present invention is not limited to these examples.

Design and evaluation of nucleic acid that suppresses bcl-2 gene expression (1)
i) Preparation of double-stranded nucleic acid As a nucleic acid sequence capable of suppressing the expression of the bcl-2 gene, (a) 4 bases of G or C from the mRNA sequence of bcl-2 (GenBank accession number: NM_000633, SEQ ID NO: 215) There were selected 53 sets of sequences satisfying the following three conditions: no sequence that continues, (b) a GC content of 20-80%, and (c) located in the 5 ′ or 3 ′ untranslated region. The sequence is represented by SEQ ID NO: N and the base sequence of SEQ ID NO: N + 53 as its complementary sequence. Here, N is an integer from 1 to 53. A DNA sequence consisting of 2 bases is appropriately added to the 3 ′ end of each RNA consisting of the sequence represented by SEQ ID NOs: 1 to 106, and a nucleic acid consisting of the base sequence represented by SEQ ID NO: N ′ and its complementary strand SiRNA was designed as a double-stranded nucleic acid consisting of a nucleic acid consisting of the base sequence represented by SEQ ID NO: N '+ 53. Here, N ′ represents an integer from 107 to 159.
Qiagen Co., Ltd. was requested to synthesize the nucleic acid constituting the double-stranded nucleic acid.

ii) Evaluation method in screening Evaluation in screening involves introducing double-stranded nucleic acid into various cancer cells together with a carrier, quantifying the expression level of Bcl-2 protein by Western blotting, and determining the amount of bcl-2 mRNA. This was carried out by quantification by RT-PCR (Reverse transcription-polymerase chain reaction).

iii) Preparation of double-stranded nucleic acid / carrier complex As a carrier, a commercially available cationic liposome, oligofectamine (Invitrogen), is used, and a double-stranded nucleic acid- oligofectamine complex is prepared according to the attached instruction manual. Prepared.

iv) Western blotting Whether or not the expression of Bcl-2 protein is suppressed by introducing a double-stranded nucleic acid into a cell using the above-mentioned double-stranded nucleic acid-oligofectamine complex is determined by Western blotting. -2 It was evaluated by measuring the change in the amount of protein.
PC-3, a prostate cancer cell, was seeded at a density of 2 × 10 ⁵ cells / dish in a 6 cm-diameter culture dish, 37% in F-12 Kaighn's medium (GIBCO, 21127) containing 10% fetal bovine serum (FBS), 37 ° C., and cultured overnight in 5% CO _2. The next day, the medium was aspirated from the culture dish and replaced with 0.8 ml of OPTI-MEM (GIBCO, 31985) which is a low serum basic medium. The double-stranded nucleic acid was introduced into PC-3 by adding 0.2 ml of the double-stranded nucleic acid-oligofectamine complex solution mixed in OPTI-MEM.
The double-stranded nucleic acid includes a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 109 and a nucleic acid comprising the base sequence represented by SEQ ID NO: 162 and a base sequence represented by SEQ ID NO: 213. A double-stranded nucleic acid (B717, positive control, Nippon Shinyaku Co., Ltd.) targeting the translation region of the bcl-2 gene consisting of the nucleic acid consisting of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 214 and the nucleic acid consisting of the base sequence represented by SEQ ID NO: 214 was used.
Further, Nontargeting siRNA # 1 (hereinafter also referred to as Nontargeting # 1) (manufactured by Dharmacon), which is a siRNA that does not cross any human gene, was separately introduced into PC-3 and used as a negative control. The final concentration of double-stranded nucleic acid was 50 nM. The concentration of the double-stranded nucleic acid is shown as a molar concentration when it is assumed that each strand forms a double strand completely.
The cells into which the double-stranded nucleic acid has been introduced are cultured in a 5% CO ₂ incubator at 37 ° C. for 48 hours, washed twice with phosphate buffered saline (PBS), and 1 using a cell scraper. Transfer to a 5 ml tube. After centrifugation at 1000 × g for 2 minutes and removing the supernatant, the cells were treated with RIPA buffer [50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 1% Nonident P-40, 0.5% sodium deoxycholate, 0.1% sodium It was dissolved and recovered in dodecyl sulfate, 1% Protease Inhibitor Cocktail Set III (Calbiochem). After leaving still on ice for 30 minutes and centrifuging at 100,000 × g for 15 minutes, the supernatant was transferred to a new tube to prepare a sample for electrophoresis.
After separation by SDS-PAGE by a conventional method, Bcl-2 protein was detected by Western blotting. For detection of Bcl-2 protein, an anti-Bcl-2 antibody (manufactured by DAKO) was used. In addition, α-actin protein was used as the internal standard of the specimen. Anti-α-actin antibody (manufactured by Sigma) was used for detection of α-actin protein.
The protein mass was measured based on the intensity of the band when the double-stranded nucleic acid was transfected compared to the intensity of the Bcl-2 protein band when transfected with the negative control Nontargeting # 1. As a result, as shown in FIG. 1, PC-3 introduced with the double-stranded nucleic acid of the present invention targeting the untranslated region of the bcl-2 gene has a decreased amount of Bcl-2 protein compared to the negative control. In the same manner as B717 targeting the translation region of the bcl-2 gene, the expression of Bcl-2 protein was suppressed.

Semi-quantitative determination of mRNA amount by RT-PCR In order to examine whether the suppression of Bcl-2 protein expression by transfection of double-stranded nucleic acid is due to the suppression of mRNA expression, the expression level of bcl-2 mRNA was determined by RT- Evaluation was performed by semi-quantification by PCR.
PC-3 was seeded at a density of 2 × 10 ⁵ cells / dish in a 6 cm-diameter culture dish, and F-12 Kaighn's medium (GIBCO, 21127) containing 10% fetal bovine serum under conditions of 37 ° C. and 5% CO _2. Incubated overnight. On the next day, the medium was aspirated from the culture dish and replaced with 0.8 ml of OPTI-MEM (GIBCO, 31985) which is a low serum basic medium. The double-stranded nucleic acid was introduced into PC-3 by adding 0.2 ml of the double-stranded nucleic acid-oligofectamine complex solution mixed in OPTI-MEM. Nontargeting # 1 (manufactured by Dharmacon) was separately introduced into PC-3 and used as a negative control. The final concentration of double-stranded nucleic acid was 50 nM.
The cells into which the double-stranded nucleic acid had been introduced were cultured for 48 hours in a 5% CO ₂ incubator at 37 ° C., washed twice with PBS, and transferred to a 1.5 ml tube using a cell scraper. After centrifuging at 1000 × g for 2 minutes and removing the supernatant, the cells are lysed and collected in RLT buffer (attached to Qiagen RNA Recovery Kit “RNeasy”), and all cells are collected according to the instructions attached to the kit. RNA was recovered.
Using 5 μg of total RNA as a template, reverse transcription reaction was performed using Superscript III First-Strand cDNA Synthesis Kit (Invitrogen) to prepare cDNA. Using this cDNA as a template for a PCR reaction, specific to the bcl-2 gene and the constitutively expressed gene GADPH (D-glyceraldehyde-3-phosphate dehydrogenase) gene by SYBR-Green PCR using ABI7900HT Fast (ABI) PCR amplification was performed, and the amount of mRNA was quantified. For PCR amplification of each gene, 250 ng of total RNA-derived cDNA was used as a template. The amount of mRNA in the specimen was expressed as a relative ratio when the amount of bcl-2 mRNA or GADPH mRNA was 1 in the double-stranded nucleic acid non-introduced group (untreated). The results for bcl-2 mRNA levels are shown in FIG.
As shown in FIG. 2, PC-3 introduced with the double-stranded nucleic acid of the present invention targeting the untranslated region of the bcl-2 gene has a decreased amount of bcl-2 mRNA compared to the negative control. It was equivalent to B717 targeting the translation region of the bcl-2 gene.

In vitro anti-cell proliferation activity of double-stranded nucleic acid PC-3 is seeded in a 96-well plate at about 2,000-2,500 cells per well and cultured overnight in F-12 Kaighn's medium containing 10% FBS did. One day later, using an oligofectamine (Invitrogen), a double-stranded nucleic acid consisting of a nucleic acid represented by SEQ ID NOs: N ′ and N ′ + 53 and having a non-translated region of the bcl-2 gene as a target gene (N 'Represents an integer of 107 to 159) was introduced into PC-3 to a final concentration of 3 nM or 30 nM. A double-stranded nucleic acid that targets the translation region of the bcl-2 gene, comprising a single-stranded nucleic acid comprising the base sequence represented by SEQ ID NO: 213 and a single-stranded nucleic acid comprising the base sequence represented by SEQ ID NO: 214 (B717, Nippon Shinyaku) was used as a positive control. Nontargeting # 1 (manufactured by Dharmacon) was separately introduced into PC-3 and used as a negative control. The double-stranded nucleic acid was introduced according to the method described in the instructions attached to the oligofectamine.
Three to six days after the introduction of the double-stranded nucleic acid, the cell viability was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega). The measuring method followed the method described in the instructions attached to the product. The relative viable cell ratio of each was calculated assuming that the viable cell ratio of PC-3 when only oligofectamine was introduced was 1.0. The results are shown in FIGS. As shown in FIGS. 3 and 4, the introduction of a double-stranded nucleic acid targeting the untranslated region of the bcl-2 gene resulted in a decrease in the viable cell rate, and in vitro of the various double-stranded nucleic acids of the present invention. Anti-cell proliferative activity in was observed.

Anti-tumor effect by double-stranded nucleic acid targeting untranslated region of bcl-2 gene as target gene From nucleic acids comprising base sequences represented by SEQ ID NOs: 109 and 162, respectively, targeting untranslated region of bcl-2 gene as target gene By measuring the antitumor effect of the double-stranded nucleic acid (hereinafter also referred to as the double-stranded nucleic acid consisting of SEQ ID NOs: 109 and 162) and B717, their efficacy was compared.
(i) Preparation of wrapped liposome (WL) containing double-stranded nucleic acid We requested Hokkaido System Science Co., Ltd. for chemical synthesis of nucleic acids consisting of the nucleotide sequences represented by SEQ ID NOs: 109, 162, 213 and 214, respectively. A double-stranded nucleic acid consisting of SEQ ID NOs: 109 and 162 and a double-stranded nucleic acid consisting of SEQ ID NOs: 213 and 214 (B717) were prepared by annealing.
DOTAP (manufactured by Avanti Polar Lipids) / PEG-DSPE (manufactured by NOF Corporation, the same shall apply hereinafter) / distilled water was mixed to 40 mg / 16 mg / mL, and the mixture was shaken and stirred with a vortex mixer. The resulting dispersion was applied to a 0.4 μm polycarbonate membrane filter (manufactured by Whatman) 10 times at room temperature, 10 times to a 0.2 μm polycarbonate membrane filter (manufactured by Whatman), 10 times to a 0.1 μm polycarbonate membrane filter (manufactured by Whatman), Further, lead particles were prepared by passing 35 times through a 0.05 μm polycarbonate membrane filter (manufactured by Whatman). To the obtained lead particle dispersion 0.624 mL, 0.208 mL of a 24 mg / mL aqueous solution of the double-stranded nucleic acid consisting of SEQ ID NOs: 109 and 162 and the double-stranded nucleic acid (B717) consisting of SEQ ID NOs: 213 and 214 is added to form composite particles Was prepared. A solution obtained by dissolving 0.8 mL of the obtained composite particle dispersion in a 62.5 vol% aqueous ethanol solution so that EPC (manufactured by NOF Corporation) / PEG-DSPE as a component of the lipid bilayer membrane is 15 mg / 3.125 mg / mL Add to 3.2 mL, then add 1 mL of distilled water, adjust the ethanol concentration to 40 vol%, and dissolve EPC / PEG-DSPE in 40 vol% ethanol aqueous solution to 62.5 mg / 62.5 mg / mL 0.32 mL of the prepared solution was added, and then 37.24 mL of distilled water was added to adjust the ethanol concentration to 5 vol% to prepare liposomes. The obtained liposome dispersion was ultracentrifuged (80 minutes, 110,000 × g, 25 ° C.), the supernatant was removed, and physiological saline was added and redispersed to obtain a preparation.
One lot of the preparation was prepared.
When the average particle size of the liposome was measured by a dynamic light scattering method (Zetasizer Nano-ZS, Malvern), it was 85 nm.

(ii) Hanks' Balanced in vivo efficacy evaluation in vitro PC-3 cells cultured in the lapped liposomes containing double-stranded nucleic acid (human prostate cancer cell line) so that 1 × 10 ⁸ cells / mL salt It was suspended in solution (HBSS), and 50 μL was transplanted into the ventral skin of Balb / c nude mice (Claire Japan, male). On day 7 after cell transplantation, the tumor diameter was measured with calipers, and the tumor volume was calculated from the following formula.

Tumor volume = minor axis x minor axis x major axis x 0.5

Individuals having a tumor volume in the range of 81 to 98 mm ³ (average 89 mm ³ ) were selected and divided into groups so that the average tumor volume was uniform, and then the following administration groups A to C were set. The day of grouping was designated as Day0.

A. Non-administration group (negative control group)
B. Double-stranded nucleic acid (SEQ ID NO: 109, 162) -WL administration group targeting untranslated region of bcl-2 gene: 150 μg of nucleic acid weight per animal on Day 0, Day 4, Day 7, Day 11 and Day 14
C. B717-WL administration group: 150 μg of nucleic acid weight per animal on Day 0, Day 4, Day 7, Day 11 and Day 14

The experiment was performed by using 5 mice in each group. Each drug was diluted with physiological saline and administered from the tail vein. The tumor volume is measured daily from Day 0, and the antitumor effect is determined by comparing the average value of the tumor volume ratio (V / V0) on each measurement day when the tumor volume on Day 0 of each group is V0. It was done by doing.
FIG. 5 shows the daily transition of the average value of V / V0 in each group. The results of a significant difference test performed between the administration group and the negative control group are shown in Table 1 as p values.

As shown in FIG. 5 and Table 1, in the double-stranded nucleic acid (SEQ ID NO: 109, 162) -WL administration group of the present invention, tumor growth was suppressed with a statistically significant difference. On the other hand, the B717-WL administration group also showed a tendency to suppress tumor growth, but its effect was not statistically significant. From the above, it was revealed that the double-stranded nucleic acid of the present invention consisting of the nucleic acids represented by SEQ ID NOs: 109 and 162 has an in vivo antitumor action superior to B717.

Design and evaluation of nucleic acids that suppress bcl-2 gene expression (2)
A double-stranded nucleic acid obtained by annealing a nucleic acid of a sense strand (Eurogentec) consisting of a base sequence represented by SEQ ID NO: 216 and a nucleic acid of an antisense strand (Eurogentec) consisting of a base sequence represented by SEQ ID NO: 217 Hereinafter, it is also referred to as a double-stranded nucleic acid consisting of SEQ ID NO: 216 and 217), and a nucleic acid having a sense strand consisting of the base sequence represented by SEQ ID NO: 109 and a nucleic acid having an antisense strand consisting of the base sequence represented by SEQ ID NO: 162 And a double-stranded nucleic acid (hereinafter also referred to as a double-stranded nucleic acid consisting of SEQ ID NOs: 109 and 162) were prepared as siRNAs using the untranslated region of the bcl-2 gene as a target gene, respectively.
SEQ ID NO: 216 is a 25-base sequence in which 6 bases are added to the 3 ′ end of the nucleic acid represented by SEQ ID NO: 3, and SEQ ID NO: 217 is the 3 ′ end of the nucleic acid represented by SEQ ID NO: 56. It is a sequence consisting of 27 bases with 8 bases added, SEQ ID NO: 109 is a sequence consisting of 21 bases with 2 bases added to the 3 ′ end of the nucleic acid represented by SEQ ID NO: 3, and SEQ ID NO: 162 is a sequence This is a sequence consisting of 21 bases with 2 bases added to the 3 ′ end of the nucleic acid represented by No. 56.
Further, the nucleic acid represented by the sequence 1s to 5s as a nucleic acid having a 2′-O-methyl (2′-OMe) modification in the sugar part (ribose) of a part of the nucleotide of the nucleic acid represented by SEQ ID NO: 216, The nucleic acid represented by the sequences 1as and 2as as the nucleic acid having a 2′-OMe modification in the ribose of some nucleotides of the nucleic acid represented by SEQ ID NO: 217, and the nucleotides of some nucleotides of the nucleic acid represented by SEQ ID NO: 109 Double-stranded nucleic acids having the sequence names shown in Table 2 were prepared using the nucleic acid represented by the sequence 6as as the nucleic acid having 2'-OMe modification in ribose as the sense strand and the antisense strand, respectively. Among the nucleotide sequences described in Table 2, nucleotides written in lower case letters are unmodified ribonucleotides, nucleotides written in upper case letters are modified ribonucleotides, dA, dC and dT are deoxyadenosine, deoxy Cytidine and deoxythymidine are shown, respectively.

In order to investigate whether these double-stranded nucleic acids have the Bcl-2 protein expression-inhibiting action, the same method as in Example 1 was used, and the expression level of bcl-2 mRNA was determined by semi-quantification by RT-PCR. Measurement and evaluation of the double-stranded nucleic acid.
The amount of bcl-2 mRNA was expressed as a relative ratio when the amount of bcl-2 mRNA in the double-stranded nucleic acid non-introduced group (untreated) was 1, and the data was corrected with the amount of GADPH mRNA. . The results are shown in FIG.
As shown in FIG. 6, the double-stranded nucleic acid consisting of SEQ ID NOs: 216 and 217, the double-stranded nucleic acid consisting of SEQ ID NOs: 109 and 162, and the 2′-OMe modification of the ribose of some nucleotides of these double-stranded nucleic acids In PC-3 cells each introduced with a double-stranded nucleic acid having a phenotype, the amount of bcl-2 mRNA was reduced compared to the negative control.

The present invention provides a nucleic acid that suppresses the expression of Bcl-2 protein, a pharmaceutical composition comprising the nucleic acid, and the like. Since the nucleic acid and pharmaceutical composition of the present invention promote apoptosis, they are useful for the treatment of cancer and the like.

This application is based on Japanese Patent Application No. 2008-213300 filed in Japan (application date: August 21, 2008), the contents of which are incorporated in full herein.

Claims (28)

1. A nucleic acid comprising a partial base sequence of the untranslated region of the gene encoding the Bcl-2 protein, and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid, and comprising the Bcl-2 protein Nucleic acid having expression suppressing activity.
2. The nucleic acid according to claim 1, wherein a part of the base sequence of the untranslated region of the gene encoding Bcl-2 protein is a sequence consisting of 15 to 27 bases.
3. A nucleic acid consisting of a partial base sequence of the untranslated region of the gene encoding Bcl-2 protein is a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 54 to 106 or at least one end of the nucleic acid is 1 The nucleic acid according to claim 1 or 2, wherein the nucleic acid has been deleted by ~ 4 bases.
4. A nucleic acid comprising a part of the base sequence of the untranslated region of the gene encoding the Bcl-2 protein according to claim 1, and at least one nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid A nucleic acid having 1-3 base substitutions, deletions or additions and having Bcl-2 protein expression-inhibiting activity.
5. A double-stranded nucleic acid comprising a nucleic acid comprising a partial base sequence of the untranslated region of the gene encoding the Bcl-2 protein and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid.
6. The double-stranded nucleic acid according to claim 5, which has a double-stranded forming part consisting of 15 to 27 base pairs.
7. A nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 54 to 106, a nucleic acid from which at least one end of the nucleic acid has been deleted by 1 to 4 bases, and a base sequence complementary to the base sequence of the nucleic acid A double-stranded nucleic acid consisting of a nucleic acid.
8. A double strand in which at least one base is substituted, deleted or added in at least one strand of the double-stranded nucleic acid according to any one of claims 5 to 7, and has Bcl-2 protein expression inhibitory activity Strand nucleic acid.
9. A double-stranded nucleic acid comprising the double-stranded nucleic acid according to any one of claims 5 to 8, wherein the double-stranded forming part has 27 base pairs or less.
10. A double-stranded nucleic acid having 1 to 4 bases added to the 3 'end or 5' end of at least one strand of the double-stranded nucleic acid according to any one of claims 5 to 9.
11. A nucleic acid consisting of a base sequence represented by any of SEQ ID NOs: 1-212, 216 and 217.
12. The nucleic acid according to claim 11, wherein 1 to 3 bases are substituted, deleted or added, and has Bcl-2 protein expression inhibitory activity.
13. A nucleic acid of 30 bases or less, comprising the nucleic acid according to claim 11 or 12.
14. A nucleic acid in which a part or all of the nucleotides constituting the nucleic acid according to any one of claims 1 to 13 are ribonucleotides and has Bcl-2 protein expression inhibitory activity.
15. A nucleic acid in which some or all of the nucleotides constituting the nucleic acid according to any one of claims 1 to 14 are deoxyribonucleotides or modified nucleotides, and has Bcl-2 protein expression inhibitory activity.
16. The nucleic acid according to claim 10 or 15, wherein at least one of 1 to 4 bases added to the 3 'end or 5' end is deoxyribonucleotide.
17. A vector encoding the nucleic acid according to any one of claims 1 to 16.
18. A pharmaceutical composition comprising the nucleic acid or vector according to any one of claims 1 to 17 as an active ingredient.
19. The pharmaceutical composition according to claim 18, further comprising a carrier effective for transferring the nucleic acid into the cell.
20. The pharmaceutical composition according to claim 19, wherein the carrier effective for transferring the nucleic acid into the cell is a cationic carrier.
21. 21. The pharmaceutical composition according to claim 20, wherein the effective carrier for transferring the nucleic acid into the cell is a liposome.
22. The pharmaceutical composition according to claim 21, wherein the liposome is a liposome that reaches a tissue or an organ containing an expression site of a gene encoding Bcl-2 protein.
23. A liposome comprising a composite particle comprising a nucleic acid or vector and a lead particle as constituents and a lipid bilayer covering the composite particle, wherein the constituents of the lipid bilayer are soluble in ethanol, and The pharmaceutical composition according to claim 18, wherein the composition is dispersed in a 5 vol% ethanol aqueous solution, and the composite particles are dispersed in a 5 vol% ethanol aqueous solution.
24. The pharmaceutical composition according to any one of claims 18 to 23, which suppresses the expression of Bcl-2 protein.
25. The pharmaceutical composition according to claim 24, which promotes apoptosis.
26. The pharmaceutical composition according to any one of claims 18 to 25, which is used for treatment or prevention of cancer.
27. A method for suppressing the expression of Bcl-2 protein in a subject, comprising administering the nucleic acid, vector or pharmaceutical composition according to any one of claims 1 to 26 to the subject.
28. Use of the nucleic acid or vector according to any one of claims 1 to 17 for the production of a pharmaceutical composition.

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