JP2013039036A - NUCLEIC ACID INHIBITING EXPRESSION OF HIF-2α - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid useful for controlling growth of a cell, to provide a nucleic acid having an expression inhibitory activity for HIF (hypoxia-inducible factor)-2α by requiring a method for utilization thereof, and to provide a pharmaceutical composition composed of the nucleic acid.SOLUTION: There is provided the nucleic acid including a part of base sequence of an hif-2α gene, and a base sequence complementary to the base sequence of the nucleic acid, and inhibiting the expression of the HIF-2α. Furthermore, there is provided the pharmaceutical composition for treatment or prophylaxis of diseases such as cancer, diabetic retinopathy or rheumatism utilizing an angiogenic inhibitory action of the nucleic acid.

Description

  The present invention relates to a nucleic acid or a derivative thereof for use in suppressing the expression of HIF-2α acting as an angiogenic factor, and a pharmaceutical composition containing the nucleic acid or a derivative thereof.

  The formation of new blood vessels from existing blood vessels is called angiogenesis. Angiogenesis is an in vivo phenomenon that usually occurs during wound healing and inflammation repair, but is involved in the establishment and malignancy of many diseases such as solid cancer, diabetic retinopathy, rheumatism, atherosclerosis, and psoriasis It is known (see Non-Patent Document 1). In particular, angiogenesis plays a major role in cancer development and tumor growth.

  HIF (hypoxia-inducible factor) is a transcription factor that functions in a hypoxic environment and promotes angiogenesis, glucose metabolism, and cell growth. Although it normally functions only in a hypoxic environment, it is constantly expressed in many tumors and is considered to be one of the causes of tumor growth (see Non-Patent Document 2). HIF is a heterodimeric transcription factor, and consists of a constitutively expressed HIF-1β subunit and a regulatory factor HIF-α subunit. Three types of HIF-α subunits, HIF-1α, HIF-2α, and HIF-3α, are known and each function redundantly or tissue-specifically.

  The tumor suppressor gene VHL (von Hippel-Lindau tumor suppressor) is responsible for the degradation of the HIF-α subunit, thereby suppressing the constant expression of HIF. VHL is mutated in most sporadic clear cell kidney cancer and VHL disease, which contributes to the constant expression of HIF in tumors. Inhibition of HIF that is constantly expressed in tumors controls tumor angiogenesis, so inhibition of HIF is effective as an anti-angiogenesis method based on the mechanism of action (see Non-Patent Document 3).

  Until now, it is known that HIF-1α is a target of antitumor action among HIF-α subunits (see Non-Patent Document 4). On the other hand, HIF-2α is also constitutively expressed in many cancer types, and is involved in tumor progression more specifically than HIF-1α in specific cancer types (see Non-Patent Documents 5 to 6), and VHL is mutated. It is known that inhibition of HIF-2α is sufficiently effective in such tumors (see Non-Patent Documents 7 to 9).

Suppressive oligonucleotide compounds configured to inhibit the expression by binding to a nucleic acid encoding a protein that causes disease such as cancer may be useful for the treatment of those diseases. As such an example, for example, an antisense oligonucleotide against the hif-2α 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 the expression of a target gene is suppressed by introducing a double-stranded RNA having a length of 21 to 23 bases into a cell. Such RNA is named small interfering RNA (siRNA).

  Antisense nucleotides and siRNAs for hif-2α mRNA are known (see Patent Documents 1 and 2, Non-Patent Documents 5, 8, and 10-12), but both are different from the double-stranded RNA of the present invention. Has a sequence or no specific sequence is shown.

  International Publication No. 04/048526

  International Publication No. 00/09657

  Journal of Molecular Medicine, 73 (7), 333-46 (1995)

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  An object of the present invention is to provide a nucleic acid capable of suppressing the expression of HIF-2α that acts as an angiogenic factor. Another object of the present invention is to provide a pharmaceutical composition for treating or preventing diseases such as cancer, diabetic retinopathy, and rheumatism, in which angiogenesis suppression is effective in healing.

The present invention relates to the following (1) to (24).
(1) A nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 22 to 40 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 nucleic acid comprising a nucleic acid comprising a base sequence and having an activity of suppressing the expression of HIF-2α.
(2) A nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 22 to 40 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 nucleic acid containing a nucleic acid in which 1 to 3 bases are substituted, deleted or added in at least one of nucleic acids having a base sequence, and having an activity of suppressing the expression of HIF-2α.
(3) The nucleic acid according to (1) or (2), which has a duplex forming part of 27 base pairs or less.
(4) The nucleic acid according to (3), which has a double-stranded forming part consisting of 15 to 19 base pairs.
(5) The double-stranded nucleic acid according to any one of (1) to (4).
(6) A double-stranded nucleic acid obtained by adding 1 to 4 bases to the 3 ′ end or 5 ′ end of at least one strand of the double-stranded nucleic acid according to (5).
(7) A nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 22-40.
(8) The nucleic acid according to (7), wherein 1 to 3 bases are substituted, deleted or added, and has HIF-2α expression inhibitory activity.
(9) A nucleic acid having 30 bases or less, including the nucleic acid according to (7) or (8).
(10) A nucleic acid according to any one of (1) to (9), wherein a part or all of the nucleotides constituting the nucleic acid or double-stranded nucleic acid are ribonucleotides and have an activity to suppress the expression of HIF-2α .
(11) Part or all of the nucleotides constituting the nucleic acid or double-stranded nucleic acid according to any one of (1) to (10) are deoxyribonucleotides or modified nucleotides, and the expression suppression of HIF-2α A nucleic acid having activity.
(12) The nucleic acid according to (11), wherein at least one of the modified nucleotides is a 2′-modified nucleotide of a ribonucleotide.
(13) The nucleic acid according to (11), wherein at least one of 1 to 4 bases added to the 3 ′ end or the 5 ′ end is deoxyribonucleotide.
(14) A vector encoding the nucleic acid according to any one of (1) to (13).
(15) A pharmaceutical composition comprising the nucleic acid or vector according to any one of (1) to (14) as an active ingredient.
(16) The pharmaceutical composition according to (15), further comprising a carrier effective for transferring the nucleic acid into cells.
(17) The pharmaceutical composition according to (16), wherein the carrier effective for transferring the nucleic acid into the cell is a cationic carrier.
(18) The pharmaceutical composition according to (17), wherein the carrier effective for transferring nucleic acid into cells is a liposome.
(19) The pharmaceutical composition according to (18), wherein the liposome reaches the tissue or organ containing the expression site of the gene encoding HIF-2α.
(20) Consists of a composite particle comprising a nucleic acid or vector and a lead particle as constituents and a lipid membrane that coats the composite particle, wherein the constituent of the lipid membrane contains a soluble polar organic solvent, The pharmaceutical composition according to (15), which is a liposome in which a liquid containing the polar organic solvent is present at a concentration at which the constituent components of the lipid membrane can be dispersed and the composite particles can also be dispersed.
(21) The pharmaceutical composition according to any one of (15) to (20), which suppresses expression of HIF-2α.
(22) The pharmaceutical composition according to (21), which suppresses angiogenesis.
(23) The pharmaceutical composition according to any one of (15) to (22), which is used for treatment or prevention of cancer, diabetic retinopathy or rheumatism.
(24) A method for suppressing the expression of HIF-2α, comprising administering the nucleic acid, vector or pharmaceutical composition according to any one of (1) to (23) to a subject.

FIG. 1 shows hif-2α after transfection of a double-stranded nucleic acid of the present invention (SEQ ID NO: N, N + 21: N is an integer of 43 to 61) into A-498 cells at a final concentration of 200 pM and culturing for 48 hours. The result of having quantified and evaluated the expression of mRNA by RT-PCR is shown. When semiquantitatively determining the amount of hif-2α mRNA, the amount of mRNA of gapdh (D-glyceraldehyde-3-phosphate dehydrogenase) of the corresponding sample was calculated as an internal control. FIG. 2 shows the mRNA of vegfa after the double-stranded nucleic acid of the present invention (SEQ ID NO: N, N + 21: N is an integer of 43 to 61) transfected into A-498 cells at a final concentration of 200 pM and cultured for 48 hours. The result of having quantified and evaluated the expression of this by RT-PCR is shown. When semi-quantifying the amount of vegfa mRNA, the amount of gapdh mRNA in the corresponding sample was used as an internal control. FIG. 3 shows the results of evaluating the inhibitory effect on cell proliferation of the double-stranded nucleic acid of the present invention (SEQ ID NO: N, N + 21: N is an integer of 43 to 61) by measuring the number of cells. A-498 cells were transfected with double-stranded nucleic acid at 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 nM, respectively, and the number of viable cells after culturing for 7 days was measured. The ratio of the number of living cells when each double-stranded nucleic acid is transfected is shown on the vertical axis when the number of living cells of A-498 cells not transfected with double-stranded nucleic acid is 100%. Continuation of Fig. 3 FIG. 5 shows that the modified double-stranded nucleic acid of the present invention (SEQ ID NO: N ′, N ′ + 6: N ′ is an integer of 85 to 90) was transfected into A-498 cells at a final concentration of 200 pM and cultured for 48 hours. The result of having quantified and evaluated the expression of mRNA of hif-2 (alpha) by RT-PCR is shown. On the right side of the graph, the result of the corresponding unmodified double-stranded nucleic acid (SEQ ID NO: N ′, N ′ + 6: N ′ is 43, 46, 48, 51, 53 or 57) is shown. When semiquantitatively determining the amount of hif-2α mRNA, the amount of gapdh mRNA in the corresponding sample was used as an internal control. FIG. 6 shows that the modified double-stranded nucleic acid of the present invention (SEQ ID NO: N ′, N ′ + 6: N ′ is an integer of 85 to 90) was transfected into A-498 cells at a final concentration of 200 pM and cultured for 48 hours. The result of having quantified and evaluated the expression of mRNA of vegfa by RT-PCR is shown. On the right side of the graph, the result of the corresponding unmodified double-stranded nucleic acid (SEQ ID NO: N ′, N ′ + 6: N ′ is 43, 46, 48, 51, 53 or 57) is shown. When semi-quantifying the amount of vegfa mRNA, the amount of gapdh mRNA in the corresponding sample was used as an internal control.

A gene encoding HIF-2α targeted by the nucleic acid of the present invention (hereinafter also referred to as hif-2α gene) is Genbank Accession No. A gene containing a DNA base sequence (SEQ ID NO: 97) corresponding to the full-length mRNA of hif-2α registered as NM_001430.3 can be mentioned. In the present invention, the nucleotide consisting of the base sequence represented by SEQ ID NO: 97 is also referred to as a target gene.
1. Nucleic acid of the present invention In the present invention, a nucleic acid containing a base sequence complementary to a nucleic acid consisting of a part of the base sequence of the hif-2α gene is referred to as an antisense strand nucleic acid, and 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 hif-2α gene.

  The nucleic acid of the present invention includes a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 22 to 40, 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 of the nucleic acid. A nucleic acid comprising a nucleic acid comprising a complementary base sequence and having an activity to suppress the expression of HIF-2α, a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 22 to 40, or at least one of the nucleic acids A nucleic acid in which 1 to 3 bases are substituted, deleted or added in at least one nucleic acid of a nucleic acid from which 1 to 4 bases have been deleted and a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid And a nucleic acid containing and inhibiting the expression of HIF-2α.

  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 with RNA or DNA, the affinity with a complementary strand nucleic acid is increased in order to improve or stabilize nuclease resistance. Therefore, in order to increase cell permeability or to make it visible, 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 in which at least one of the sugar moiety, phosphodiester bond and base is modified.
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.

The 2'-modified nucleotides, such as 2'-OH group of the 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 carbons, and R 'is alkylene, preferably alkylene having 1 to 6 carbons) 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'-modification 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 nucleotides.

  Examples of the sugar-modified nucleotide include a crosslinked nucleic acid (BNA) having two cyclic structures by introducing a crosslinked structure into the sugar, and specifically, the 2′-position. Locked Nucleic Acid (LNA) in which the oxygen atom and 4'-position carbon atom are cross-linked via methylene, Ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32 , e175 (2004)] and the like, and peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acid (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 an oxygen atom is substituted with a sulfur atom, a hydrogen atom is substituted with an alkyl group having 1 to 6 carbon atoms, a methyl group is substituted with hydrogen or an alkyl group 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 Examples include an added nucleotide derivative, a Cy3 added nucleotide derivative, a 6-FAM added nucleotide derivative, and a biotin added nucleotide derivative.

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 may be any nucleotide as long as it is a nucleic acid having a function equivalent to a nucleic acid consisting of a partial base sequence of the hif-2α gene or a nucleic acid consisting of a base sequence complementary to the base sequence of the nucleic acid. Or you may be comprised from the derivative (s). That is, in a nucleic acid consisting of a partial base sequence of the hif-2α gene or a nucleic acid consisting of a base sequence complementary to the base sequence of the nucleic acid, the nucleotide constituting the base sequence has a function equivalent to that of the nucleotide. The substituted ribonucleotide, deoxyribonucleotide or derivative thereof may be used.

  The nucleic acid of the present invention has any length as long as a nucleic acid consisting of a partial base sequence of the hif-2α gene and a nucleic acid consisting of a base sequence complementary to the base sequence of the nucleic acid can form a duplex. However, the length of the sequence capable of forming a double chain is usually 15 to 27 bases, preferably 15 to 25 bases, more preferably 15 to 23 bases, further preferably 15 to 21 bases, and 15 to 25 bases. 19 bases are particularly preferred.

As the nucleic acid of the present invention, a nucleic acid consisting of a partial base sequence of the hif-2α gene is used. Among the nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base is deleted. A substituted or added one may be used.
The nucleic acid that suppresses the expression of HIF-2α includes a part of the base sequence of the hif-2α gene and a nucleic acid consisting of a base sequence complementary to the base sequence of the nucleic acid, and the expression of HIF-2α Any nucleic acid such as a single-stranded nucleic acid and a double-stranded nucleic acid can be used as long as the nucleic acid is suppressed, but a double-stranded nucleic acid is preferably used.

  In the present invention, the term “double-stranded nucleic acid” refers to a nucleic acid in which two strands are paired and have a duplex 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 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 having a protruding portion, one having a protruding portion consisting of 1 to 4 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used. What has the protrusion part which becomes is preferably used, and what has the protrusion part which consists of dTdT or UU is used more preferably. The overhang can have only the antisense strand, only the sense strand, and both the antisense strand and the sense strand, but a double-stranded nucleic acid having an overhang on both the antisense strand and the sense strand is preferably used. . In addition, a sequence that partially or entirely 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. Furthermore, the double-stranded nucleic acid of the present invention has, for example, a nucleic acid molecule (WO2005 / 089287) that generates the above-mentioned double-stranded nucleic acid by the action of a ribonuclease such as Dicer, or a protruding portion at the 3 ′ end or 5 ′ end. A double-stranded nucleic acid that has not been used can also be used.

  As the double-stranded nucleic acid of the present invention, a nucleic acid having the same sequence as the base sequence of the target gene or its complementary strand may be used, but the 5 ′ end or 3 ′ of at least one strand of the nucleic acid may be used. You may use the double stranded nucleic acid which consists of a nucleic acid by which the terminal 1-4 base was deleted, and the nucleic acid which consists of a base sequence complementary to the base sequence of this nucleic acid. Examples of the double-stranded nucleic acid include a double-stranded nucleic acid whose double-stranded forming part is composed of, for example, 15 to 19 base pairs.

  Moreover, a single-stranded nucleic acid can also be used as the nucleic acid of the present invention. As an example of such a nucleic acid, a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 22 to 40, or a substitution of 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base in these nucleic acids. Also included are nucleic acids that are deleted or added and that have the activity of suppressing the expression of HIF-2α. Examples of the nucleic acid containing these nucleic acids include 30 bases or less, preferably 29 bases or less, more preferably 27 bases or less, still more preferably 25 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 double-stranded nucleic acid described above 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 nucleic acid of the present invention is designed to produce the above-mentioned single-stranded nucleic acid or double-stranded nucleic acid by the action of ribonuclease or the like, 70 base length or less, preferably 50 base length or less, more preferably 30 base length or less. It may be a nucleic acid.

  A method for producing the nucleic acid of the present invention is not particularly limited, and examples thereof include a method using known chemical synthesis, an enzymatic transcription method, and the like. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method [Nucleic Acid Research, 35, 3287 (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 strand and the antisense strand synthesized and purified are in an appropriate ratio, for example, 0.1 to 10 equivalents, preferably 0.5 to 1 sense strand with respect 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 used after annealing, 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.
Instead of the nucleic acid of the present invention, a vector that is introduced into a cell and expressed therein may 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.
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 can also be used. Examples of virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like.
2. Nucleic acid having HIF-2α expression inhibitory activity
As a first selection criterion for a double-stranded nucleic acid having an activity of suppressing the expression of HIF-2α, a nucleic acid comprising a partial base sequence of the hif-2α gene and a base sequence complementary to the base sequence of the nucleic acid A double-stranded nucleic acid consisting of a nucleic acid consisting of The nucleic acid comprising the partial base sequence may be selected as a second selection criterion that (a) no G or C sequence has 4 or more base sequences, and (b) the GC content is 20 to 80%. preferable.

A nucleic acid consisting of a partial base sequence of the hif-2α gene is Genbank Accession No. It can be designed based on the cDNA base sequence (SEQ ID NO: 97) of the full-length mRNA of hif-2α registered as NM_001430.3.
Among the partial base sequences of the hif-2α gene, the base sequence used for designing the nucleic acid having the activity of suppressing the expression of HIF-2α is, for example, the base sequence represented by any one of SEQ ID NOs: 1 to 19 The base sequence represented by any of SEQ ID NOs: 1, 6 and 15 is more preferred, and the base sequence represented by SEQ ID NO: 1 is particularly preferred.

  The nucleic acid thus selected includes, for example, a nucleic acid consisting of a partial base sequence of the hif-2α gene, and a nucleic acid consisting of a base sequence complementary to the base sequence of the nucleic acid, and HIF A double-stranded nucleic acid comprising a nucleic acid having an activity of inhibiting the expression of -2α. The single-stranded nucleic acid constituting the double-stranded nucleic acid usually comprises 15 to 30 bases, preferably 15 to 29, more preferably 15 to 27, and preferably 15 to 25. More preferably, it is particularly preferably 17-23, and most preferably 19-21. 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: 22 to 40 corresponding to a complementary sequence of hif-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 a base sequence represented by any of SEQ ID NOs: 22 to 40 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′-end or 3′-end of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 22 to 40 has been deleted at 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 the base sequence represented by any of SEQ ID NOs: 1 to 19 and an antisense strand nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 22 to 40 (here, the sense strand) In at least one strand of a double-stranded nucleic acid consisting of SEQ ID NO: N (N is 1 to 19) and the antisense strand is SEQ ID NO: N + 21), 1 to 3 bases, preferably 1 to A double-stranded nucleic acid having 2 bases, more preferably 1 base substituted, deleted or added, and having HIF-2α expression-suppressing activity.
(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) In the double-stranded nucleic acid according to any one of (a) to (e), a nucleotide or nucleotide derivative is added to 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 having 1 to 4 bases, preferably 1 to 3 bases, more preferably 1 to 2 bases, and even more preferably 2 bases.

As a specific example of the double-stranded nucleic acid of (f) above, a nucleic acid comprising the base sequence represented by SEQ ID NO: N ′ and a nucleic acid comprising the base sequence represented by SEQ ID NO: N ′ + 21 as its complementary strand A double-stranded nucleic acid consisting of Here, N ′ represents any integer of 43 to 61.
As the single-stranded nucleic acid having the activity of suppressing the expression of HIF-2α, an antisense strand nucleic acid consisting of a partial base sequence of the hif-2α gene can also be used. As an example of such a nucleic acid, a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 22 to 40, and in these nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base Examples also include nucleic acids which are substituted, deleted or added and have the activity of suppressing the expression of HIF-2α. Examples of the nucleic acid containing these nucleic acids include 30 bases or less, preferably 29 bases or less, more preferably 27 bases or less, still more preferably 25 bases or less, and particularly preferably 23 bases or less.

As a single nucleic acid having the activity of suppressing the expression of HIF-2α, a nucleic acid consisting of a partial base sequence of the hif-2α gene and a base sequence complementary to the base sequence of the nucleic acid, the following (A) The single-stranded nucleic acid of-(G) is also mentioned.
(A) an antisense strand nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 22 to 40 corresponding to a complementary sequence of hif-2 mRNA, and a sense strand nucleic acid complementary to the base sequence; A single-stranded nucleic acid obtained by linking each other via a spacer sequence.
(B) An antisense strand nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 22 to 40 and a sense strand nucleic acid comprising a base sequence complementary to the base sequence are linked via a spacer sequence. A single-stranded nucleic acid.
(C) an antisense strand nucleic acid in which at least one end is deleted from 1 to 4 bases out of the 5 ′ end or 3 ′ end of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 22 to 40, and A single-stranded nucleic acid obtained by linking a sense strand nucleic acid comprising a base sequence complementary to a nucleic acid base sequence via a spacer sequence.
(D) A sense strand nucleic acid consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 19 and an antisense strand nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 22 to 40 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 the single-stranded nucleic acid formed by ligation And a double-stranded nucleic acid having an activity of suppressing the expression of HIF-2α.
(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 in any one of (A)-(E) which forms a double strand formation part by a stem loop structure.
(G) The single stranded nucleic acid in any one of (A)-(F) which consists of 50-70 base length.

  By introducing these single-stranded nucleic acid or double-stranded nucleic acid into cells, the expression of HIF-2α can be suppressed. In the suppression of hif-2α mRNA expression using antisense DNA, compared to the case where antisense DNA is generally required at a concentration of several tens of nM to several hundred nM, for example, the double strand of the present invention The nucleic acid can suppress the expression of hif-2α mRNA even at a concentration of several hundreds pM to several nM even after 24 hours of introduction, for example, 48 hours after introduction into the cell.

In addition, the evaluation of the activity of suppressing the expression of hif-2α mRNA of single-stranded nucleic acid or double-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid etc. into cultured cancer cells etc. using cationic liposomes, etc. After culturing for a period of time, the expression level of hif-2α mRNA in the cancer cells can be quantified by RT-PCR. The effect of suppressing the expression of angiogenic factors is evaluated by quantifying the amount of angiogenic factor VEGF protein produced by the cells into which the single-stranded nucleic acid or double-stranded nucleic acid of the present invention has been introduced. be able to. 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 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 caused by cancers that are effective in inhibiting angiogenesis, diseases such as diabetic retinopathy and rheumatism, diseases caused by overexpression of HIF-2α, and mutations in VHL. It can be used for the treatment or prevention of certain diseases. Examples of the cancer include solid cancers such as kidney cancer, liver cancer, skin cancer, breast cancer, lung cancer, digestive organ cancer, head and neck cancer, prostate cancer, uterine cancer, and bladder cancer.

  Examples of the carrier effective for transferring the nucleic acid into the cell include a cationic carrier. 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. Examples of the cationic liposome include liposomes containing 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol (hereinafter also referred to as liposome A), oligofectamine (Invitrogen), lipofectin ( Invitrogen), Lipofectamine (Invitrogen), Lipofectamine 2000 (Invitrogen), DMRIE-C (Invitrogen), GeneSilencer (Gene Therapeutics Systems), TransMessenger (QIAGEN M), TransMus, Trans Ks, M Etc. are preferably used. As the cationic polymer, JetSI (Qbiogene), Jet-PEI (polyethyleneimine; Qbiogene) and the like are preferably used. As the carrier utilizing the virus envelope, GenomeOne (HVJ-E liposome; Ishihara Sangyo Co., Ltd.) is preferably used.

  A composition comprising the carrier in a single-stranded nucleic acid, a double-stranded nucleic acid or a vector contained in the pharmaceutical composition 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 having an appropriate concentration and 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. For example, in the case of liposome A, an oligo double-stranded RNA solution in a 10% maltose aqueous solution is gradually added to a 16 mg / ml liposome dispersion in a 10% maltose aqueous solution with stirring at pH 7.4 and 25 ° C. Can be prepared.

If necessary, the composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersing device or a high-pressure emulsifying device. A person skilled in the art uses an optimal method and conditions for preparing a composition comprising a carrier and a single-stranded nucleic acid, a double-stranded nucleic acid or a vector, without depending on the above-mentioned method, because it depends on the carrier used. The optimum method for the carrier can be selected.
The pharmaceutical composition of the present invention comprises a single-stranded nucleic acid, a double-stranded nucleic acid, or a composite particle comprising a vector and a lead particle as constituent components, and a lipid membrane covering the composite particle, and the constituent components of the lipid membrane Liposomes in which a liquid containing the polar organic solvent is present in a concentration that can disperse the components of the lipid membrane and the composite particles can also be dispersed in a liquid containing a polar organic solvent that is soluble in water. . The lead particles are, for example, fine particles containing lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations and the like as constituent components, and preferably include fine particles containing liposomes as constituent components. The lead particles in the present invention may be composed of a complex obtained by combining two or more lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle formulations, etc., and lipid aggregates, liposomes, emulsion particles, A complex formed by combining a polymer, 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.

Examples of the lipid membrane that coats the composite particles include neutral lipids and polyethylene glycol-phosphatidylethanolamine as constituent components.
The liposome can be prepared according to the method described in WO2006 / 080118, for example.
The compounding ratio of 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 parts by weight of carrier per 1 part by weight of 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 is provided in a form that contains an effective amount of the complex for treating or preventing a disease and 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 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 freeze-dried preparation can be prepared by dispersing a single-stranded nucleic acid, double-stranded nucleic acid or vector and carrier, followed by freeze-drying. The lyophilization treatment can be performed by a conventional method. For example, a predetermined amount of the complex solution after the dispersion treatment described above is dispensed into a vial in an aseptic state, preliminarily dried for about 2 hours under conditions 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 of the solution 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 by way of examples. However, the present invention is not limited to these examples.

Design and evaluation of nucleic acid that suppresses hif-2α gene expression (1) Preparation of double-stranded nucleic acid
As a nucleic acid sequence capable of suppressing the expression of the hif-2α gene, from the mRNA sequence of hif-2α (GenBank accession number: NM_001430.3, SEQ ID NO: 97), (a) there is no sequence in which G or C continues for 4 bases or more, Nineteen sequences were selected that met three conditions: (b) a GC content of 20-80%, more preferably (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 + 21 as its complementary sequence. Here, N is an integer from 1 to 19. A DNA sequence comprising 2 bases is appropriately added to the 3 ′ end of each RNA comprising the sequence represented by SEQ ID NOs: 1 to 19, and a nucleic acid comprising the base sequence represented by SEQ ID NO: N ′ and its complementary strand SiRNA, which is a double-stranded nucleic acid consisting of a nucleic acid consisting of the base sequence represented by SEQ ID NO: N ′ + 21, was designed. Here, N ′ represents an integer from 43 to 61.

The synthesis of the nucleic acid constituting the double-stranded nucleic acid was requested to Gene Design Co., Ltd.
(2) Evaluation method in screening Evaluation in screening involves introducing double-stranded nucleic acid into various cancer cells together with a carrier, and quantifying the amount of mRNA of hif-2α using RT-PCR (Reverse transcription-polymerase chain reaction). It went by.
(3) Preparation of complex of double-stranded nucleic acid and carrier As a carrier, a commercially available cationic liposome, Hyperfect (manufactured by QIAGEN), is used, and double-stranded nucleic acid-high according to the method described in the attached instruction manual. A perfect complex was prepared.
(4) Inhibition of hif-2α mRNA expression by double-stranded nucleic acid To examine whether transfection of double-stranded nucleic acid suppresses expression of hif-2α mRNA, the expression level of hif-2α mRNA was determined. Evaluation was performed by semi-quantification by RT-PCR.

  As the double-stranded nucleic acid, a nucleic acid consisting of the base sequence represented by SEQ ID NO: N ′ and a nucleic acid consisting of the base sequence represented by SEQ ID NO: N ′ + 21 were used. Here, N ′ represents an integer from 43 to 61.

  As shown in Table 1, a nucleic acid consisting of a base sequence represented by SEQ ID NO: N ′ (43 to 61) and a nucleic acid consisting of a base sequence represented by SEQ ID NO: N ′ + 21 (64 to 82) The double-stranded nucleic acids are also referred to as HIF-2 siRNA # 1 to # 19, respectively. In Table 1, uppercase letters indicate ribonucleotides, and lowercase letters indicate deoxyribonucleotides.

  In addition, a 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 used as a negative control, and a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 62 Nucleic acid consisting of the base sequence represented by No. 83 (hereinafter also referred to as Control # 1. Journal of Cellular Biochemistry, 92, 491-501 (2004)), and nucleic acid and sequence comprising the base sequence represented by SEQ ID No. 63 A nucleic acid consisting of the base sequence represented by No. 83 (hereinafter also referred to as Control # 2; PLoS Biology, 1 (3), 439-444 (2003)) was used as a positive control.

These double-stranded nucleic acids were respectively converted into human kidney cells A-498 cells (
1260933853765_1.html
HTB 44).
Hyperfect diluted with MEM so that it becomes the amount described in the attached instruction manual after dispensing 10μL each of double-stranded nucleic acid diluted with MEM (manufactured by GIBCO, 11095) to 96-well culture plate 10 μL each was added to prepare a double-stranded nucleic acid-hyperfect complex. Next, A-498 cells suspended in MEM containing 1.11% fetal bovine serum (FBS), Non-Essential Amino-acids (NEAA), and pyruvate were seeded so that the number of cells was 5000 cells / 180 μL / well. The double-stranded nucleic acid was introduced into A-498 cells by culturing at 37 ° C. under 5% CO ₂ conditions. The final concentration of double-stranded nucleic acid was adjusted to 200 pM.

Cells introduced with double-stranded nucleic acid are cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours, washed with ice-cold PBS, and total RNA is recovered using Cells-to-Ct Kit (ABI) Then, reverse transcription was performed according to the method described in the instructions attached to the product to prepare cDNA.
By using the obtained cDNA as a template, using Universal Probe Library (Roche Applied Science) as a probe, using ABI7900HT Fast (ABI), and performing a PCR reaction according to the method described in the attached instruction manual, Semi-quantification of the amount of mRNA of hif-2α and the amount of mRNA of gapdh from the amplification amount obtained by PCR reaction of the hif-2α gene and gapdh (D-glyceraldehyde-3-phosphate dehydrogenase) gene, which is a constitutive expression gene, respectively. The value was calculated. When semiquantitatively determining the amount of hif-2α mRNA, the amount of gapdh mRNA in the corresponding sample was used as an internal control.

The results regarding the amount of hif-2α mRNA are shown in FIG. The amount of mRNA in the specimen was expressed as a relative ratio when the amount of mRNA for hif-2α and the amount of mRNA for gapdh were 1 in the group treated with carrier alone (mock group).
As shown in FIG. 1, A-498 cells into which the double-stranded nucleic acid of the present invention targeting the hif-2α gene was introduced had a decreased amount of hif-2α mRNA compared to the negative control.

Suppression of vegfa mRNA expression by double-stranded nucleic acid
Whether or not the expression of VEGF, which is an angiogenic factor whose transcription is promoted by HIF-2, is suppressed along with the suppression of expression of hif-2α mRNA, RT- described in Example 1 (4). Evaluation was performed in the same manner as in the semi-quantification of mRNA amount by PCR. The results regarding the amount of mRNA of vegfa which is a gene encoding VEGF are shown in FIG.

  As shown in FIG. 2, the amount of vegfa mRNA was decreased in A-498 cells into which the double-stranded nucleic acid of the present invention targeting the hif-2α gene was introduced, compared to the negative control.

In vitro anti-cell proliferation activity of double-stranded nucleic acids
Hyperfect diluted to 384-well culture plate with double-stranded nucleic acid diluted with MEM (GIBCO, 11095) by 6 μL each, and then diluted to MEM with the amount described in the attached instruction manual 10 μL each was added to prepare a double-stranded nucleic acid-hyperfect complex. Next, A-498 cells suspended in MEM containing 1.47% fetal bovine serum (FBS), Non-Essential Amino-acids (NEAA), and pyruvate were seeded so that the number of cells was 2000 cells / 34 μL / well. The double-stranded nucleic acid was introduced into A-498 cells by culturing at 37 ° C. under 5% CO ₂ conditions. The final concentration of the double-stranded nucleic acid was adjusted to 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 nM, respectively. 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 had been introduced were cultured in a 37 ° C. 5% CO ₂ incubator for 7 days, and the viable cell ratio 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 was calculated assuming that the viable cell ratio of A-498 cells when only the carrier was treated was 100%. The results are shown in FIGS.

  As shown in FIG. 3 and FIG. 4, there is no decrease in the viable cell rate due to the introduction of the double-stranded nucleic acid targeting the hif-2α gene, and the various double-stranded nucleic acids of the present invention do not inhibit cell proliferation. It was shown that.

Suppression of hif-2α and vegfa mRNA expression by modified double-stranded nucleic acid Modification of double-stranded nucleic acid is expected to improve nuclease resistance and reduce immunogenicity, while RNAi The effect may be reduced. In order to examine whether the activity was changed by the modification, the expression levels of hif-2α and vegfa mRNA were evaluated by semi-quantification by RT-PCR.

  As a modified nucleotide of a modified double-stranded nucleic acid, refer to Oligonucleotides, 18 (2), 187-200 (2008), and substitute the 2'-OH group of ribose ribose with a 2'-methoxy group. Using. As the modified double-stranded nucleic acid, a nucleic acid consisting of the base sequence represented by SEQ ID NO: N ′ and a nucleic acid consisting of the base sequence represented by SEQ ID NO: N ′ + 6 are used, and the corresponding unmodified double-stranded nucleic acid is used. Compared with the results. Here, N ′ represents an integer from 85 to 90.

  As shown in Table 2, a nucleic acid consisting of a nucleotide sequence represented by SEQ ID NO: N ′ (85 to 90) and a nucleic acid consisting of a nucleotide sequence represented by SEQ ID NO: N ′ + 6 (91 to 96). This double-stranded nucleic acid is also referred to as HIF-2 siRNA # 1-OMe, # 4-OMe, # 6-OMe, # 9-OMe, # 11-OMe, # 15-OMe, respectively. Unmodified double-stranded nucleic acids corresponding to these are HIF-2 siRNAs # 1, # 4, # 6, # 9, # 11, and # 15 shown in Table 1, respectively. In Table 2, uppercase letters indicate ribonucleotides, lowercase letters indicate deoxyribonucleotides, and italic letters indicate nucleotides in which the 2'-OH group of ribose is substituted with a 2'-methoxy group.

The results of evaluation of the mRNA levels of hif-2α and vegfa by the same method as the semi-quantification of mRNA levels by RT-PCR described in Example 1 (4) are shown in FIGS. 5 and 6, respectively.
As shown in FIG. 5 and FIG. 6, A-498 cells into which the modified double-stranded nucleic acid of the present invention targeting the hif-2α gene is introduced have a higher amount of hif-2α and vegfa mRNA than the negative control. Declined. Among them, HIF-2 siRNA # 1-OMe, # 6-OMe, # 15-OMe retained high inhibitory activity.

  According to the present invention, a nucleic acid having an activity of suppressing the expression of HIF-2α, a pharmaceutical composition comprising the nucleic acid, and the like are provided. The nucleic acid and pharmaceutical composition of the present invention are useful for the treatment of diseases such as cancer, diabetic retinopathy, rheumatism and the like in order to suppress angiogenesis.

Claims (24)

From a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 22 to 40 or 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 And a nucleic acid having an activity to suppress the expression of HIF-2α. From a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 22 to 40 or 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 nucleic acid containing a nucleic acid in which 1 to 3 bases are substituted, deleted or added in at least one of the nucleic acids and having an activity of suppressing the expression of HIF-2α. The nucleic acid according to claim 1 or 2, which has a duplex forming part of 27 base pairs or less. The nucleic acid according to claim 3, which has a duplex forming part consisting of 15 to 19 base pairs. The double-stranded nucleic acid according to any one of claims 1 to 4. A double-stranded nucleic acid obtained by adding 1 to 4 bases to the 3 'end or 5' end of at least one strand of the double-stranded nucleic acid according to claim 5. A nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 22 to 40. The nucleic acid according to claim 7, wherein 1 to 3 bases are substituted, deleted or added, and has an activity of suppressing expression of HIF-2α. A nucleic acid having 30 bases or less, comprising the nucleic acid according to claim 7 or 8. A nucleic acid having part or all of the nucleotides constituting the nucleic acid or the double-stranded nucleic acid according to any one of claims 1 to 9 being a ribonucleotide and having an activity of suppressing the expression of HIF-2α. A nucleic acid in which part or all of the nucleotides constituting the nucleic acid or double-stranded nucleic acid according to any one of claims 1 to 10 are deoxyribonucleotides or modified nucleotides, and has an activity of suppressing the expression of HIF-2α. 12. The nucleic acid of claim 11, wherein at least one of the modified nucleotides is a ribonucleotide 2'-modified nucleotide. The nucleic acid according to claim 11, wherein at least one of 1 to 4 bases added to the 3 'end or the 5' end is deoxyribonucleotide. A vector encoding the nucleic acid according to any one of claims 1 to 13. The pharmaceutical composition which uses the nucleic acid or vector of any one of Claims 1-14 as an active ingredient. The pharmaceutical composition according to claim 15, further comprising a carrier effective for transferring the nucleic acid into the cell. The pharmaceutical composition according to claim 16, wherein the carrier effective for transferring the nucleic acid into the cell is a cationic carrier. The pharmaceutical composition according to claim 17, wherein the effective carrier for transferring the nucleic acid into the cell is a liposome. The pharmaceutical composition according to claim 18, wherein the liposome is a liposome that reaches a tissue or organ containing an expression site of a gene encoding HIF-2α. The lipid membrane is composed of a composite particle comprising a nucleic acid or vector and a lead particle and a lipid membrane that coats the composite particle, and the lipid membrane contains a polar organic solvent in which the component of the lipid membrane is soluble. The pharmaceutical composition according to claim 15, wherein the composition is a liposome in which a liquid containing the polar organic solvent is present at a concentration capable of dispersing the composite particles. The pharmaceutical composition according to any one of claims 15 to 20, which suppresses the expression of HIF-2α. The pharmaceutical composition according to claim 21, which suppresses angiogenesis. The pharmaceutical composition according to any one of claims 15 to 22, which is used for treatment or prevention of cancer, diabetic retinopathy or rheumatism. A method for suppressing the expression of HIF-2α, comprising administering the nucleic acid, vector or pharmaceutical composition according to any one of claims 1 to 23 to a subject.

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