JP2004129650A - New antisense compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for designing an antisense nucleotide having a high expression inhibitory effect on a target gene without affecting expression of other genes, and to provide a compound of the antisense nucleotide. <P>SOLUTION: This method for designing the antisense compound includes processes (1) to (7) as follows: a process (1) for inputting data on base sequences of oligonucleotides having an antisense effect on the target gene; a process (2) for searching homology of the input data on the base sequences; a process (3) for outputting the data on the genes of which the homology is high; a process (4) for extracting an entire RNA fraction of a cell incubated by adding the oligonucleotide having the base sequence specified in the process (1) to the cell and another entire RNA fraction of the cell incubated by not adding the oligonucleotide; a process (5) for analyzing a difference of expression levels of the genes output in the process (3) between the entire RNA fractions; a process (6) for modifying the base sequence of the oligonucleotide specified in the process (1) based on the difference of the expression levels of the genes; and a process (7) for selecting the oligonucleotide having the antisense effect on the target gene without affecting the expression levels of the genes output in the process (3). <P>COPYRIGHT: (C)2004,JPO

Description

(4) The present invention relates to an oligonucleotide compound that suppresses the function of a specific gene and does not affect the function of another gene, and a method for designing the compound.

Antisense oligonucleotides function by a unique mechanism that suppresses the expression of target proteins by forming Watson-Crick type base pairs with mRNA in a sequence-specific manner, and therefore have higher selectivity than existing drugs. And low side effects are expected (for example, see Non-Patent Document 1). However, on the other hand, if another gene having a similar complementary sequence is present, it may bind to the mRNA and inhibit the expression of the gene. In such a case, unexpected side effects may occur. Therefore, it is important to use an antisense oligonucleotide that suppresses only the expression of the target gene and does not affect the expression of other genes.

As a method for finding a gene whose expression is suppressed besides a target gene by administering an antisense oligonucleotide, a method using a DNA chip technology such as a Stanford microarray and an Affymetrix Genechip (hereinafter, referred to as a “DNA chip”) It has been known. With this method, it is possible to observe the expression fluctuation of many genes at once, and Fisher et al. Confirmed by DNA microarray that the antisense oligonucleotide of the MDR1 gene suppressed the expression of genes other than MDR1. (For example, see Non-Patent Document 2). However, the detection sensitivity of a DNA chip currently in practical use is not sufficient, so that it may be extremely difficult to detect the amount of mRNA expressed only in a small amount in cells (for example, see Non-Patent Document 3). .). Also, due to sensitivity issues, discussions on reproducibility are often accompanied. Further, in a microarray, when a plurality of highly homologous genes are present, there is a problem that the specificity is reduced due to cross-linking (for example, see Non-Patent Document 3). That is, in a DNA chip, it is possible in principle to narrow down suppressed genes from analysis of gene expression such as thousands or tens of thousands at a time, but the obtained data is not always highly reliable.

Therefore, there has been a need for a highly reliable method for designing an antisense oligonucleotide that acts on a target gene and does not act on other genes.

Vascular endothelial growth factor (VEGF) is a growth factor that acts specifically on vascular endothelial cells and promotes angiogenesis. Angiogenesis is performed by proliferating and proliferating vascular endothelial cells under stimulation from growth factors, physiologically active substances or mechanical damage, and plays an important role in development, wound healing, inflammation and the like. In solid cancer, in order for cancer tissue to grow beyond 1-2 mm in diameter, it is necessary for new blood vessels to extend from existing blood vessels to reach cancer tissue in order to supply oxygen and nutrients necessary for the tumor. Yes (see, for example, Non-Patent Document 4), it is known that the growth of cancer tissue explosively accelerates when blood vessels reach the cancer tissue, and further, cancer cell metastasis is performed through this blood vessel. VEGF is thought to play a central role in tumor angiogenesis. Regarding the involvement of VEGF in cancer, 1) many cancer cells secrete VEGF (for example, see Non-Patent Document 5). When a cancer tissue section is stained with an anti-VEGF antibody, the cancer tissue and new blood vessels around it are strongly stained. (See, for example, Non-Patent Documents 6 and 7.) In mice in which one of the VEGF receptors is genetically inactivated, the growth of transplanted cancer is suppressed (for example, see Non-Patent Document 8). It has been reported that neutralizing antibodies exhibit antitumor activity against cancer-bearing mice (for example, see Non-Patent Documents 5 and 9). Based on the above facts, it is considered that VEGF secreted by cancer cells plays a major role in tumor angiogenesis, and it is expected that substances that suppress the angiogenic activity of VEGF can suppress cancer growth and metastasis Have been. In addition, diabetic retinopathy, which is an eye disease, is recognized as a complication in nearly half of diabetic patients. In diabetic retinopathy, it is considered that the formation of microcapillaries is promoted due to lack of oxygen, which eventually ruptures and bleeds to form scar tissue, which eventually leads to retinal detachment and blindness. Therefore, it is expected that suppression of angiogenesis can suppress the severity of retinopathy. (See, for example, Non-Patent Document 10), based on the results of experiments using monkeys, report that VEGF is very closely related to the development of proliferative retinopathy. Therefore, a substance that suppresses the angiogenic activity of VEGF is considered to be useful for preventing or treating diabetic retinopathy. Further, diseases such as rheumatoid arthritis, psoriasis, hemangiomas, scleroderma and neovascular glaucoma are accompanied by pathological angiogenesis, which is one of the main symptoms (for example, see Non-Patent Document 11). . Therefore, a substance that suppresses the function of VEGF may be applicable to the treatment of the aforementioned various diseases in which angiogenesis is involved. VEGF is known to be involved not only in angiogenesis but also in vascular hyperpermeability. It has been suggested that VEGF-induced vascular hyperpermeability is involved in pathological symptoms such as cerebral edema during cancerous ascites retention and ischemia-reperfusion injury (for example, see Non-Patent Document 12). Therefore, a substance that suppresses the function of VEGF is considered to be useful for treating the above-mentioned diseases in which angiogenesis or vascular hyperpermeability by VEGF is involved.

As a compound that inhibits the action of VEGF, the following sequence:
5'-cccaagacagcagaaagttcat-3 '(SEQ ID NO: 1 in Sequence Listing)
And an antisense molecule (hereinafter, referred to as "compound A") in which each base is bound by phosphorothioate (for example, see Non-patent Document 13). Compound A corresponds to the complementary strand of the VEGF sequence (GenBank Accession No. AF022375.1: nucleotide numbers 702 to 723 of SEQ ID NO: 2 in the sequence listing).

However, compound A has insufficient VEGF inhibitory activity, and a compound having stronger VEGF inhibitory activity has been expected. In addition, a method for designing a compound that has a stronger inhibitory activity on VEGF and does not affect the expression of other genes has been expected.
R. Schlingensiepen and K.-H. Schinggensiepen, Eds, R. Schlingensiepen, W. Brysch and K.-H. 3-28 "Journal of Biological Chemistry", 2002, Vol. 277, p. 22980-22984 Masaaki Matsumura, Hiroyuki Nami, "Cell Engineering Separate Volume Genome Science Series 1," DNA Microarrays and the Latest PCR Method "", Shujunsha, p. 8-9 "Journal of National Cancer Institute", 1990, Vol. 82, p. 4-6 "Biochemical and Biophysical Research Communications", 1993, vol. 194, p. 1234-1241. "Journal of Experimental Medicine", 1991, Vol. 174, pp. 1275-1278. "Cancer Research", 1993, vol. 53, p. 4727-4735 "Nature", 1994, Vol. 367, p. 576: "Nature", 1993, vol. 362, p. 841: "American Journal of Pathology", 1994, Vol. 145, p. 574-584 "Journal of Medicine", 1989, vol. 320, p. 1211: "The Journal of Clinical Investigation", 1999, Vol. 104, p. 1613-1620 "The Journal of Biological Chemistry", 1995, Vol. 270, p. 28316-28324

The present inventor studied a substance that suppresses the action of VEGF, and as a result, found that compound (I)
HO−C ^e2 −C ^e2 −C ^e2 −A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^s −G ^s −C ^s −A ^s −G ^s −A ^e2 −A ^e2 −A ^e2 − ^{G m -T e2 -T e2 -C e2} -A e2 -T e2t -H ( compound I: sequence Listing SEQ ID NO: compounds having the nucleotide sequence of 3)
Have potent VEGF inhibitory activity and are useful for treating diseases associated with VEGF-induced angiogenesis or vascular hyperpermeability.

The present inventor further found a method for designing an antisense oligonucleotide having a high expression inhibitory effect on a target gene and having no effect on the expression of other genes. The present inventors have found that it has a strong VEGF expression inhibitory activity and that the expression inhibitory effect on other genes is lower than that of the original antisense oligonucleotide, and thus completed the present invention.

The present invention
(1) A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting a total RNA fraction from cells incubated with the oligonucleotide having the nucleotide sequence shown in step 1) and cells incubated without the oligonucleotide;
5) analyzing the difference in the expression level of each gene output in step 3) between the total RNA fractions obtained in step 4);
6) a step of modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each gene obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and not affecting the expression level of each gene output in step 3) The process of choosing,
(2) A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting a total RNA fraction from each of the animal to which the oligonucleotide having the nucleotide sequence shown in step 1) has been administered and the animal to which the oligonucleotide has not been administered;
5) analyzing the difference in the expression level of each gene output in step 3) between the total RNA fractions obtained in step 4);
6) a step of modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each gene obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and not affecting the expression level of each gene output in step 3) The process of choosing,
(3) A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting all polypeptide fractions from cells incubated with the oligonucleotide having the nucleotide sequence shown in step 1) and cells incubated without the oligonucleotide;
5) analyzing the difference in the expression level of the polypeptide encoded by each gene output in step 3) among the total polypeptide fractions obtained in step 4);
6) modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each polypeptide obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and not affecting the expression level of each gene output in step 3) The process of choosing,
(4) A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence and base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting all polypeptide fractions from animals to which the oligonucleotide having the nucleotide sequence shown in step 1) has been administered and animals to which the oligonucleotide has not been administered, respectively;
5) analyzing the difference in the expression level of the polypeptide encoded by each gene output in step 3) among the total polypeptide fractions obtained in step 4);
6) modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each polypeptide obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and not affecting the expression level of each gene output in step 3) The process of choosing,
(5) The modification of the base sequence is i) reducing the number of bases constituting the oligonucleotide, ii) transferring the nucleotide sequence of the oligonucleotide to the 3 ′ or 5 ′ side in the target gene, or iii) transferring the oligonucleotide. The number of constituent bases is reduced, and the nucleotide sequence of the oligonucleotide is transferred to the 3 ′ or 5 ′ side in the target gene, characterized in that the method is performed by any one of i) to iii). The method according to any one of (1) to (4),
(6) The method according to (1) or (2), wherein the expression level of the gene is measured by Northern blotting, dot blotting, slot blotting, or RT-PCR.
(7) The expression according to (3) or (4), wherein the expression level of the polypeptide is measured by Western blotting, dot blotting, slot blotting or enzyme-linked immunosorbent assay (ELISA). the method of,
(8) The method according to any one of (1), (3) and (5) to (7), wherein the cells are cultured cells.
(9) The method according to (8), wherein the cultured cells are animal cells,
(10) The method according to any one of (2), (4) and (5) to (7), wherein the animal is a mammal;
(11) The method according to (10), wherein the mammal is a rodent.
(12) The method according to (11), wherein the mammal is a mouse or a rat.
(13) the method according to any one of (1) to (12), wherein the target gene is a VEGF gene;
(14)
The following general formula (I)
HO-B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-H (I)
(Where B1 to B3, B8 and B11 are the same or different

(Hereinafter referred to as “C ^p ”), a formula

(Hereinafter referred to as “C ^s ”) or a general formula C ^e

(In the formula, l represents an integer of 1 to 5.)
B4, B5, B7, B9, B12, B14 to B16 are the same or different

(Hereinafter referred to as “A ^p ”), a formula

(Hereinafter referred to as “A ^s ”) or a general formula A ^e

(In the formula, m represents an integer of 1 to 5.)
B6, B10, B13 and B17 are the same or different

(Hereinafter referred to as “G ^p ”), a formula

(Hereinafter referred to as “G ^s ”), a formula

(Hereinafter referred to as “G ^m ”) or a general formula

(In the formula, n represents an integer of 1 to 5.)
B18 and B19 are the same or different

(Hereinafter referred to as “T ^p ”), a formula

(Hereinafter referred to as “T ^s ”) or a general formula

(In the formula, o represents an integer of 1 to 5.)
B20 is the formula

(Hereinafter referred to as “E”)
And / or a salt thereof,
In (15) (14), B1 to B3, B8 and B11 are the same or different C ^p or C ^e,
B4, B5, B7, B9, B12, B14 to B16 are the same or different and A ^p or A ^e,
B6, B10, B13 and B17 are the same or different and G ^p, a G ^m or G ^e, and,
Compound is a T ^p or T ^e B18 and B19 are identical or different, and / or, a salt thereof,
(16) In any one of (14) or (15), compound B1 to B3 is C ^e, and / or a salt thereof,
(17) In (16), the compound wherein B4 is A ^e and / or a salt thereof,
(18) In (17), the compound wherein B5 is A ^e and / or a salt thereof,
In (19) (18), compound B6 is G ^m or G ^e, and / or, a salt thereof,
(20) In (19), the compound wherein B7 is A ^e and / or a salt thereof,
(21) In (20), compound B8 is C ^e, and / or, a salt thereof,
In (22) (21), compound B19 is T ^e, and / or, a salt thereof,
In (23) (22), compound B18 is T ^e, and / or, a salt thereof,
In (24) (23), compound B17 is a G ^m or G ^e, and / or, a salt thereof,
(25) The compound according to (24), wherein B16 is ^Ae , and / or a salt thereof,
(26) The compound according to (25), wherein B15 is ^Ae , and / or a salt thereof,
(27) The compound according to (26), wherein B14 is ^Ae , and / or a salt thereof,
(28) The compound according to any one of (14) to (27), wherein l, m, n and o are the same or different and are 1 or 2, and / or a salt thereof,
(29) In (28), the compound wherein l, m, n and o are the same and are 1 or 2, and / or a salt thereof,
(30) In (29), the compound wherein l, m, n and o are 2, and / or a salt thereof,
Consists of

According to the present invention, it has become possible to design an antisense compound that suppresses the expression level of a target gene and has little effect on the expression of other genes.

Among the compounds of the present invention, the most preferred compounds are compounds selected from the following compound group 1, and / or salts thereof.
Compound group 1
HO−C ^e2 −C ^e2 −C ^e2 −A ^e2 −A ^e2 −G ^e2 −A ^e2 −C ^e2 −A ^p −G ^p −C ^p −A ^p −G ^p −A ^e2 −A ^e2 −A ^e2 − G ^e2 −T ^e2 −T ^e2 −E−H,
HO−C ^e2 −C ^e2 −C ^e2 −A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^p −G ^p −C ^p −A ^p −G ^p −A ^e2 −A ^e2 −A ^e2 − G ^m −T ^e2 −T ^e2 −E−H,
HO-C ^e1 -C ^e1 -C ^e1 -A ^e1 -A ^e1 -G ^e1 -A ^e1 -C ^e1 -A ^p -G ^p -C ^p -A ^p -G ^p -A ^e1 -A ^e1 -A ^e1- G ^e1 −T ^e1 −T ^e1 −E−H, and
HO-C ^e1 -C ^e1 -C ^e1 -A ^e1 -A ^e1 -G ^m -A ^e1 -C ^e1 -A ^p -G ^p -C ^p -A ^p -G ^p -A ^e1 -A ^e1 -A ^e1- G ^m −T ^e1 −T ^e1 −E−H.

In the above description and in the present specification, A ^e1 , G ^e1 , C ^e1 , T ^e1 , A ^e2 , G ^e2 , C ^e2 , T ^e2 and T ^e2t represent groups having the following chemical structures.

The "salt" refers to a salt of the compound of the present invention, which can be converted into a salt. Such a salt is preferably an alkali metal salt such as a sodium salt, a potassium salt, and a lithium salt. , Calcium salts, alkaline earth metal salts such as magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, metal salts such as cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts , Dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt , Chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylene Amine salts such as organic salts such as ruamine salt, piperazine salt, tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt; hydrofluoric acid, hydrochloride, hydrobromide and hydroiodide Inorganic acid salts such as hydrohalide, nitrate, perchlorate, sulfate and phosphate; lower alkanesulfonic acids such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate Salts, arylsulfonates such as benzenesulfonate, p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate Organic acid salts such as acid salts; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamates, and aspartates.

The compounds of the present invention represented by the general formula (I) and salts thereof can also exist as hydrates, and the present invention also includes those hydrates.

Another aspect of the present invention is a pharmaceutical composition containing the compound represented by the general formula (I) of the present invention and / or a salt thereof, preferably angiogenesis or vascular permeability by VEGF. Pharmaceutical composition for use in the treatment and prevention of diseases associated with hypertension, more preferably cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, hemangiomas, scleroderma, neovascular glaucoma, cancerous ascites retention Or a pharmaceutical composition used for treatment and prevention of cerebral edema at the time of ischemia-reperfusion injury.

In the present invention, an antisense compound refers to an oligonucleotide designed to suppress the expression of a specific gene, and an oligonucleotide having a sequence complementary to the base sequence of the mRNA of the target gene. Means The target gene in the present invention is a gene selected as a target of gene expression suppression by an antisense compound when designing an antisense compound. The antisense effect in the present invention means an effect in which the expression of a target gene is suppressed by administration or addition of an antisense compound. The match ratio in the present invention is a numerical value indicating a ratio of forming a complete base pair (adenine and thymine, guanine and cytosine) with a base sequence complementary to nucleotides constituting an antisense oligonucleotide, and is represented by the following formula. be able to. (Match ratio = (the number of nucleotides constituting the antisense oligonucleotide−the number of mismatched nucleotides) / (the number of nucleotides constituting the antisense oligonucleotide) × 100) In the present invention, unless otherwise specified, the oligonucleotides are naturally occurring. The method described in the literature (Nucleic Acids Research, 12, 4539 (1984)) using a DNA synthesizer, for example, a model 392 based on the phosphoramidite method of PerkinElmer, in addition to those having a phosphoric acid ester bond of the type nucleotide. Also includes those synthesized and modified according to In the present invention, the oligonucleotide base sequence having complementarity means that the base sequence of the complementary strand of one oligonucleotide and the base sequence of the other oligonucleotide completely match in the corresponding portion, or partially match. Even if there are no sequences, those that form base pairs under incubation conditions are also included.

Phosphoramidite reagents used in the phosphoramidite method, the natural nucleosides and 2'-O- methyl guanosine (G ^m) can be used commercially available reagents. The non-natural phosphoramidite is described in Example 5 of JP-A-2000-297097 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-4-N-benzoylcytidine-3'-O-). (2-cyanoethyl N, N-diisopropyl) phosphoramidite), Example 9 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-5-methyluridine-3'-O- ( 2-cyanoethyl N, N-diisopropyl) phosphoramidite, Example 14 (5′-O-dimethoxytrityl-2′-O, 4′-C-ethylene-6-N-benzoyladenosine-3′-O- ( 2-cyanoethyl N, N-diisopropyl) phosphoramidite, or 5′-O-dimethoxytrityl-2′-O-methyl-2-N-isobutyrylphenoxyacetylguanosine-3′-O- (2 -Cyanoethyl N, N-diisopropyl) phosphoramidite (Pharmacia) For other nucleosides, WO99 / 14226 for nucleosides where l, m, n, o and p are 1 and WO 00/47599 for nucleosides where l, m, n, o and p are 2 to 5. It can be obtained according to the method described.

If desired, if thioate is used, a reagent that reacts with trivalent phosphorous acid such as tetraethylthiuram disulfide (TETD, Applied Biosystems) or Beaucage reagent (Glen Research) in addition to sulfur to form thioate And a thioate derivative can be obtained according to the method described in the literature (Tetarhedron Letters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 1253 (1990)). Modified oligonucleotides described in the literature (SM Freier et al, Nucleic Acids Res., 25, 4429 (1997)) can also be synthesized by the method cited in the literature.
1. Determination of Target Gene The antisense compound suppresses the expression of the target protein by forming a Watson-Crick base pair with the mRNA of the target gene. The method for designing an antisense compound of the present invention is applicable to any gene. First, a target gene is determined, and an oligonucleotide having an antisense effect on the target gene is selected. The antisense compound may be an oligonucleotide that is already known to have an effect of suppressing the expression of the target gene. Even if the base sequences of the parts are different, they may have an antisense effect. Alternatively, an oligonucleotide whose antisense effect has not been confirmed in advance may be used as long as it is an oligonucleotide uniquely determined to determine a target gene and have a sequence complementary to the base sequence of mRNA of the gene. The oligonucleotide usually consists of 10 to 50 nucleotides, preferably 15 to 25 nucleotides.

The nucleotide sequence of the oligonucleotide having an antisense effect used in this step can be determined by a known method based on the nucleotide sequence of the target gene, for example, by mixing a random short-chain DNA fragment with the RNA of the target gene, RNase H (RNase H), and the cleavage site is converted to an antisense sequence (Lloyd, BH et al. (2001) Nucleic Acids Res. 29, 3664-3673. Ho, SP et al. (1996) Nucleic Acids Res. 24, 1901-1907. Matveeva, O. (1997) Nucleic Acids Res. 25, 5010-5016.), Random short DNA fragment and RNA of target gene were mixed, and the bound product was separated by gel shift to determine the binding site. (Bruce, TW and Lima, WF (1997) Biochemistry, 36, 5004-5019., Bruce, TW and Lima, WF US patent, 6,022,691.), A method of preparing a 20-mer oligonucleotide having an antisense sequence into a glass Create an array immobilized on a plate, etc. A method in which hybridization is performed using RNA of a target gene labeled with a sotope or the like to determine a sequence to which RNA binds (Sohail, M. et al. (2001) Nucleic Acids Res. 29, 2041-2051), etc. Can also be determined. The nucleotide sequence of an oligonucleotide having an antisense sequence can also be determined using a computer program (Scherr, M. (2000) Nucleic Acids Res. 28, 2455-2461., Patzel et al. (1999). Nucleic Acids Res. 27, 4328-4334).

For example, when the target gene is VEGF, the following nucleotide sequence can be input as the oligonucleotide nucleotide sequence.
5'-cccaagacagcagaaagttcat-3 '(SEQ ID NO: 1 in Sequence Listing)
2. Measurement of gene expression level Whether or not the added oligonucleotide actually has a gene expression suppressing effect can be confirmed by measuring the expression level of the target gene. The expression level of the target gene can be measured by a well-known method. Also, by measuring the expression level of the polypeptide corresponding to the target gene, it can be confirmed whether or not the added oligonucleotide has the effect of suppressing the expression of the target gene. Hereinafter, a method for measuring the gene expression-suppressing effect of the oligonucleotide will be described, divided into a method for measuring the expression level of the gene and a method for measuring the expression level of the polypeptide.

A. Method for Measuring Gene Expression Level The following method will be described as a method for measuring the gene expression level, but is not limited to these methods as long as the gene expression level can be measured.

When extracting total RNA, cells washed with a medium containing no oligonucleotide are directly lysed with a solvent for RNA extraction (for example, phenol or the like containing a component having an activity of inactivating ribonuclease). Is preferred. Alternatively, the cells in the tissue are carefully scraped with a scraper or gently extracted from the tissue using a protease such as trypsin so as not to destroy the cells in the tissue. Then, the process proceeds to the RNA extraction step.

RNA extraction methods include guanidine thiocyanate / cesium chloride ultracentrifugation, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N., ( 1987) Anal. Biochem., 162, 156-159), but the guanidine acid thiocyanate-phenol-chloroform method is preferred. In addition, commercially available reagents for RNA extraction (for example, ISOGEN (manufactured by Nippon Gene Co., Ltd.), TRIZOL reagent (manufactured by Gibco BRL) or RNA-Bee (manufactured by Tel-Test, Inc.)) are attached to the reagents. It can also be used according to the protocol. The sample containing the total RNA thus obtained is referred to as a total RNA fraction.

全 It is preferable that the obtained total RNA be further purified only to mRNA if necessary. Although the purification method is not particularly limited, it is known that many mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3′-terminal. The mRNA is adsorbed on the oligo (dT) probe thus obtained, and the mRNA is captured on the paramagnetic particles on which streptavidin is immobilized using the binding between biotin / streptavidin, and after washing, the mRNA is eluted. , MRNA can be purified. Alternatively, a method in which mRNA is adsorbed on an oligo (dT) cellulose column and then eluted and purified may be employed. Further, mRNA can be further fractionated by sucrose density gradient centrifugation or the like.

Incidentally, only the mRNA can be purified without going through the step of extracting the total RNA from the cultured cells. For example, using a commercially available mRNA extraction kit (Quick Prep Micro mRNA Purification Kit; Amersham Pharmacia Biotech Co., Ltd.), mRNA can be directly purified from cultured cells.

(1) Method using nucleic acid hybridization i) Immobilization of RNA Using nucleic acid hybridization, total RNA sample obtained from cultured cells cultured with addition of oligonucleotides or mammalian individuals to which oligonucleotides were administered Northern blotting, dot blotting and slot blotting can be used to specifically observe the expression level of the target gene in the cells. Examples of a membrane for transferring or blotting RNA include a nitrocellulose membrane (for example, Hybond-C Pure (manufactured by Amersham Pharmacia)) and a positively charged nylon membrane (for example, Hybond-N + (manufactured by Amersham Pharmacia)). Or a hydrophilic nylon membrane (for example, High Bond-N / NX (manufactured by Amersham Pharmacia)).

Agarose gel electrophoresis for Northern blots includes agarose formamide gel electrophoresis, a method in which a sample is treated with glyoxal and dimethyl sulfoxide, denatured, and then electrophoresed on an agarose gel prepared with a phosphate buffer, and agarose gel. Examples include, but are not limited to, methylmercury electrophoresis (Maniatis, T. et al. (1982) in "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory, NY.).

As a so-called blotting method for transferring RNA from the gel after electrophoresis to the membrane, a capillary transfer method (Maniatis, T. et al. (1982) in “Molecular Cloning A Laboratory Manual” Cold Spring Harbor Laboratory, NY.), Vacuum And electrophoresis (Maniatis, T. et al. (1989) in "Molecular Cloning A Laboratory Manual, 2nd ed", Cold Spring Harbor Laboratory, NY.). Equipment for dot blotting and slot blotting is also commercially available (for example, Biodot (manufactured by Bio-Rad)).

After blotting, perform the operation of fixing the RNA transferred to the membrane to the membrane (this operation differs depending on the material of the membrane, and depending on the product, the fixing operation may not be necessary).

ii) Labeling and detection of probe In the detection by nucleic acid hybridization, a labeling method and a detection method of a probe for detecting a specific mRNA in the RNA sample immobilized as described above are described below. :
a) Radioisotope labeling A nick translation method (for example, using a nick translation kit (manufactured by Amersham Pharmacia) or the like) using a DNA fragment or a vector or the like carrying the same as a material or template, a random prime method (for example, Multi-prime DNA labeling system (Amersham Pharmacia) or the like), end labeling method (for example, Mega Label (Takara Shuzo Co., Ltd.), 3'-end labeling kit (Amersham Pharmacia) or the like) To prepare a labeled DNA probe, or a labeled RNA probe by an in vitro transcription method using an SP6 promoter or a T7 promoter in a vector into which a template DNA has been subcloned. Detection of these probes can be carried out by autoradiography using an X-ray film or an imaging plate. In the case of an X-ray film, densitometry (for example, GS-700 imaging densitometer (manufactured by Bio-Rad)) In the case of an imaging plate, quantification using a BAS2000II (manufactured by Fuji Film) system is also possible.

b) Enzyme labeling DNA or RNA fragments are directly enzyme-labeled. Enzymes used for labeling include, for example, alkaline phosphatase (using AlkPhos Direct system for chemiluminescence (manufactured by Amersham Pharmacia)), horseradish peroxidase (Horseradish Peroxidase) (ECL Direct Nucleic Acid Labeling and Detection, Inc.). System (Amersham Pharmacia) or the like). The detection of the probe is performed by immersing the membrane in an enzyme reaction buffer containing a substrate capable of detecting the catalytic reaction of the labeled enzyme, for example, a chromogenic substance or a substrate that emits light by the catalytic reaction. . When a chromogenic substrate is used, it can be detected visually, and when a luminescent substrate is used, autoradiography using an X-ray film or an imaging plate or a photograph using an instant camera as in the case of radioisotope labeling It can be detected by photographing. Furthermore, when a luminescent substrate is used, quantification using densitometry or a molecular imager Fx (manufactured by Bio-Rad) can be performed.

c) Labeling with other molecules Fluorescein labeling: DNA fragments are labeled by a nick translation method, a random prime method, or a 3 ′ end labeling method (such as ECL 3′-oligolabeling system commercially available from Amersham Pharmacia). Alternatively, label the RNA by in vitro transcription with the SP6, T7 promoter;
Biotin labeling: Labeling the 5 'end of DNA (using an oligonucleotide biotin labeling kit commercially available from Amersham Pharmacia) or labeling a DNA fragment by a nick translation method, a random prime method, or the like;
Digoxigenin-modified dUTP labeling: A DNA fragment is labeled by a nick translation method, a random prime method, or the like.

検 出 Detection of these labeled molecules includes, in each case, an operation of binding a molecule that specifically binds to the labeled molecule, labeled with a radioisotope or an enzyme, to a probe. Specific binding molecules include, for example, an anti-fluorescein antibody or an anti-digoxigenin antibody in the case of fluorescein or digoxigenin, and avidin or streptavidin in the case of biotin. After these are bound to the probe, detection can be carried out using the labeled radioisotope or enzyme according to the same method as described in a) or b) above.

iii) Hybridization and detection Hybridization can be performed by a method well known in the art to which the present invention belongs. The relationship between the composition of the hybridization solution or the washing solution and the hybridization temperature or the washing temperature in the present invention can be in accordance with, for example, the description in the literature (Bio-Experiment Illustrated 4, p148, published by Shujunsha). Is as follows:
Hybridization solution: ExpressHyb Hybridization Solution (Clontech);
Final concentration of probe (for radiolabeled probes): 1 to 2 × 10 ⁶ cpm / ml (preferably 2 × 10 ⁶ cpm / ml);
Hybridization temperature and time: 68 ° C, 1 to 24 hours.

Cleaning conditions for the membrane:
a) 0.1-5 × SSC (most preferably 2 × SSC), 0.05-0.1% SDS (most preferably 0.05%) in sodium dodecyl sulfate (hereinafter “SDS”) at room temperature to Shaking at 42 ° C. (most preferably room temperature) for 20 to 60 minutes (most preferably 20 minutes) is performed 2 to 6 times (most preferably 3 times) with the exchange of the washing solution;
b) After the above a), the operation of shaking in 0.1 × SSC, 0.1% SDS at 50 to 65 ° C. for 20 to 60 minutes is performed 2 to 6 times by changing the washing solution. Alternatively, the operation of shaking in 0.2 to 0.5 × SSC, 0.1% SDS at 62 to 65 ° C. for 20 to 60 minutes is performed 2 to 6 times by changing the washing solution. Most preferably, the operation of shaking in 0.1 × SSC, 0.1% SDS at 50 ° C. for 20 minutes is performed three times by changing the washing solution.

(4) After the completion of washing, detection and quantification according to the labeling method of the probe are performed as described in 7) above. Also, since the expression level of the β-actin gene per cell is known to be stable, the β-actin gene in each sample was corrected for the purpose of correcting the variation caused by the difference in the amount of RNA between the samples. The expression level of the gene was measured at the same time, and the relative value of the expression level of the gene to be detected (oligonucleotide) based on the β-actin expression level was compared with the cell group to which the test substance was added and the cell group to which no test substance was added. By making a comparison between and, a more precise evaluation can be performed.

As a result, the compound that reduces the expression level of the target gene can be said to have an antisense effect, and can be an oligonucleotide that is an antisense compound for the target gene.

(2) RT-PCR
In another embodiment of the present invention for detecting a nucleic acid, a reverse transcriptase reaction using mRNA as a template is first performed, and then PCR is performed to specifically amplify a DNA fragment, that is, so-called RT-PCR is performed. Is the way. In this method, in order to specifically amplify a target nucleotide sequence, an antisense primer complementary to a specific partial sequence of the target mRNA and a cDNA sequence generated by the reverse transcriptase from the antisense primer are used. A sense primer complementary to a specific partial sequence therein is used. RT-PCR can be performed using a commercially available kit (for example, RNA PCR kit AMV ver2.1 (Takara Shuzo), Ready to Go RT-PCR Beads (Amersham Pharmacia Biotech), etc.) according to the manual attached to the kit. The following method is also possible.

The antisense primer used in both the reverse transcriptase reaction and the PCR is substantially 15 to 40 nucleotides, preferably at least 18 to 35 nucleotides, more preferably in the antisense sequence of the base sequence of the target gene. Consists of a nucleotide sequence of 21 to 30 nucleotides.

On the other hand, the sequence of the sense primer used in the PCR is more 5'-terminal than the most 5'-terminal position in the sequence corresponding to the complementary strand of the antisense primer in the base sequence of the target gene of the oligonucleotide of the present invention. The sequence consists of any partial sequence of 15 to 40 nucleotides, preferably 18 to 35 nucleotides, more preferably 21 to 30 nucleotides in the sequence present in the region. However, if the sense primer and the antisense primer have mutually complementary sequences, annealing of the primers will amplify non-specific sequences, which may hinder specific gene detection. It is preferable to design primers that avoid various combinations.

Both the antisense primer and the sense primer may have a base sequence unrelated to the base sequence of the target gene of the oligonucleotide added as a linker at the 5 'end of the base sequence defined above. However, it is preferable that the linker does not cause non-specific annealing with the nucleic acid in the reaction solution during the reaction so as not to hinder the specific gene detection.

The reaction conditions for RT-PCR are as described below. In addition, the sample for detection by RT-PCR does not always need to be usually purified to poly (A) ⁺ RNA.

i) Reverse transcriptase reaction Example of reaction solution composition (total volume 20 μl):
Total RNA as appropriate;
Magnesium chloride 2.5 to 5 mM (preferably 5 mM);
1 × RNA PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 (preferably 8.3) at 25 ° C., 50 mM potassium chloride));
dNTPs 0.5 to 1 mM (preferably 1 mM);
Antisense primer 1 μM (2.5 μM of a commercially available random primer or oligo (dT) primer (12-20 nucleotides) can be added as a substitute for the antisense primer);
Reverse transcriptase 0.25 to 1 unit / μl (preferably 0.25 unit / μl);
Adjust to 20 μl with sterile water.

Reaction temperature conditions:
After incubating at 30 ° C for 10 minutes (only when using a random primer), incubating at 42 to 60 ° C (preferably 42 ° C) for 15 to 30 minutes (preferably 30 minutes), and further heating at 99 ° C for 5 minutes, the enzyme And then cooled at 4-5 ° C (preferably 5 ° C) for 5 minutes.

ii) PCR
Example of reaction solution composition:
Magnesium chloride 2 to 2.5 mM (preferably 2.5 mM);
1 × PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 (preferably 8.3) at 25 ° C.)), 50 mM potassium chloride;
dNTPs 0.2 to 0.25 mM (preferably 0.25 mM);
Antisense and sense primers 0.2-0.5 μM (preferably 0.2 μM);
1 to 2.5 units (preferably 2.5 units) of Taq polymerase;
The total volume is adjusted to 80 μl by adding sterile water, and the total volume is added to the total volume of the reaction solution after the completion of the reverse transcription reaction before PCR is started.

Reaction temperature conditions: First, heating at 94 ° C. for 2 minutes, and then at 90 to 95 ° C. (preferably 94 ° C.) for 30 seconds, 40 to 60 ° C. (preferably, the dissociation temperature (Tm) calculated from the characteristics of the primer) A temperature cycle of 70 to 75 ° C. (preferably 72 ° C.) for 1.5 seconds at 28 to 50 cycles (preferably 28 cycles) at a temperature lower than 20 ° C. for 30 seconds, and then cooling to 4 ° C. I do.

(4) After the PCR is completed, the reaction solution is subjected to electrophoresis to detect whether or not a band of a desired size has been amplified. In order to perform quantitative detection, PCR is performed under the same conditions using a serially diluted cDNA clone as a standard template DNA, and the number of temperature cycles at which quantitative detection can be performed is determined. A part of the reaction solution is sampled and subjected to electrophoresis. Also, for example, by using radiolabeled dCTP during the PCR reaction, quantification can be performed using the amount of radioactivity incorporated in the band as an index. As methods for improving the reliability of gene quantification, competitive RT-PCR methods (Souaze et al., BioTechniques 21, 280-285 (1996)), and TaqMan @ PCR methods (Heid et al. , Genom. Res. 6, 986-994 (1996)).

The detection results were compared between a sample derived from cells cultured in the presence of the oligonucleotide and a sample derived from cells cultured in the absence of the oligonucleotide. It is a substance that suppresses the expression and can be an antisense compound against the target gene. By suppressing the expression of the target gene, it can be a therapeutic drug for a disease caused by an increase in the expression level of the target gene.

(3) Analysis method using immobilized sample i) Immobilized sample Examples of the immobilized sample include the following.
a) Gene chip:
A gene chip on which an antisense oligonucleotide synthesized based on an EST (expressed sequence tag) sequence or an mRNA sequence on a database is immobilized can be used. As such a gene chip, a gene chip manufactured by Affymetrix (Lipshutz, RJ et al. (1999) Nature genet. 21, suppliment, 20-24) can be used. It may be produced based on the method. It is most preferable that the EST sequence or mRNA sequence serving as the base of the antisense oligonucleotide immobilized on the gene chip be derived from an animal of the same species as the animal or animal cell to be analyzed. When a mouse or a mouse-derived cultured cell is used, a mouse-derived cell is desirable, and when a human specimen or a human cultured cell is used, a human-derived cell is desirable. When analyzing mRNA derived from a KK mouse, those derived from a mouse are most preferable. ) Can be used. When analyzing mRNA derived from human white adipocytes, those derived from human are preferable. For example, human U95 set or U133 set manufactured by Affymetrix can be used. However, the present invention is not limited thereto, and for example, those derived from animals of closely related species can also be used. Closely related species include, for example, mammals, and include, but are not limited to, combinations of humans and mice and humans and monkeys.

b) Array or membrane filter on which cDNA or RT-PCR product prepared from mouse or human, total RNA or total RNA obtained from a specific tissue is immobilized:
The cDNA or RT-PCR product is cloned by performing a reverse transcriptase reaction or PCR using primers prepared based on sequence information such as a mouse or human EST database. As the array or the filter, a commercially available one (for example, Inteligene: manufactured by Takara Shuzo Co., Ltd.) may be used. It may be produced by using the solid phase.

ii) Preparation and analysis of probe Not a specific mRNA clone but a label of all expressed mRNAs is used as a labeled probe. Unpurified mRNA may be used as a starting material for preparing the probe, but it is preferable to use poly (A) ⁺ RNA purified by the above-described method. Hereinafter, a method for preparing a labeled probe and a method for detecting and analyzing a labeled probe when various types of immobilized samples are used will be described.

a) Gene chip manufactured by Affymetrix:
A biotin-labeled cRNA probe is prepared according to the protocol (Affymetrix Expression Analysis Technical Manual) attached to the gene chip manufactured by Affymetrix. Next, hybridization and analysis were performed using an analysis device (GeneChip Fluidics Station 400) manufactured by Affymetrix in accordance with the protocol (expression analysis technology manual) attached to the gene chip manufactured by Affymetrix, and luminescence by avidin was detected and analyzed. Do.

b) Array:
When cDNA is prepared from poly (A) ⁺ RNA by the reverse transcriptase reaction, it is necessary to label the cDNA so that the cDNA can be detected. For example, cDNA is fluorescently labeled by adding d-UTP or the like labeled with Cy3, Cy5, or the like. In this case, it is used as a mixture of both the if and labeled from oligonucleotide added cell poly (A) + ^RNA to control cells derived from poly (A) + ^RNA with different dyes, after hybridization when it can. For example, when a commercially available array of Takara Shuzo Co., Ltd. is used as the array, hybridization and washing are performed according to the protocol of the company, and the fluorescence signal is detected by a fluorescent signal detector (for example, GMS418 array scanner (Takara Shuzo Co., Ltd.) or the like). After detecting, analysis is performed. However, the array to be used is not limited to a commercially available array, and may be a homemade array or a specially manufactured array.

c) Membrane filter:
When preparing cDNA from poly (A) ⁺ RNA using reverse transcriptase, a labeled probe is prepared by adding a radioisotope (for example, d-CTP, etc.), and hybridization is performed by a conventional method. After hybridization and washing are performed using an atlas system (manufactured by Clontech), which is a filter microarray, detection and analysis are performed using an analyzer (for example, Atlas image: manufactured by Clonetech).

も In any of the methods a) to c), the probe derived from the target gene is hybridized to the solid-phased sample of the same lot. At this time, the conditions other than the hybridization other than the probe to be used are the same. In the case of using fluorescently labeled probes, if each probe is labeled with a different fluorescent dye, a mixture of both probes can be hybridized to one immobilized sample at a time, and the fluorescence intensity can be read (Brown, PO et al. (1999) Nature genet, 21, supplement, 33-37).

(4) Other Methods Other methods for measuring the effect of the oligonucleotide on the expression level of the target gene include those described below.

i) RNase protection assay: Labeled probe only in mRNA having the nucleotide sequence described in any one of 1) to 5) above (where t in the sequence is replaced by u) in the RNA sample After hybridizing to form a double-stranded oligonucleotide and incubating the sample with ribonuclease, the probe-hybridized mRNA is not digested by ribonuclease due to its double-stranded form. Since the other RNA is digested, only the double-stranded oligonucleotide remains (if the probe is shorter than the mRNA to be detected, the double-stranded oligonucleotide corresponding to the length of the probe remains). By quantifying the double-stranded oligonucleotide, the expression level of the target gene is measured. Specifically, for example, the method described below is used.

In order to easily separate the surplus labeled probe without forming a double strand from the double-stranded oligonucleotide to facilitate quantification, the surplus labeled probe is preferably digested with ribonuclease. The labeling probe may be DNA or RNA as long as it is capable of digesting the main-stranded DNA. The method for preparing a labeled probe can be performed according to the method described in the section “(ii) {Labeling and detection of probe” ”in the above section“ (1) Method using nucleic acid hybridization ”, but in the case of ribonuclease assay. The length of the probe used is preferably about 50 to 500 nucleotides. Further, a probe in which complementary strands are mixed, such as a probe obtained by directly labeling a double-stranded DNA and thermally denaturing it, is not suitable for this method.

RNA probes are prepared, for example, according to the following method. First, the template DNA is inserted into a plasmid vector (for example, pGEM-T (promega)) having a bacteriophage promoter (T7, SP6, T3 promoter, etc.). Next, this recombinant plasmid vector is digested with a restriction enzyme so that it is cleaved only one point immediately downstream of the inserted fragment. Using the obtained linear DNA as a template, an in vitro transcription reaction is performed in the presence of radiolabeled ribonucleotides. In this reaction, an enzyme such as T7, SP6 or T3 polymerase is used in accordance with the promoter in the vector. The above operation can be performed using, for example, Riboprobe System-T7, -SP6 or -T3 (all manufactured by Promega).

It is desirable that total RNA extracted from cells cultured with the addition of the oligonucleotide be further purified and used as mRNA. A ribonuclease protection assay is performed using 10 to 20 μg of the prepared total RNA sample and an excess amount of the labeled probe corresponding to 5 × 10 ⁵ cpm. This operation can be performed using a commercially available kit (HybSpeed RPA Kit, manufactured by Ambion). The obtained ribonuclease digested sample is electrophoresed on a 4 to 12% polyacrylamide gel containing 8 M urea, the gel is dried, and autoradiography is performed with an X-ray film. By the above operation, the band of the double-stranded oligonucleotide which escaped the ribonuclease digestion can be detected, and its quantification can be determined by the method described in “(1) Method using nucleic acid hybridization” in the section “(iii) Hybridization”. And detection ”. Further, as in the case of the Northern blot analysis, if the expression level of the β-actin gene is simultaneously measured for the purpose of correcting the variation caused by the difference in the RNA amount between the samples, more precise evaluation can be performed. be able to.

In this manner, the detection results were compared between a sample derived from cells cultured with the addition of the oligonucleotide and a sample derived from cells cultured in the absence of the oligonucleotide, and the expression level of the target gene was reduced. Oligonucleotides can be antisense compounds for target genes and can be therapeutic agents for diseases caused by increased expression of target genes.

ii) Run-on assay, Greenberg, ME and Ziff, EB
B. (1984) Nature 311, 433-438, and Groudine, M. et al. (1981) Mol. Cell Biol. 1, 281-288): This method involves isolating the nucleus from a cell and generating the gene of interest. This is a method for measuring the transcriptional activity of, and is not a method for detecting intracellular mRNA as described above, but is included in the "method for detecting the expression level of a gene" in the present invention. When a transcription reaction is performed in a test tube using the isolated cell nucleus, only the reaction in which transcription is already started before the nucleus is isolated and the one where the mRNA chain is being generated elongates proceeds. I do. At the time when the nucleus was isolated by adding a radiolabeled ribonucleotide during this reaction to label the elongating mRNA and detecting the mRNA contained therein that hybridized to the unlabeled probe. The transcription activity of the target gene in can be measured. In order to use the data of the time at which the influence of the test substance appears most remarkably, the time from the addition of the test substance to the cultured cells to the isolation of the nucleus, for example, 30 minutes after addition, 1 hour after, 2 hours , 4 hours, 8 hours, and 24 hours later, the nuclei are isolated from the cells and assayed, respectively. The specific operation method is the same as that described in the above-mentioned reference except that a probe that is not labeled is prepared according to the description in the above (1). In this manner, the detection results were compared between a sample derived from cells cultured in the presence of the oligonucleotide and a sample derived from cells cultured in the absence of the oligonucleotide, and the oligos with reduced transcription activity of the target gene were compared. The nucleotide decreases the expression level of the target gene and can be a therapeutic or preventive agent for a disease caused by an increase in the expression level of the target gene.
B. Method for Measuring Polypeptide Expression A method using an antibody will be described as a method for measuring the gene expression suppressing effect of an oligonucleotide. First, the polypeptide in the sample is immobilized on the inner bottom of a well of a multi-well plate such as a 96-well plate or a 384-well plate, or on a membrane, and then an antibody that specifically recognizes the target gene product of the oligonucleotide is obtained. The used detection is performed. Among them, a multi-well plate such as a 96-well plate or a 384-well plate is generally used as a method called an enzyme-linked immunosorbent assay (ELISA) or a radioisotope immunoassay (RIA). On the other hand, as a method for immobilizing a sample on a membrane, a method of transferring a polypeptide to a membrane via polyacrylamide gel electrophoresis of a sample (Western blotting method), or a method of directly impregnating a sample or a dilution thereof into the membrane, a so-called method. The dot blot method and the slot blot method are mentioned.

(1) Preparation of sample The sample is preferably human whole blood, serum, liver or cultured cells. The whole blood, liver or adipose tissue extract is subjected to high-speed centrifugation as necessary to remove insoluble substances, and then prepared as a sample for ELISA / RIA or a sample for western blot as follows.

As a sample for ELISA / RIA, for example, the collected serum, liver or cultured cell extract is used as it is, or a sample appropriately diluted with a buffer is used. The sample for Western blotting (for electrophoresis) is, for example, a sample containing 2-mercatorethanol for SDS-polyacrylamide gel electrophoresis, using serum, liver or cultured cell extract as it is, or appropriately diluting with a buffer solution. Mix with a buffer (eg, Sigma). In the case of the dot / slot blot, for example, the collected serum, liver or cultured cell extract itself, or appropriately diluted with a buffer solution is directly adsorbed to the membrane using a blotting device or the like.

(2) Immobilization of Sample In order to specifically detect the polypeptide in the sample obtained as described above, the sample is precipitated by immunoprecipitation, a method utilizing ligand binding, or the like. Detection can be performed without immobilization, or the sample to be detected can be immobilized as it is. When the polypeptide is immobilized, a nitrocellulose membrane (for example, manufactured by Bio-Rad Co., Ltd.), a nylon membrane (for example, Hybond-ECL ( Amersham Pharmacia), a cotton membrane (for example, a blot absorbent filter (manufactured by Bio-Rad), etc.) or a polyvinylidene difluoride (PVDF) membrane (for example, manufactured by Bio-Rad, etc.).

As a so-called blotting method for transferring the polypeptide from the gel after electrophoresis to the membrane, a wet blotting method (CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 ed by JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strober), semi-drying Expression blotting (see CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 above) and the like. Equipment for dot blotting and slot blotting is also commercially available (for example, Biodot (Biorad)).

On the other hand, in order to perform detection and quantification by the ELISA method / RIA method, a sample or a diluent thereof (for example, 0 μl plate) (for example, Immunoplate Maxisorp (manufactured by Nunc)) or a 384-well plate is required. 0.05% sodium azide in phosphate-buffered saline (hereinafter, referred to as “PBS”) and allowed to stand at 4 ° C. to room temperature overnight or at 37 ° C. for 1 to 3 hours. Then, the polypeptide is adsorbed on the inner bottom surface of the well and solidified.

(3) Antibody The antibody used specifically recognizes the target gene product of the oligonucleotide or a part thereof. Such antibodies include, for example, antibodies that bind to any of the target gene product polypeptides of the oligonucleotides of the present invention, but do not bind to any other mouse or human protein.

The antibody can be prepared by subjecting an animal to a protein serving as an antigen or an arbitrary polypeptide selected from the amino acid sequence of the antibody using standard methods (for example, Shinsei Kagaku Koza Kozai, Protein 1, p. 389-397, 1992). It can be obtained by immunizing, collecting and purifying an antibody produced in the living body. Also, according to a known method (for example, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody p. 365-367, 1980, Prenum Press, NY), the target gene of the oligonucleotide is used. A hybridoma can be established by fusing an antibody-producing cell that produces an antibody against the product protein with a myeloma cell to obtain a monoclonal antibody.

As an antigen for producing an antibody, a polypeptide of a target gene product of an oligonucleotide or a polypeptide comprising at least six consecutive partial amino acid sequences thereof, or a derivative obtained by adding an arbitrary amino acid sequence or a carrier to these polypeptides is used. Can be mentioned.

The antigen polypeptide can be obtained by synthesizing a polypeptide encoded by the target gene of the oligonucleotide in vitro, or by producing it in a host cell by genetic manipulation. Specifically, after incorporating the target gene into a vector capable of expressing the polypeptide that is the product of the oligonucleotide target gene, the target gene is synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other The target polypeptide can be obtained by transforming a prokaryotic or eukaryotic host cell to express the target gene.

InIn vitro synthesis of the polypeptide includes, for example, Rapid Translation System (RTS) manufactured by Roche Diagnostics Co., Ltd., but is not limited thereto. For example, when RTS is used, the gene of interest is cloned into an expression vector controlled by a T7 promoter and added to an in vitro reaction system. First, mRNA is transcribed from a template DNA by T7 RNA polymerase, Thereafter, translation is performed by ribosomes or the like in an Escherichia coli lysate, and the target polypeptide is synthesized in the reaction solution (Biochemica, 1, 20-23 (2001), Biochemica, 2, 28-29 (2001)).

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cells are transformed with a plasmid vector that contains replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

For example, K12 strain or the like is often used as Escherichia coli, and pBR322 or a pUC-type plasmid is generally used as a vector, but is not limited thereto, and any of various known strains and vectors can be used.

Examples of the promoter include, in Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, and the like. Any promoter can be used to produce the polypeptide of interest.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used, but it is not limited thereto. is not. By ligating a DNA sequence encoding the signal peptide sequence of Bacillus subtilis α-amylase, extracellular secretory expression becomes possible.

Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include monkey COS cells (Gluzman, Y. (1981) Cell 23, 175- 182, ATCC @ CRL-1650) or a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells, ATCC @ CCL-61) (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA) 77, 4126-4220) and the like are often used, but are not limited thereto.

As a vertebrate cell expression promoter, a promoter having an upstream site of a gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used. May be provided. Examples of the expression vector include pCR3.1 (Invitrogen) having a cytomegalovirus early promoter and pSV2dhfr having an SV40 early promoter (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864), but are not limited thereto.

As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and further has a transcription promoter, a transcription termination signal, and an RNA splice site. Can be used. The expression vector is prepared by a diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308), a calcium phosphate-DNA coprecipitation method (Graham, FL and van der). Eb, AJ (1973) Virology 52, 456-457), and electric pulse perforation (Neumann, E. et al. (1982) EMBO J. 1, 841-845), etc. Thus, a desired transformed cell can be obtained. When CHO cells are used as host cells, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, J. et al. (1989): “Molecular Cloning A Laboratory Manual “Cold Spring Harbor Laboratory, NY” or pSV2neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341), etc. By selecting a colony, a transformed cell stably producing the desired polypeptide can be obtained.

When insect cells are used as host cells, ovarian cell-derived cell lines (Sf-9 or Sf-21) of Spodoptera frugiperda of the Lepidoptera Noctuidae and High Five cells derived from Trichoplusia ni egg cells (Wickham, TJ et al, ( 1992) Biotechnol. Prog. I: 391-396) is often used as a host cell, and as a baculovirus transfer vector, pVL1392 / 1393 using a polyhedrin protein promoter of autographa nucleopolyhedrovirus (AcNPV) is often used. (Kidd, IM and VC Emery (1993) The use of baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 42, 137-159). In addition, vectors using baculovirus P10 or a promoter of the same basic protein can also be used. Furthermore, the recombinant protein can be expressed as a secreted protein by linking the secretory signal sequence of the envelope surface protein GP67 of AcNPV to the N-terminal side of the target protein (Zhe-mei Wang, et al. (1998)). Biol. Chem., 379, 167-174).

Yeast is generally well known as an expression system using eukaryotic microorganisms as host cells, and among them, yeast of the genus Saccharomyces, such as baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris, are preferred. Examples of expression vectors for eukaryotic microorganisms such as the yeast include an alcohol dehydrogenase gene promoter (Bennetzen, JL and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025) and an acid phosphatase gene. A promoter (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5) can be preferably used. When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site of an endogenous protease or a known protease possessed by the host cell at the N-terminal side. For example, in a system in which human mast cell tryptase of a trypsin-type serine protease is expressed in petroleum yeast, the activity is achieved by connecting the secretory signal sequence of α-factor of yeast and the cleavage site of KEX2 protease possessed by petroleum yeast to the N-terminal side and expressing it. It is known that type tryptase is secreted into the medium (Andrew, L. Niles, et al. (1998) Biotechnol. Appl. Biochem. 28, 125-131).

形 質 The transformant obtained as described above can be cultured according to a conventional method, and the culture produces the target polypeptide inside or outside the cell. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used. For example, in the case of the above-mentioned COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) ), To which a serum component such as fetal bovine serum may be added as necessary.

Recombinant proteins produced in the cells of the transformants or outside the cells by the above culture can be separated and purified by various known separation procedures utilizing the physical properties and chemical properties of the proteins. it can. As the method, specifically, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography ( Examples thereof include various liquid chromatography such as HPLC, dialysis, and combinations thereof. In addition, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column. By combining the above methods, the polypeptide of the present invention can be easily produced in large quantities with high yield and high purity.

抗体 The antibody obtained as described above can be used for various immunological measurement methods such as RIA, ELISA, fluorescent antibody method, passive hemagglutination, immunohistological staining, and the like.

(4) Detection The antibody obtained by the method described in (3) above can be directly labeled, or can be used as a primary antibody to specifically recognize the antibody (antibody derived from the animal in which the antibody is produced is used). It is used for detection in cooperation with a labeled secondary antibody.

Preferred examples of the type of label include, but are not limited to, an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin (however, an operation of further binding enzyme-labeled streptavidin to biotin of the secondary antibody is added). Various pre-labeled antibodies (or streptavidin) are commercially available for methods that use labeled secondary antibodies (or labeled streptavidin). In the case of RIA, an antibody labeled with a radioisotope such as I ¹²⁵ is used, and the measurement is performed using a liquid scintillation counter or the like.

量 By detecting the activity of these labeled enzymes, the amount of the polypeptide which is the antigen is measured. In the case of alkaline phosphatase or horseradish peroxidase, substrates that emit color or emit light by the catalyst of those enzymes are commercially available.

場合 When a substrate that develops color is used, it can be visually detected by Western blotting or dot / slot blotting. In the ELISA method, quantification is preferably performed by measuring the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader. Also preferably, a dilution series of the antigen used for the production of the antibody in the above (3) is prepared, and this is used as a standard antigen sample, and the detection operation is performed simultaneously with other samples, and the standard antigen concentration and the measured value are plotted. By creating a curve, it is possible to quantify the antigen concentration in another sample.

On the other hand, when a substrate that emits light is used, it can be detected by Western radiography or dot / slot blotting by autoradiography using an X-ray film or an imaging plate, or photography using an instant camera, Quantitation using densitometry, Molecular Imager Fx system (manufactured by Bio-Rad), or the like is also possible. When a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).

(5) Measurement operation i) In the case of Western blot, dot blot or slot blot First, in order to prevent nonspecific adsorption of the antibody, the membrane must be previously treated with a substance that inhibits such nonspecific adsorption (skim milk, casein, bovine). An operation (blocking) of dipping in a buffer solution containing serum albumin, gelatin, polyvinylpyrrolidone, etc. for a certain period of time is performed. The composition of the blocking solution is, for example, phosphate buffered saline (PBS) or Tris buffered saline (TBS) containing 5% skim milk, 0.05 to 0.1% Tween 20. Instead of skim milk, Block Ace (Dainippon Pharmaceutical), 1 to 10% bovine serum albumin, 0.5 to 3% gelatin, 1% polyvinylpyrrolidone, or the like may be used. The blocking time is 16 to 24 hours at 4 ° C. or 1 to 3 hours at room temperature.

Next, the membrane is washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove an excess blocking solution, and then manufactured by the method described in (3) above. The antibody thus obtained is immersed in a solution appropriately diluted with a blocking solution for a certain period of time to bind the antibody to the antigen on the membrane. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary Western blotting experiment using a sample obtained by serially diluting the recombinant antigen described in 3) above. This antibody reaction operation is preferably performed at room temperature for 2 hours. After completion of the antibody reaction operation, the membrane is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. When using a labeled secondary antibody, for example, when a commercially available product is used, it is used after diluting 2,000 to 20,000-fold with a blocking solution (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, the membrane is immersed in a secondary antibody solution at room temperature for 45 minutes to 1 hour, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed, for example, by first shaking the membrane in the washing solution for 15 minutes, exchanging the washing solution for a new one, shaking for 5 minutes, exchanging the washing solution again, and shaking for 5 minutes. If necessary, the washing solution may be further exchanged for washing.

ii) ELISA / RIA
First, as in the case of Western blotting, blocking is performed in advance to prevent nonspecific adsorption of the antibody to the bottom surface in the well of the plate on which the sample is immobilized by the method (2). The blocking conditions are as described in the section of Western blot.

Next, the wells are washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove an excess blocking solution, and then manufactured by the method described in (3) above. A solution obtained by appropriately diluting the washed antibody with a washing solution is dispensed and incubated for a certain period of time to allow the antibody to bind to the antigen. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary ELISA experiment using a serially diluted recombinant antigen described in (3) above as a sample. This antibody reaction operation is preferably performed at room temperature for about 1 hour. After completion of the antibody reaction operation, the inside of the well is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when a commercially available product is used, the labeled secondary antibody is diluted 2,000 to 20,000 times with a washing solution and used (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, a secondary antibody solution is dispensed into the wells, incubated at room temperature for 1 to 3 hours, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed by, for example, dispensing a washing solution into a well and shaking for 5 minutes, replacing the washing solution with a new one, shaking for 5 minutes, exchanging the washing solution again and shaking for 5 minutes. If necessary, the washing solution may be further exchanged for washing.

In the present invention, the so-called sandwich ELISA can be carried out, for example, by the method described below. First, in any one of the amino acid sequences of the polypeptide encoded by the target gene, two regions with high hydrophilicity are selected, and a partial peptide consisting of 6 or more amino acids in each region is synthesized. Two kinds of antibodies using the partial peptide as an antigen are obtained. One of the antibodies is labeled as described in (4) above. The unlabeled antibody is immobilized on the bottom surface of a 96-well or 384-well ELISA plate in a well according to the method described in (2) above. After blocking, the sample solution is placed in a well and incubated at room temperature for 1 hour. After washing the wells, the labeled antibody diluent is dispensed into each well and incubated. After washing the inside of the well again, a detection operation according to the labeling method is performed.

(6) The target protein can also be quantified by using a ligand such as a substrate, a coenzyme, or a regulator that specifically binds to the polypeptide encoded by the target gene, in addition to the ligand antibody.

3. Method for Designing Antisense Compound The antisense compound in the present invention can be designed by any one of the following methods A to D.

A. A method comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting a total RNA fraction from cells incubated with the oligonucleotide having the nucleotide sequence shown in step 1) and cells incubated without the oligonucleotide;
5) analyzing the difference in the expression level of each gene output in step 3) between the total RNA fractions obtained in step 4);
6) a step of modifying the nucleotide sequence shown in step 1) based on the difference in the expression level of each gene obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and having no effect on the expression level of each gene output in step 3) The process of selecting.

B. A method comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting a total RNA fraction from each of the animal to which the oligonucleotide having the nucleotide sequence shown in step 1) has been administered and the animal to which the oligonucleotide has not been administered;
5) analyzing the difference in the expression level of each gene output in step 3) between the total RNA fractions obtained in step 4);
6) a step of modifying the nucleotide sequence shown in step 1) based on the difference in the expression level of each gene obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and having no effect on the expression level of each gene output in step 3) The process of selecting.

C. A method comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting a total polypeptide fraction from cells incubated with the oligonucleotide having the nucleotide sequence shown in step 1) and animal cells incubated without the oligonucleotide;
5) analyzing the difference in the expression level of the polypeptide corresponding to each gene output in step 3) between the total polypeptide fractions obtained in step 4);
6) a step of modifying the nucleotide sequence shown in step 1) based on the difference in the expression level of each polypeptide obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and having no effect on the expression level of each gene output in step 3) The process of selecting.

D. A method comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting all polypeptide fractions from animals to which the oligonucleotide having the nucleotide sequence shown in step 1) has been administered and animals to which the oligonucleotide has not been administered, respectively;
5) analyzing the difference in the expression level of the polypeptide corresponding to each gene output in step 3) between the total polypeptide fractions obtained in step 4);
6) a step of modifying the nucleotide sequence shown in step 1) based on the difference in the expression level of each polypeptide obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and having no effect on the expression level of each gene output in step 3) The process of selecting.

Hereinafter, each step will be described in detail.
(1) Step 1) Step 1) comprises input means for inputting the base sequence of the complementary strand of an oligonucleotide having an antisense effect on the target gene determined in the above section "1. Determination of target gene". The antisense compound suppresses the expression of the protein encoded by the target gene by forming a Watson-Crick base pair with the mRNA of the target gene. Therefore, when the oligonucleotide has high homology with the complementary strand of the gene sequence data on the base sequence database, there is a possibility that the expression of the gene is suppressed. Some computer programs do not perform homology search for the complementary strand of the antisense compound base sequence even when the base sequence of the antisense compound is input. In such a case, input the base sequence data of the oligonucleotide. Instead, the base sequence data of the complementary strand of the oligonucleotide may be input.

塩 基 The base sequence input here becomes the key data of the search key in the following steps.

(2) Regarding step 2) As the base sequence database in step 2), any database containing known and / or unknown base sequence information of genes may be used. A database of sequence information can be used. Published databases of gene sequence information include the National Institute of Genetics in Japan, the NCBI (National Center for Biotechnology) GenBank in the United States, and the EBI (European Bioinfromatics Institite; formerly EMBL) gene database in Europe. The data of these databases can be used through a computer memory, a hard disk, an external storage medium such as a CD-ROM, a DVD-ROM, a magnetic tape, and the Internet. The base sequence data of the gene registered in the base sequence database is the target data described below.

に は A homology search program generally used can be used to determine the homology between the nucleotide sequence data of the oligonucleotide input in step 1) and the nucleotide sequence data of the gene registered in the nucleotide sequence database. Such a homology search program may be a program capable of searching for a sequence that matches or is similar to the nucleotide sequence of the oligonucleotide input in step 1) and the nucleotide sequence of the gene in the nucleotide sequence database. Such programs include, for example, FASTA (WR Pearson, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988)) and BLAST (SF Altschul, Nucleic Acids Res., 25: 3389 (1997)). And CLUSTAL @ W (JD Thompson, Nucleic Acids Res., 22: 4673 (1994)). These homology search programs can be executed by being incorporated into a user's computer, or programs registered on a computer of a database providing organization via a network such as the Internet.

When a homology search program is executed, if a search parameter is included in the program, a gene having a desired homology can be detected by appropriately setting the parameter.

For example, a Blast search is performed on a gene sequence in the base sequence database, and a gene group having an E value (Expectation value) of 10 or less or an alignment using CLUSTALW is performed to extract a gene group having a score of 80 or more.

Alternatively, a Blast search is performed on the gene sequences in the base sequence database, and a gene group having an E value (Expectation value) of 1000 or less or an alignment using CLUSTALW is performed. By calculating ΔG (free energy change) with the compound, it is possible to obtain a gene which is ranked in the order of small ΔG (the order in which energetically it is expected to form a stable double strand with the antisense compound).

(3) Step 3) Step 3) is a step of outputting data of the highly homologous gene obtained in step 2). An arbitrary number of genes can be output in descending order of homology determined in step 2). Although the format of the data output is based on the homology search program, any format may be used as long as it can identify a gene having a base sequence having homology with the base sequence of the complementary strand of the input oligonucleotide. An accession number assigned to each gene may be output, or the name of the gene may be output. Alternatively, a format may be used in which gene sequence data is simultaneously output to indicate a region of a base sequence having homology with the antisense compound.

(4) Steps 1) to 3) The hardware configuration of steps 1) to 3) will be described with reference to FIG. FIG. 1 is a diagram showing a basic configuration of a homology gene search system. In FIG. 1, reference numeral 101 denotes a workstation or a personal computer (hereinafter, the workstation includes a personal computer); 102, an external storage device; 103, a device such as a modem or a router for connecting the workstation to a network; , 105 are base sequence databases. In this system, the workstation 101 is a part serving as a man-machine interface, is installed at a site where a researcher is present, and executes a process of searching a local base sequence database (homology search).

An external storage device 102 as a base sequence database is connected to the workstation 101, and a storage medium such as a CD-ROM, a DVD-ROM, or a hard disk in which sequence data of gene information is stored by the external storage device 102 Then, the target data of the gene sequence data is obtained, the homology of the search key to the key data is evaluated, and the homology search of the gene sequence to be extracted as a candidate from the target data is performed. Further, the workstation 101 is connected to a public base sequence database 105 via a device such as a modem or a router. The workstation 101 obtains information of the base sequence database, and uses the information as target data in a local base sequence database. Similar to the data search processing, a homology search of the sequence data of the gene information with respect to the key data of the search key is performed. In FIG. 1, the system is completed at the upper part of the broken line. However, instead of directly inputting the key data of the search key to the workstation 101, a client terminal 106 shown below the broken line is used as 101 as a server. It is also possible to input the key data of the search key, cause the homology search to be performed at 101, and output it to 106.

配 列 The sequence data of the gene sequence is input to the workstation 101 directly from the keyboard or via a recording medium such as a flexible disk or a network. A search process (homology search) of the base sequence database is performed using this sequence data as key data of a search key. The homology search can be performed by using the aforementioned homology search program.

The workstation for performing the homology search may have any configuration capable of performing the above-described homology search, and includes a WINDOWS operating system (a trademark of Microsoft Corporation) having a central processing unit, a memory, an external storage device, an external output device, and the like. If the computer has a configuration in which an operating program such as a Linux system or a Macintosh (Apple trademark) operation system is operated, the homology search can be performed by incorporating the above-described homology search program into a personal computer. Further, it is also possible to obtain a search result by performing a homology search on an external computer via a network without incorporating a homology search program in a workstation.

結果 The result of homology search can be output to any output means such as a computer display and a printer.

In addition, in order to perform such homology search of the gene and obtain the data of the homologous gene, besides assembling the system such as the workstation described above, a commercially available gene analysis system can be used. An example of such a system is a Decypher system manufactured by TimeLogic (USA).

For example, when using the above Decypher system, enter the nucleotide sequence of the oligonucleotide into a computer, select a base sequence database, select a homology search program, set search parameters, and perform a search. A matched homologous gene can be output. In addition, homology gene data can be obtained using a public homology search service via a network. For example, a BLAST search in the GenBank database on the NCBI (National Center for Biotechnology Information) website (http://www.ncbi.nlm.nih.gov/) can be used to obtain the search results. is there.

(5) Step 4) The compound having the base sequence shown in step 1) can be prepared by using a DNA synthesizer, for example, a model 392 by the phosphoramidite method of Perkin Elmer (Nucleic Acids Research, 12, 4539 ( 1984)).

Its phosphoramidite reagents used in, for native nucleoside and 2'-O- methyl guanosine (G ^m) can be used commercially available reagents. The non-natural phosphoramidite is described in Example 5 of JP-A-2000-297097 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-4-N-benzoylcytidine-3'-O-). (2-cyanoethyl N, N-diisopropyl) phosphoramidite), Example 9 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-5-methyluridine-3'-O- ( 2-cyanoethyl N, N-diisopropyl) phosphoramidite, Example 14 (5′-O-dimethoxytrityl-2′-O, 4′-C-ethylene-6-N-benzoyladenosine-3′-O- ( 2-cyanoethyl N, N-diisopropyl) phosphoramidite, or 5′-O-dimethoxytrityl-2′-O-methyl-2-N-isobutyrylphenoxyacetylguanosine-3′-O- (2 -Cyanoethyl N, N-diisopropyl) phosphoramidite (Pharmacia) For other nucleosides, WO99 / 14226 for nucleosides where l, m, n, o and p are 1 and WO 00/47599 for nucleosides where l, m, n, o and p are 2 to 5. It can be obtained according to the method described.

If desired, a thioate can be formed by reacting with trivalent phosphorous acid such as tetraethylthiuram disulfide (TETD, Applied Biosystems) or Beaucage reagent (Glen Research) in addition to sulfur to form a thioate. And a thioate derivative can be obtained according to the method described in the literature (Tetarhedron Letters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 1253 (1990)). Modified oligonucleotides described in the literature (S. M. Freier et al, Nucleic Acids Res., 25, 4429 (1997)) can also be synthesized by the method cited in the literature.

The cell used in this step may be any cell that expresses the target gene. For example, for example, to see the effect of suppressing expression of c-raf, A549 cells (ATCC number CCL-185) (Brett P. Monia et al., Proc. Natl. Acad. Sci. USA, (1996) 93, 15481-15484), in order to observe the expression-suppressing effect on ras, T24 cells (ATCC number HTB-4), MRC- 5 cells (ATCC number CCL-171) and J82 cells (ATCC number HTB-1) (Guan Chen et al., J. Biol. Chem., (1996) 271, 28259-28265) can be used.

In order to examine the expression inhibitory effect on VEGF, cells expressing VEGF can be used in a normal state. Examples of such cells include human lung cancer cell line A549 cells (American Type Culture Collection; ATCC No. CCL-185). These cells can be cultured by a culture method usually used by those skilled in the art. In the present invention, an animal individual can be used instead of a cultured cell, and a mammal, such as a rat or a mouse, can be arbitrarily selected as long as the target gene is expressed. When the oligonucleotide is added to the cultured cells, it is added together with an intracellular introduction (transfection) reagent such as EPEI (Gene-tools) and Lipofectin (Invitrogen).

i) Method for measuring mRNA expression level In step 4), when using cultured cells as cells, the cell line is cultured under certain conditions, and then an oligonucleotide having the nucleotide sequence shown in step 1) is added together with an intracellular transfection reagent such as EPEI or lipofectin. And incubate for a certain period of time, after which the total RNA fraction is extracted from the cells. The extraction of the total RNA fraction and the purification of the mRNA can be carried out according to the method described in the section “A. Method for measuring the gene expression level” in the section “2. Measurement of gene expression level” above. .

全 The total RNA fraction or mRNA derived from cells cultured without adding the oligonucleotide used as a control can be extracted and purified in the same manner as described above.

On the other hand, when animals are used, they can be administered by an appropriate method such as diet, subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection and the like. After sacrifice and blood collection, appropriate tissues, blood, etc. are taken out from the animal individual after administration so as not to contaminate other tissues, and the total RNA fraction is subjected to a solvent for RNA extraction (for example, phenol or other ribonuclease inactive). It is preferable to dissolve it directly in a solution containing a component having an action of forming Alternatively, the cells in the tissue are carefully scraped with a scraper or gently extracted from the tissue using a protease such as trypsin so as not to destroy the cells in the tissue. Then, the process proceeds to the RNA extraction step. Extraction and purification of the total RNA fraction can be performed by the same method as described above.

ii) Method for measuring the expression level of polypeptide In the step 4) of the above B., the cell line is cultured under constant conditions by the same method as the above i), and then the oligonucleotide having the base sequence shown in the step 1) is added. And incubate for a period of time, after which the total polypeptide is extracted from the cells. When using an animal, an oligonucleotide having the nucleotide sequence shown in step 1) is administered, and after a certain period of time, appropriate tissues, blood, and the like are extracted in the same manner as in i). The preparation of the polypeptide fraction is performed according to the method described in “(1) Preparation of sample” in the section “B. Method for measuring the expression level of polypeptide” in the section “2. Measurement of gene expression level”. It can be performed according to it.

(6) Step 5) The analysis of the difference in gene expression level in step 5) can be performed using a known method as long as the gene expression level can be analyzed. -PCR and TaqMan PCR are preferred.
The RT-PCR method is performed by the method described in the section “(2) RT-PCR” in “A. Method for measuring the expression level of gene” in the section “2. Measurement of gene expression level” above. Can be. As methods for improving the reliability of gene quantification, competitive RT-PCR methods (Souaze et al., BioTechniques 21, 280-285 (1996)) and TaqMan PCR methods (Heid et al. , Genom. Res. 6, 986-994 (1996)).

In step 5), the detection results are compared between a sample derived from cells cultured with the addition of the oligonucleotide and a sample derived from cells cultured without the addition of the oligonucleotide. When the amount is reduced, it can be determined that the oligonucleotide has suppressed the expression of the gene. Similarly, the detection results are compared between a sample derived from an animal to which the oligonucleotide was administered in step 5) and a sample derived from an animal to which the oligonucleotide was not administered, and the expression level of each gene was reduced by the addition of the oligonucleotide. When it decreases, it can be determined that the oligonucleotide has suppressed the expression of the gene.

Note that the difference between the expression levels can be analyzed by measuring the expression levels of the gene and the target gene output in step 3) by the method described in the above section “2. Measurement of gene expression level”. It is also possible to combine a plurality of measurement methods.

遺 伝 子 Alternatively, the expression level of the gene can be determined by measuring the expression level of the mRNA translation product polypeptide. The expression level of the polypeptide can be measured by the method described in the section "B. Method for measuring the expression level of polypeptide" in the section "2. Measurement of gene expression level".

(7) Step 6) When the difference in the expression level of homologous genes other than the target gene is remarkably observed depending on the presence or absence of the oligonucleotide in step 5), side effects and the like may be caused. Therefore, if a difference in the expression level is observed, the nucleotide sequence shown in step 1) is modified. Here, "when the difference in the expression level of the homologous gene other than the target gene is remarkably recognized depending on the presence or absence of the oligonucleotide" means that the expression level is significantly reduced by the addition of the oligonucleotide, When the amount is reduced by 50% or more, it can be said that the expression amount is significantly reduced. However, in the present invention, even if the decrease in the expression level of the gene is less than 50%, the nucleotide sequence shown in step 1) can be modified as long as the decrease in the expression level is observed.

改 変 The modification of the base sequence can be performed by any of the following methods i) to iii).

I) Reduce the number of bases constituting the oligonucleotide. By this method, a stricter sequence specificity with the target gene is obtained. That is, if the number of bases constituting the oligonucleotide is reduced, a sequence having an affinity for a gene other than the target gene is removed, and the affinity for a gene other than the target gene can be reduced. When reducing the number of bases constituting the oligonucleotide, it is necessary to reduce the number of nucleotides by 1 to 5 from the 5 ′ side or from the 3 ′ side or from both the 5 ′ side and the 3 ′ side of the base sequence. it can. FIG. 2 shows a conceptual diagram of this method. For example, in FIG. 2, if the length of the oligonucleotide having an antisense effect on the target gene is reduced by 5 nucleotides, the match rate for the target gene remains at 100% and the match rate for the non-target gene decreases from 75% to 67%. It shows that the effect of suppressing the expression of a homologous gene other than the target gene can be reduced. If the affinity with the target gene is significantly reduced instead of shortening the base sequence and increasing the sequence specificity, a non-natural nucleoside may be newly introduced into the oligonucleotide to improve the affinity. .

(Ii) Move the nucleotide sequence of the oligonucleotide to the 3 'or 5' side in the target gene. Specifically, the base sequence selected as an oligonucleotide having an antisense effect on the target gene was moved 1 to 10 bases, preferably 1 to 5 bases to the 3 ′ side of the target gene without changing the length of the base sequence. An oligonucleotide corresponding to the base sequence or an oligonucleotide corresponding to the base sequence shifted by 1 to 10 bases, preferably 1 to 5 bases on the 5 ′ side is designed.

When the base sequence of the oligonucleotide is moved to the 5 ′ side, the oligonucleotide having a base sequence complementary to the base sequence from the 5 ′ side of the target gene to the nucleotide number (X) to (X + Y) is From the 5 'side of the target gene, from the (X-1) th to the (X-1 + Y) th to the (X-10) th to the (X-10 + Y) th, preferably from the (X-1) th to (X-1) th An oligonucleotide having a base sequence complementary to the (X-1 + Y) th to (X-5) th to (X-5 + Y) th base sequences is designed (where X is an integer and Y is 10 to 50). , Preferably an integer of 15 to 25. The same applies hereinafter.)

When the base sequence of the oligonucleotide is shifted to the 3 'side, the antisense compound having a base sequence complementary to the base sequence from the (X) to (X + Y) from the 5' side of the target gene can be used. Is (X + 1) th to (X + 1 + Y) th to (X + 10 + Y) th from the (X + 1) th to (X + 10 + Y) th from the 5 ′ side of the target gene, preferably from (X + 1 + Y) th to (X + 5 + Y) th to (X + 5 + Y) th ) Design an oligonucleotide having a base sequence complementary to the base sequence.

オ リ ゴ Thus, the oligonucleotide whose base sequence has been moved to the 5 'side or 3' side can reduce the affinity for a gene other than the target gene while maintaining the affinity for the mRNA of the target gene.

By shifting the position of the nucleotide sequence to the 5 'side or 3' side, the homology may be shown to a gene other than the gene searched by the homology search in step 3). The homology search can be performed again to select the direction in which the sequence is to be moved and the number of the sequence to be moved. FIG. 3 shows a conceptual diagram of this method. In FIG. 3, when the oligonucleotide having an antisense effect on the target gene is moved 4 bases to the 5 ′ side, the match rate for the target gene remains unchanged at 100%, whereas the match rate for the non-target gene changes from 75% to 55%. This shows that the expression is reduced and the effect of suppressing the expression of the non-target gene is reduced.
iii) By shortening the number of bases constituting the oligonucleotide, a stricter (higher match rate) sequence specificity with the target gene is provided. Then, in the oligonucleotide thus designed, the nucleotide sequence of the oligonucleotide is further shifted to the 3 ′ side or 5 ′ side in the target gene. Specifically, the length of the base sequence selected as the oligonucleotide having an antisense effect on the target gene was shortened, and 1 to 10 bases, preferably 1 to 5 bases were shifted to the 3 ′ side of the target gene. An oligonucleotide corresponding to a base sequence or an oligonucleotide corresponding to a base sequence shifted by 1 to 10 bases, preferably 1 to 5 bases on the 5 ′ side is designed. The length of the oligonucleotide base sequence can be reduced by 1 to 10 bases, preferably 1 to 5 bases.

(4) The oligonucleotide base sequence is modified by any of the above methods i) to iii).

(8) Step 7) In step 7), using the oligonucleotide having a base sequence obtained in the base sequence modification step in step 6), the oligonucleotide has an antisense effect on the target gene, and The oligonucleotide which does not affect the expression level of each gene output in step (1) is selected.

That is, steps 4) and 5) are performed using the oligonucleotide having the base sequence obtained in step 6) instead of the oligonucleotide having the base sequence shown in step 1), and the gene output in step 3) An oligonucleotide that does not affect the expression level of is selected.

In step 7), if necessary, i) steps 1) to 6) are repeated using the oligonucleotide having the base sequence obtained in step 6), or ii) steps 4) to 6). Oligonucleotides that have an antisense effect repeatedly on the target gene and do not affect the expression level of each gene output in step 3) can be selected. In the case of i) or ii), the following steps are performed.
i) repeating steps 1) to 6) using the oligonucleotide having the nucleotide sequence obtained in step 6), having an antisense effect on the target gene, and expressing each gene output in step 3) Select an oligonucleotide that does not affect the amount.
Or
ii) The steps 4) to 6) are repeated using the oligonucleotide having the nucleotide sequence obtained in the step 6) to have an antisense effect on the target gene, and the expression of each gene output in the step 3) Select an oligonucleotide that does not affect the amount.

Here, in the case of i), when the step 1) is the (n + 1) th time, the base sequence of the oligonucleotide designed in the nth step 6) is inputted in the step 1) by a method of inputting the base sequence of the oligonucleotide in the step 1). Step 6) is repeated, and an oligonucleotide which does not affect the expression level of each gene output in Step 3) is selected (where "n" represents an integer of 1 or more).

In the case of ii), in the step 4), when the step 4) is (n + 1) -th, the oligonucleotide designed in the n-th step 6) is used instead of the nucleotide sequence of the oligonucleotide shown in the step 1) Is used.

When step 6) is the (n + 1) -th time (where “n” represents an integer of 1 or more), in the step 6), instead of the base sequence shown in the step 1), the n-th time in the step 6) The designed nucleotide sequence of the oligonucleotide is used.

In the present step 7), the “oligonucleotide having no effect on the expression level of each gene output in step 3” refers to, for example, the expression level of the gene whose gene expression level has decreased by 50 or more in step 5) Is reduced to less than 50%. However, depending on the gene, it may be sufficient that the rate of decrease in the expression level of the gene is reduced.In such a case, if the rate of decrease in the expression level of the gene is reduced, the expression level of the gene before modification is reduced. May not be 50% or more, and the rate of decrease in the expression level of the modified gene may not be less than 50%.

Thus, by the steps 1) to 7), compounds having an antisense effect on the target gene and having no influence on the expression levels of other genes are selected.

{Circle around (1)} According to the method for designing an oligonucleotide of the present invention, for example, when the steps 1) to 7) are performed using the oligonucleotide base sequence of the compound (I), a compound having the general formula (I) is selected.

The compound of the present invention represented by the general formula (I) or a salt thereof exhibits a VEGF inhibitory activity and has a lower inhibitory activity on the expression level of other genes than the compound (I). Further, the compound represented by the general formula (I) and / or a salt thereof of the present invention has excellent pharmacokinetics such as absorption, distribution in the body and half-life in blood, and has low toxicity to organs such as kidney and liver. . Therefore, the compounds represented by the general formula (I) of the present invention and / or salts thereof are useful, for example, as medicaments, and in particular, treat or prevent various diseases involving angiogenesis or vascular hyperpermeability. It is useful as a medicament.

When the compound of the present invention is used as a prophylactic or therapeutic agent for the above-mentioned diseases, the compound having the general formula (I), or a salt or ester thereof, can be used as such or an appropriate pharmacologically acceptable compound. Mixed with excipients, diluents, etc., orally with tablets, capsules, granules, powders or syrups, or parenterally with injections, suppositories, patches, or external preparations. Can be administered in a controlled manner.

These formulations include excipients (eg, sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Inorganic excipients such as carbonates such as calcium; sulfates such as calcium sulfate; and lubricants (eg, metal stearate such as stearic acid, calcium stearate, magnesium stearate). Salt; talc; colloidal silica; beeswax Waxes such as gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; Silicic acids such as silicic acid hydrate; and the above-mentioned starch derivatives.), Binders (for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and the same as the above-mentioned excipients) And disintegrants (eg, cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, and internally cross-linked sodium carboxymethylcellulose; carboxymethyls). Mention may be made of chemically modified starch and celluloses such as tart, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone; emulsifiers (e.g. colloidal clays such as bentonite, veegum; magnesium hydroxide, aluminum hydroxide Metallic hydroxides, such as sodium lauryl sulfate, anionic surfactants, such as calcium stearate; cationic surfactants, such as benzalkonium chloride; and polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters And non-ionic surfactants such as sucrose fatty acid esters.) Stabilizers (paraoxybenzoic acid esters such as methyl paraben and propyl paraben; chlorobutanol, benzyl alcohol, phenyl ether). Alcohols such as alcohol; benzalkonium chloride; phenols, phenols such as cresol; thimerosal; dehydroacetic acid; and sorbic acid and the. ), Flavoring agents (for example, commonly used sweeteners, sour agents, flavors, etc.) and diluents can be used in a well-known method.

コ ロ イ ド As for the method of introducing the compound of the present invention into a patient, a colloidal dispersion system can be used in addition to the above. The colloidal dispersion system is expected to have the effect of increasing the stability of the compound in vivo and the effect of efficiently transporting the compound to a specific organ, tissue or cell. Colloidal dispersions are not limited as long as they are commonly used, but are based on polymer complexes, nanocapsules, microspheres, beads, and lipids including oil-in-water emulsifiers, micelles, mixed micelles, and liposomes. Dispersion systems can be mentioned, and it is preferably a plurality of liposomes and artificial membrane vesicles having an effect of efficiently transporting a compound to a specific organ, tissue or cell (Mannino et al., Biotechniques, 1988). , 6,682; Blume and Cevc, Biochem. Et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech., 1995, 6, 698).

Unilamellar liposomes, ranging in size from 0.2-0.4 μm, can encapsulate a significant proportion of the aqueous buffer containing macromolecules, and the compound is encapsulated in this aqueous inner membrane, and the biologically active form (Fraley et al., Trends Biochem. Sci., 1981, 6, 77). The composition of the liposome is usually a complex of a lipid, especially a phospholipid, especially a phospholipid having a high phase transition temperature, with one or more steroids, especially cholesterol. Examples of lipids useful for liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipids, phosphatidylethanolamine, cerebroside and ganglioside. Particularly useful are diacylphosphatidylglycerols, wherein the lipid moiety contains 14-18 carbon atoms, especially 16-18 carbon atoms, and is saturated (with a double bond within the 14-18 carbon atom chain). Lack). Representative phospholipids include phosphatidylcholine, dipalmitoyl phosphatidylcholine, and distearoyl phosphatidylcholine.

標的 The targeting of colloidal dispersions, including liposomes, can be either passive or active. Passive targeting is achieved by taking advantage of the liposome's natural tendency to distribute to cells of the reticuloendothelial system of organs containing sinusoidal capillaries. Active targeting, on the other hand, includes, for example, protein coats of viruses (Morishita et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 8744), monoclonal antibodies (or appropriate binding portions thereof). Binding specific ligands, such as sugars, glycolipids or proteins (or suitable oligopeptide fragments thereof) to liposomes, or to achieve distribution to organs and cell types other than the naturally occurring localization sites Examples of the method include modifying the liposome by changing the composition of the liposome. The surface of the targeted colloidal dispersion can be modified in various ways. In liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the target ligand in intimate association with the lipid bilayer. Various linking groups can be used to link the lipid chain to the targeting ligand. Target ligands that bind to specific cell surface molecules that are predominantly found on cells where delivery of the oligonucleotides of the invention are desired are, for example, (1) predominantly expressed by cells where delivery is desired Polyclonal or monoclonal antibodies that specifically bind to hormones, growth factors or suitable oligopeptide fragments thereof, or (2) antigenic epitopes predominantly found on target cells, which bind to specific cell receptors Or an appropriate fragment thereof (eg, Fab; F (ab ') 2). Two or more bioactive agents (eg, a compound of general formula (I) or a salt thereof and another drug) can be complexed and administered within a single liposome. Agents that increase the intracellular stability and / or targeting of the contents can also be added to the colloidal dispersion.

The dosage varies depending on the condition, age, etc., but in the case of oral administration, the lower limit is 1 mg (preferably 30 mg) and the upper limit is 2000 mg (preferably 1500 mg), and in the case of intravenous administration, It is desirable that the lower limit of 0.5 mg (preferably 5 mg) and the upper limit of 500 mg (preferably 250 mg) be administered to an adult 1 to 6 times per day depending on the symptoms.

Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples, and Formulation Examples, but the present invention is not limited to these Examples.

(Example 1)
HO−C ^e2 −C ^e2 −C ^e2 −A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^s −G ^s −C ^s −A ^s −G ^s −A ^e2 −A ^e2 −A ^e2 − VEGF sequence as oligonucleotides having antisense effect on the synthesis VEGF gene ^{G m -T e2 -T e2 -C e2} -A e2 -T e2t -H (GenBank Accetion number AF022375.1, of SEQ ID NO: 2) An antisense compound consisting of the nucleotide sequence of the complementary strand of nucleotide numbers 702 to 723 was synthesized.

Using a nucleic acid synthesizer (ABI model 392 DNA / RNA synthesizer manufactured by PerkinElmer), the reaction was performed on a 1.0 μmol scale. The concentrations of the solvent, the reagent, and the phosphoramidite in each synthesis cycle were the same as those in the synthesis of the natural oligonucleotide, and the solvent, the reagent, and the phosphoramidite of the natural nucleoside were those manufactured by PE Biosystems. The non-natural phosphoramidite is described in Example 5 of JP-A-2000-297097 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-4-N-benzoylcytidine-3'-O-). (2-cyanoethyl N, N-diisopropyl) phosphoramidite), Example 9 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-5-methyluridine-3'-O- ( 2-cyanoethyl N, N-diisopropyl) phosphoramidite, Example 14 (5′-O-dimethoxytrityl-2′-O, 4′-C-ethylene-6-N-benzoyladenosine-3′-O- ( 2-cyanoethyl N, N-diisopropyl) phosphoramidite, or 5′-O-dimethoxytrityl-2′-O-methyl-2-N-isobutyrylphenoxyacetylguanosine-3′-O- (2 -Cyanoethyl N, N-diisopropyl) phosphoramidite (Pharmacia) The sulfurization reaction was performed using a sulfurizing reagent from Glen Research at a concentration of 1 g / 100 ml, the compound of Reference Example 1 (1.0 μmol) was used as a solid support, and the DMTr group was deprotected with trichloroacetic acid. A condensation reaction was repeatedly performed using a natural phosphoramidite unit and a non-natural phosphoramidite unit for the 5′-hydroxyl group to synthesize the title compound.
The synthesis cycle is as follows.

Synthesis cycle
1) detritylation trichloroacetic acid / dichloromethane; 85sec
2) coupling phosphoramidite (25eq), tetrazole / acetonitrile; 10-20min
3) capping 1-methylimidazole / tetrahydrofuran, acetic anhydride / pyridine / tetrahydrofuran; 15 sec
4) sulfurization sulfurization reagent; 5min (× 2) (A s, when G ^S, C ^S, synthesis cycle condensing T ^S)
Or, Oxidation iodine / water / pyridine / tetrahydrofuran; 15sec (A ^p, when ^{G p, C p, T p} , A e2, G e2, C e2, T e2, synthesis cycle condensing G ^m)
Using 1 mol of modified control pore glass (CPG) (described in Example 12b of JP-A-7-87982) as a solid support, the title compound was obtained using a DNA synthesizer.

After synthesizing a protected oligonucleotide analog having a target sequence with a 5′-DMTr group attached thereto, the oligomer is cut out from the support by treating with concentrated aqueous ammonia, and a protecting group cyanoethyl group on a phosphorus atom is removed. The protecting group on the nucleobase was removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (LC-10VP, manufactured by Shimadzu Corporation; column (GL Science Inertsil Prep-ODS (20 × 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; % → 60% CH ₃ CN (30 min, linear gradient); 40 ° C .; 10 ml / min; 254 nm), and the fraction eluted at 9.0 minutes was collected. After evaporating the solvent under reduced pressure, 80% aqueous acetic acid was added and the mixture was allowed to stand for 30 minutes to remove the DMTr group. After distilling off the solvent, about 5 ml of H ₂ O was added, and the aqueous layer was washed three times with about 5 ml of ethyl acetate to obtain the target compound after distilling off the aqueous solvent.

This compound is ion-exchange HPLC (column (Tosoh TSKgel DEAE-2SW (4.6 × 250 mm)); solution A: 20% CH ₃ CN, solution B: 67 mM phosphate buffer (pH 6.8), 1.5 M NaCl; gradient: B The solution was eluted at 13.8 minutes when analyzed with liquid 20 → 80% (20 min, linear gradient); 60 ° C .; 1 ml / min). (144 A ₂₆₀ units) (λmax (H ₂ O) = 260 nm)
The base sequence of this compound is as follows.

5'-cccaagacagcagaaagttcat-3 '(SEQ ID NO: 3 in Sequence Listing)
This compound was designated as compound a.

(Example 2) Synthesis of mismatched compound The following compound having eight mismatch sequences with respect to the base sequence of compound a was synthesized.
HO−C ^e2 −C ^e2 −A ^e2 −A ^e2 −C ^e2 −G ^m −T ^e2 −C ^e2 −A ^s −G ^s −A ^s −C ^s −G ^s −A ^e2 −A ^e2 −C ^e2 − G ^m −A ^e2 −T ^e2 −A ^e2 −A ^e2 −T ^e2t −H
The compound was synthesized and purified according to the method described in Example 1.

After synthesizing the protected oligonucleotide analog having the target sequence with the 5'-DMTr group removed, the oligomer is cut out from the support by treating with concentrated aqueous ammonia, and the protecting group cyanoethyl group on the phosphorus atom is removed. And the protecting group on the nucleobase was removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse-phase HPLC (LC-10VP, manufactured by Shimadzu Corporation; column (GL Science Inertsil Prep-ODS (20 × 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; % → 25% CH ₃ CN (30 min, linear gradient); 40 ° C .; 10 ml / min; 254 nm), and fractions eluted at 14.1 minutes were collected. This compound is ion-exchange HPLC (column (Tosoh TSKgel DEAE-2SW (4.6 × 250 mm)); solution A: 20% CH ₃ CN, solution B: 67 mM phosphate buffer (pH 6.8), 1.5 M NaCl; gradient: B The solution was eluted at 14.0 minutes when analyzed with liquid 20 → 80% (20 min, linear gradient); 60 ° C .; 1 ml / min). (39 A ₂₆₀ units) (λmax (H ₂ O) = 259 nm)
The base sequence of this compound is as follows.

5'-ccaacgtcagacgaacgataat-3 '(SEQ ID NO: 4 in Sequence Listing)
This compound was designated as compound b.

(Example 3) In order to find mRNA of a gene having a complementary sequence similar to the nucleotide sequence of the homology search compound a (SEQ ID NO: 3 in the sequence listing), the nucleotide sequence data of compound a was input to a computer, and TimeLogic was used. A homology search using the BLAST program was performed on the GenBank human gene database (hs · fna) using the Decypher system (US) (parameter conditions: Expectation: 10; Extension threshold: 11; Word size: 11; Nucleic Match: 1; Nucleic Mismatch: -1 or -3; Gapped Alignments: On; Gap Oopen Penalty: 5; Gap Extension Penalty: 2; Reporting Threshold: 1; Show GI numbers: On). As a result, 13 candidate genes having a base sequence similar to the complementary strand of the base sequence of compound a and capable of binding to compound a were found (Table 1). In the following table, transcription variants are also described, but the number of genes was analyzed as one gene even if there were multiple transcription variants.

(Table 1)
In Table 1, 10 genes having an Expectation Score of 4.9 or more (Table 2) excluding hypothetical proteins and genomic sequences whose functions were unknown on the database were analyzed.

(Table 2)
Using ClustalW, a gene containing a similar sequence was searched from the Genebank human gene database (parameter conditions were Ktuple size: 2, Window size: 4, Pairwise gap penalty: 5, Gap opening penalty: 15, Gap extension penalty: 6.66, Weight matrix: IUB, Gap separation distance: 8, End gaps: OFF, Transition weight: 0.5, Top diagonals: 5, Score: PERCENTAGE), and 100 genes with a score of 77 or more (Table 3) were extracted. .

(Table 3)
In addition, out of about 100 extracted genes, 10 genes with a score of 88 or more excluding putative proteins (hypothetical proteins) and genomic sequences whose functions are unknown on the database, and 9 consecutive bases in a gene group with a score of 77 or more A total of 12 genes were selected, two genes whose sequences were perfectly complementary (Table 4).

(Table 4)
(Example 4) Fluctuation analysis of expression of genes other than VEGF gene of compound a
Gene expression in human lung cancer cell line A549 cells was examined by RT-PCR for 10 genes (Table 2) output by BLAST search and 14 genes (Table 4) out of genes output by ClustalW search.

As a result, expression was confirmed for 6 genes (Table 5) among the 10 genes output by BLAST search. In addition, among the 14 genes output by the ClustalW search, expression was confirmed for 7 genes (Table 6). With respect to these 13 genes in total, fluctuations in mRNA expression among the untreated cell group, the compound a-added group, and the compound b-added group were analyzed by RT-PCR.

(Table 5)
(Table 6)
Human lung cancer cell line A549 cells (ATCC number: CCL-185) were plated on a 12-well plate (manufactured by IWAKI) coated with collagen type I at 0.6 × 10 ⁵ cells / well / 1 ml RPMI1640 (GIBCO BRL) (10% Fetal Bovine Serum (containing Hyclone)), and cultured overnight at 37 ° C under 5% CO ₂ . Preparation of each oligonucleotide-EPEI complex is required by adding 20 μL of various oligonucleotide aqueous solutions (50 μM) and 2 μL of EPEI (Gene Tool, 200 μM) to 178 μL of water, vortexing immediately for 10 seconds, and allowing to stand at room temperature for 20 minutes. When done. Immediately before adding the specimen, the cell culture solution in each well was replaced with 0.8 ml of a fresh medium (RPMI1640), and 200 μL of the oligonucleotide-EPEI complex prepared as needed was added thereto. After incubating for 4 hours at 37 ° C. and 5% CO ₂ , the medium was replaced with 1 ml of oligonucleotide-free RPMI1640 (containing 10% Fetal Bovine Serum), and further incubated for 20 hours. Next, the cells in each well were peeled off the plate using 400 μL of trypsin-EDTA (0.05% Trypsin, 0.53 mM EDTA · 4Na) (GIBCO BRL), and 1 ml of RPMI1640 (containing 10% Fetal Bovine Serum) was added. After being collected in an Eppendorf tube by centrifugation, it was washed twice with PBS (-) buffer (Nissui Pharmaceutical Co., Ltd.). Extraction of total mRNA from the collected cells was performed using the Quick Prep Micro mRNA Purification Kit (Amersham Pharmacia Biotech Co., Ltd.) as per the manual, and 200 μL of an aqueous solution containing total mRNA was obtained. Using 10 μL of these, a reverse transcription reaction using oligo dT _(12-18) as a primer was performed using Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech Co., Ltd.) according to the manual, and total cDNA solution was used. 50 μL was obtained. Using this as a template, β-actin mRNA was similarly amplified. By using the mRNA amount of β-actin, which is a housekeeping gene, as an internal standard, using the amount of template cDNA corrected so that the amount of β-actin mRNA in each sample becomes equal, the mRNA of VEGF and other 13 genes was used. Amplification by quantitative PCR was performed. The conditions for the PCR reaction are shown below.
VEGF (VEGF165: AF022375.1, VEGF121: E15157.1)
primer 1: 5'-tgcattggagccttgccttg-3 '(SEQ ID NO: 5 in Sequence Listing)
primer 2: 5'-ctcaccgcctcggcttgt-3 '(SEQ ID NO: 6 in Sequence Listing)
Expected amplified fragment (base sequence number in gene database: same hereafter 724-1278 555 bp (VEGF165), 1061-1483, 445 bp (VEGF121)
phospholipase C, beta4 (PLCβ4) (NM_000933.1)
primer 3: 5'-aatgcccacgagcagcaaac-3 '(SEQ ID NO: 7 in Sequence Listing)
primer 4: 5'-ccatctcggcttccaatttcacc-3 '(SEQ ID NO: 8 in Sequence Listing)
Expected amplified fragment: 2937-3273 (337bp)
polymerase iota (POLI) (NM_007195.1)
primer 5: 5'-ctgccaaatgtcttgaagcactgg-3 '(SEQ ID NO: 9 in Sequence Listing)
primer 6: 5'-tagggcactgacgactctcacg-3 '(SEQ ID NO: 10 in Sequence Listing)
Expected amplified fragment: 801-1154 (354bp)
TATA box binding protein (TBP) -associated factor, RNA polymerase II, C1 (TAF2C1) (NM_003185.1)
primer 7: 5'-catctggcaagcagtctacagag-3 '(SEQ ID NO: 11 in Sequence Listing)
primer 8: 5'-gcacaaggtaaggttgaggtgaag-3 '(SEQ ID NO: 12 in Sequence Listing)
Expected amplified fragment: 1817-1957 (141bp)
transducin (beta) −like 1 (TBL1) (NM_005647.1)
primer 9: 5'-tttccttttcattcagcccctgccc-3 '(SEQ ID NO: 13 in Sequence Listing)
primer 10: 5'-ccgtagaatcaaacgaagcacttgcc-3 '(SEQ ID NO: 14 in Sequence Listing)
Expected amplified fragment: 1745-2105 (361bp)
cell division cycle 25B (CDC25B) (NM_021872.1, 021873.1, 402187.1)
primer 11: 5'-tcacgatgagatcgagaacctcc-3 '(SEQ ID NO: 15 in Sequence Listing)
primer 12: 5'-cccacgctcagatgagaattcac-3 '(SEQ ID NO: 16 in Sequence Listing)
Expected amplified fragment: 1416-1767 (352bp)
signal transducer and activator of transcription 1 (STAT1) (NM_007315.1)
primer 13: 5'-atctccaacgtcagccagctcc-3 '(SEQ ID NO: 17 in Sequence Listing)
primer 14: 5'-tgatgaagcccatgatgcaccc-3 '(SEQ ID NO: 18 in Sequence Listing)
Expected amplified fragment: 1568-1943 (376bp)
Rab escort protein 1 (NM_000390.1)
primer 15: 5'-tgacagtgccagcagaggaac-3 '(SEQ ID NO: 19 in Sequence Listing)
primer 16: 5'-gggccagagcagacataaacg-3 '(SEQ ID NO: 20 in Sequence Listing)
Expected amplified fragment: 1454-1763 (310bp)
tubby super-family protein (TUSP) (NM_020245.2)
primer 17: 5'-ctctgccatgttacacggtcttc-3 '(SEQ ID NO: 21 in Sequence Listing)
primer 18: 5'-tcttagaaccctacccctcccc-3 '(SEQ ID NO: 22 in Sequence Listing)
Expected amplified fragment: 10124-10497 (374bp)
deoxyguanosine kinase (DGUOK) (NM_001929.1)
primer 19: 5'-cgaaaacttacccagaatggcacg-3 '(SEQ ID NO: 23 in Sequence Listing)
primer 20: 5'-cccacaggagaaaagaatgccag-3 '(SEQ ID NO: 24 in Sequence Listing)
Expected amplified fragment: 136-495 (360bp)
nuclear receptor subfamily 2, group C, member 1 (NR2C1) (NM_003297.1)
primer 21: 5'-cctgatctgtctgcacaacacc-3 '(SEQ ID NO: 25 in Sequence Listing)
primer 22: 5'-tgaagcggcacagttggaag-3 '(SEQ ID NO: 26 in Sequence Listing)
Expected amplified fragment: 315-665 (351bp)
E74−like factor 2 (ELF2) (NM_006874.1)
primer 23: 5'-tccaacaacagcgacctctcc-3 '(SEQ ID NO: 27 in Sequence Listing)
primer 24: 5'-tgccactgcttccgatttaacc-3 '(SEQ ID NO: 28 in Sequence Listing)
Expected amplified fragment: 1213-1564 (352bp)
v-raf-1 murine leukemia viral oncogene homolog 1 (RAF1) (NM_002880.1)
primer 25: 5'-ccacttttctgctccctttctcc-3 '(SEQ ID NO: 29 in Sequence Listing)
primer 26: 5'-actgcctgctaccttacttcctc-3 '(SEQ ID NO: 30 in Sequence Listing)
Expected amplified fragment: 2131-2433 (303bp)
glucose regulated protein, 58kD (GRP58) (NM_005313.1)
primer 27: 5'-ttttatgccccttggtgtggtc-3 '(SEQ ID NO: 31 in Sequence Listing)
primer 28: 5'-tcctcctgtgccttcttcttcttc-3 '(SEQ ID NO: 32 in Sequence Listing)
Expected amplified fragment: 1201-1511 (311bp)
Enzyme: Takara Taq premix (Takara Shuzo)
Reaction conditions: 95 ° C (15 sec), 55 ° C (30 sec), 72 ° C (30 sec)
Cycle number: VEGF: 30 times, PLC β4: 27 times, POLI: 30 times, TAF2C1: 30 times, TBL1: 27 times, CDC25B: 25 times, STAT1: 21 times, Rab escort protein 1: 28 times, TUSP: 28 Times, DGUOK: 28 times, NR2C1: 26 times, ELF2: 28 times, RAF1: 28 times, GRP58: 26 times Each of the obtained PCR products was subjected to 10% acrylamide gel electrophoresis (1x TBE solution (0.89 M Tris, Acid, EDTA solution (pH 8.3, Takara Shuzo) 240V, 30 minutes), and using 100bp DNA Ladder (Takara Shuzo) as a molecular weight marker, fluorescently stain the PCR product with SYBR Green I (FMC), The PCR products were visualized with an image analyzer (Molecular Imager FX (Bio-Rad)), and the amount of the products was quantified.The result of visualization and reversing the black and white of the image is shown in FIG. Inhibition of VEGF expression was observed by the addition of compound a. In 3 gene (CDC25B, STAT1, NR2C1), marked expression inhibition in the case of adding the compound a as compared with the case of adding the compound b was observed.

(Example 5) Design of oligonucleotide (1)
1. Based on the results of Example 4, the following compounds were designed under the policy of shortening the length of the nucleotide sequence of the oligonucleotide. This compound corresponds to the complementary strand of the nucleotide sequence of nucleotide numbers 705 to 723 of SEQ ID NO: 2 in the sequence listing.
HO−C ^e2 −C ^e2 −C ^e2 −A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^p −G ^p −C ^p −A ^p −G ^p −A ^e2 −A ^e2 −A ^e2 − according ^{G m -T e2 -T e2 -E-} H the same manner as in synthesis example 1, the title compound was obtained using a DNA synthesizer.

After synthesizing a protected oligonucleotide analog having a target sequence with a 5′-DMTr group attached thereto, the oligomer is cut out from the support by treating with concentrated aqueous ammonia, and a protecting group cyanoethyl group on a phosphorus atom is removed. The protecting group on the nucleobase was removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (Gilson model 305; Applied Biosystems Japan Poros R3 (4.6 × 100 mm)) under flowing conditions (solution A: 0.1 M triethylamine acetate). Aqueous solution (TEAA) (pH 7), solution B: 100% acetonitrile solution, solution C: 2% trifluoroacetic acid aqueous solution, 0 min → 5 min (solution A 95% → 90%, solution B 5% → 10%, solution C 0 %), 5min → 10min (95% of solution A, 5% of solution B, 0% of solution C), 10min → 15min (100% → 0% of solution A, 0% of solution B, 0% of C solution → 100%), 15min → 19min (A solution 0% → 100%, B solution 0%, C solution 100% → 0%), 19min → 24min (A solution 100% → 68%, B solution 0% → 32%, C solution 0%) 40 ° C .; 10 ml / min; detection 254 nm), and the peak fractions detected between 22 and 26 minutes were collected to obtain a crude purified product from which the 5′-DMTr group had been removed. After evaporating under reduced pressure, the remaining residue was subjected to reverse phase HPLC (LC-10VP, manufactured by Shimadzu Corporation; column (Merck chromolith RP-18e (4.6 × 100 mm)); 0.1 M triethyl acetate. Aqueous amine solution (TEAA), pH7; 6% → 15% CH 3 CN (15min, linear gradient); 60 ℃;. Repurified by 2 ml / min), fractions were collected eluting the 9.0-10.0 minutes present The compound was reverse phase HPLC (LC-10VP, manufactured by Shimadzu Corporation; column (Merck chromolith RP-18e (4.6 × 100 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 6% → 15% CH ₃ CN (10 min, linear When analyzed by (gradient); 60 ° C .; 2 ml / min), it was eluted at 7.90 minutes (151 nmol) (λmax (H ₂ O) = 257 nm)
The base sequence of this compound is as follows.

5'-cccaagacagcagaaagtt-3 '(SEQ ID NO: 33 in Sequence Listing)
This compound was designated as compound c.

2. Based on the results of Example 4, the following compounds were designed under the policy of shortening the length of the nucleotide sequence of the oligonucleotide. This compound corresponds to the complementary strand of the nucleotide sequence of nucleotide numbers 702 to 720 of SEQ ID NO: 2 in the sequence listing.
HO−A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^s −G ^s −C ^s −A ^s −G ^s −A ^e2 −A ^e2 −A ^e2 −G ^m −T ^e2 −T ^e2 − C ^e2 −A ^e2 −T ^e2 −E−H
The synthesis and purification of the compound Was performed in the same manner as described above. This compound is reverse-phase HPLC (LC-10VP, manufactured by Shimadzu Corporation; column (Merck chromolith RP-18e (4.6 × 100 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 6% → 15% CH ₃ CN (10 min, When analyzed by linear gradient); 60 ° C; 2 ml / min), it was eluted at 8.70 minutes. (176 nmol) (λmax (H ₂ O) = 258 nm)
The base sequence of this compound is as follows.

5'-agacagcagaaagttcat-3 '(SEQ ID NO: 34 in Sequence Listing)
This compound was designated as compound d.

3. Based on the results of Example 4, the following compounds were designed under the policy of shortening the length of the nucleotide sequence of the oligonucleotide. This compound corresponds to the complementary strand of the nucleotide sequence of nucleotide numbers 702 to 718 of SEQ ID NO: 2 in the sequence listing.
HO−C ^e2 −C ^e2 −C ^e2 −A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^p −G ^p −C ^p −A ^p −G ^p −A ^e2 −A ^e2 −A ^e2 − G ^m −E−H
The synthesis and purification of the compound Was performed in the same manner as described above.
The product was eluted at 7.29 minutes. (146 nmol) (λmax (H ₂ O) = 256 nm)
The base sequence of this compound is as follows.
5'-cccaagacagcagaaag-3 '(SEQ ID NO: 35 in Sequence Listing)
This compound was designated as compound e.

4. Based on the results of Example 4, the following compounds were designed under the policy of shortening the length of the nucleotide sequence of the oligonucleotide. This compound corresponds to the complementary strand of the nucleotide sequence of nucleotides 705 to 720 of SEQ ID NO: 2 in the sequence listing.
HO− A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^p −G ^p −C ^p −A ^p −G ^p −A ^e2 −A ^e2 −A ^e2 −G ^m −T ^e2 −T ^e2 − E−H
The synthesis and purification of the compound Was performed in the same manner as described above.
The base sequence of this compound is as follows. This compound is reverse-phase HPLC (LC-10VP, manufactured by Shimadzu Corporation; column (Merck chromolith RP-18e (4.6 × 100 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 6% → 15% CH ₃ CN (10 min, When analyzed by linear gradient); 60 ° C; 2 ml / min), it was eluted at 8.57 minutes. (135 nmol) (λmax (H ₂ O) = 257 nm)
5'-agacagcagaaagtt-3 '(SEQ ID NO: 36 in Sequence Listing)
This compound was designated as compound f.

(Example 6) Measurement of gene expression suppressing effect The gene expression suppressing effects of compound a, compound c, compound d, compound e and compound f were examined.

MRNA expression levels in untreated cell group, compound a, compound c, compound d, compound e and compound f added groups for a total of 4 genes of VEGF and CDC25B, STAT1 and NR2C1 whose expression fluctuation was observed in Example 3. Was analyzed by the RT-PCR method.

化合物 Compound a, compound c, compound d, compound e and compound f were added to A549 cells in the same manner as in Example 4, and mRNA was extracted 24 hours later. The amount of each cDNA was corrected by quantifying β-actin mRNA. RT-PCR was performed using the same primers under the same conditions as in Example 4 to measure the expression levels of VEGF, NR2C1, STAT1, and CDC25B genes (FIG. 5).

ΔVEGF gene expression-suppressing activity was observed in compound a, compound c and compound d. Compound a exhibited the strongest expression suppressing activity, and compound c exhibited approximately half the expression suppressing activity of compound a. The strength of the expression inhibitory activity depended on the number of sequences of the compound complementary to the mRNA.

NR2C1 gene expression inhibitory activity was observed in the compound a and compound d-added groups, and both reduced the expression level of NR2C1 to 20% or less. Compound d was considered to have no effect on the expression-suppressing activity since it has a shorter 5'-side sequence that is not complementary to NR2C1 mRNA as compared to compound a. In compounds c, e and f, the 3'-side sequence complementary to NR2C1 mRNA was shortened by 3 mer or more, so that the affinity for mRNA was reduced and almost no inhibitory activity was exhibited.

Static gene expression-suppressing activity was comparable between Compound a and Compound d. Compound d only shortened the 5 'side of compound a that was not complementary to STAT1 mRNA, and thus was considered to have exhibited the same level of inhibitory activity as compound a. In compound c, compound e and compound f, the 3 ′ sequence complementary to STAT1 mRNA is shorter than compound a by 2 mer or more, so that the affinity for mRNA is reduced and is lower than compound a and compound d. It was considered that the expression was suppressed.

CDC25B gene expression inhibitory activity was observed only in compound a. It is considered that the reason is that the compound c, the compound d, the compound e, and the compound f lost at least 1 mer of the complementary sequence on both the 3 'and 5' sides of the CDC25B gene.

From the above results, among compound c, compound d, compound e, and compound f, compound c has an expression inhibitory activity on VEGF and almost has an inhibitory activity on non-target genes NR2C1, STAT1, and CDC25B. It was found to be an antisense oligonucleotide having no properties.

(Example 7) Design of oligonucleotide (2)
1. Redesign of Antisense Compound The following compound was designed in which the nucleotide sequence of the oligonucleotide was 3mer shifted to the 3 ′ side in the target gene. This compound corresponds to the complementary strand of the nucleotide sequence of nucleotides 705 to 726 of SEQ ID NO: 2 in the sequence listing.
HO−G ^m −C ^e2 −A ^e2 −C ^e2 −C ^e2 −C ^e2 −A ^e2 −A ^e2 −G ^m −A ^e2 −C ^e2 −A ^s −G ^s −C ^s −A ^s −G ^s − A ^e2 −A ^e2 −A ^e2 −G ^m −T ^e2 −T ^e2 −E−H
The synthesis and purification of the compound are described in Example 1. This is performed in the same manner as described above.
This compound has the following nucleotide sequence.
5'-gcacccaagacagcagaaagtt-3 '(SEQ ID NO: 37 in Sequence Listing)
This compound was designated as compound g.

2. Homology search In order to find mRNA having a complementary sequence similar to the nucleotide sequence of compound g, a homology search was performed using the BLAST program on the GenBank human gene database based on the method described in Example 3. . Parameter conditions were the same as in Example 3. As a result, Homo sapiens hypothetical protein FLJ13993 (FLJ13993), mRNA. (Genbank accession No.NM_024695)
Homo sapiens solute carrier family 6 (neurotransmitter transporter, GABA), member 13 (SLC6A13), mRNA. (Genbank accession No.NM_016615)
Could be obtained. Further, ClustalW analysis was performed under the conditions described in Example 3, and three genes were found (Table 7).

(Table 7)
By measuring the expression level of the above genes in the cells to which the compound g has been added by RT-PCR, the effect of the compound g on the expression levels of these genes can be measured.

用 い Using the oligonucleotides described above, RT-PCR measures expression fluctuations in three genes (STAT1, NR2C1, CDC25B) and the five genes in which fluctuations have been observed.

(Reference Example 1) Synthesis of CPG carrier binding compound

Compound 2′-O, 4′-C-ethylene-5′-O-dimethoxytrityl-5-methyluridine (580 mg, 0.988 mmol) of Example 8 of JP-A-2000-297097 was treated with anhydrous pyridine (580 mg, 0.988 mmol) in a nitrogen stream. 10 ml), succinic anhydride (178 mg, 1.78 mmol) and 4-dimethylaminopyridine (217 mg, 1.78 mmol) were added, and the mixture was stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the residue was purified using silica gel chromatography (dichloromethane: methanol = 25: 2) to obtain a colorless amorphous compound (580 mg, 0.844 mmol, yield 85%). Of these, 515 mg was dissolved in anhydrous dimethylformamide (6 ml), pentachlorophenol (330 mg, 1.12 mmol) and N, N'-dicyclohexylcarbodiimide (231 mg, 1.12 mmol) were added, and the mixture was stirred at room temperature for 24 hours. After the precipitate was filtered, the filtrate was concentrated under reduced pressure, benzene was added, and the precipitate was filtered again. The filtrate was concentrated under reduced pressure, anhydrous dimethylformamide (3 ml) was added, and triethylamine (280 μl, 2.0 mmol), long chain alkylamino glass (500 °, particle size: 120/200 mesh) (manufactured by CPG Inc) ( 6 g), and the mixture was left at 60 ° C. for 8 hours and at room temperature for 72 hours. After filtering the CPG and further washing with dichloromethane, 20 ml of each of two capping reagents (acetic anhydride / pyridine / tetrahydrofuran) (1-methylimidazole / tetrahydrofuran) for an Applied Biosystems DNA synthesizer ABI392 was added to CPG, and the mixture was added for 10 minutes. I left it. CPG was filtered, washed with pyridine and dichloromethane in that order, and dried under reduced pressure to obtain the desired product (about 6 g, dimethoxytrityl group introduction amount = 62 μmol / g).

(Formulation example)
Formulation Example 1 Soft Capsules A mixture of the compound of Example 1 in a digestible oil, such as soybean oil, cottonseed oil or olive oil, is prepared and injected into gelatin with a positive displacement pump to give 100 mg. A soft capsule containing the active ingredient is obtained, washed and dried.

(Formulation Example 2) Tablet According to a conventional method, 100 mg of the compound of Example 2, 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch and 98.8 mg of Produced using lactose.

剤 If desired, a coating is applied.

(Formulation Example 3) 100 mg of the compound of Example 1, 100 mg of sodium carboxy group methylcellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution (Japanese Pharmacopoeia) in 5 ml of a suspension, and Produced to contain 0.025 ml of vanillin.

(Formulation Example 4) Injection 1.5% by weight of the compound of Example 2 was stirred in 10% by volume of propylene glycol, then made up to a constant volume with water for injection, and then sterilized to produce it.

Homology search computer system diagram Conceptual diagram of design of antisense oligonucleotide for target gene i) Conceptual diagram of antisense oligonucleotide design for target gene ii) Diagram of gene expression suppression effect of compounds a and b Diagram of gene expression suppressing effects of compounds a, c, d, e and f

SEQ ID NO: 1: Nucleotide sequence of antisense compound against VEGF165 SEQ ID NO: 3: Compound a
SEQ ID NO: 4: Compound b
SEQ ID NO: 5: PCR sense primer for VEGF165 SEQ ID NO: 6: PCR antisense primer for VEGF165 SEQ ID NO: 7: PCR sense primer for phospholipase C, beta4 SEQ ID NO: 8: PCR antisense primer for phospholipase C, beta4 SEQ ID NO: 9: polymerase iota PCR sense primer for
SEQ ID NO: 10: PCR antisense primer for polymerase iota SEQ ID NO: 11: TATA box binding protein (TBP) -associated factor, RNA polymerase II, PCR sense primer for C1 SEQ ID NO: 12: TATA box binding protein (TBP) -associated factor, PCR antisense primer for RNA polymerase II, C1 SEQ ID NO: 13: PCR sense primer for transducin (beta) -like 1 SEQ ID NO: 14: PCR antisense primer for transducin (beta) -like 1 SEQ ID NO: 15: For cell division cycle 25B PCR sense primer SEQ ID NO: 16: PCR antisense primer for cell division cycle 25B SEQ ID NO: 17: PCR sense primer for signal transducer and activator of transcription 1 SEQ ID NO: 18: PCR antisense primer for signal transducer and activator of transcription 1 SEQ ID NO: 19 : PCR sense primer for Rab escort protein 1 SEQ ID NO: 20: PCR primer for Rab escort protein 1 Sense primer SEQ ID NO 21: tubby super-family protein for PCR sense primer SEQ ID NO 22: tubby super-family protein for PCR antisense primer b
SEQ ID NO: 23: PCR sense primer for deoxyguanosine kinase SEQ ID NO: 24: PCR antisense primer for deoxyguanosine kinase SEQ ID NO: 25: PCR sense primer for nuclear receptor subfamily 2, group C, member 1 SEQ ID NO: 26: nuclear receptor subfamily 2, group C , member 1 for PCR antisense primer SEQ ID NO: 27: PCR sense primer for E74-like factor 2 SEQ ID NO: 28: PCR antisense primer for E74-like factor 2 SEQ ID NO: 29: v-raf-1 murine leukemia viral oncogene homolog 1 PCR sense primer for SEQ ID NO: 30: PCR antisense primer for v-raf-1 murine leukemia viral oncogene homolog 1 SEQ ID NO: 31: PCR sense primer for glucose regulated protein, 58kD SEQ ID NO: 32: PCR antisense for glucose regulated protein, 58kD Primer SEQ ID NO: 33: Compound c
SEQ ID NO: 34: Compound d
SEQ ID NO: 35: Compound e
SEQ ID NO: 36: Compound f
SEQ ID NO: 37: Compound g

Claims (30)

A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting a total RNA fraction from cells incubated with the oligonucleotide having the nucleotide sequence shown in step 1) and cells incubated without the oligonucleotide;
5) analyzing the difference in the expression level of each gene output in step 3) between the total RNA fractions obtained in step 4);
6) a step of modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each gene obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and having no effect on the expression level of each gene output in step 3) The process of selecting. A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) Step of outputting data of a highly homologous gene obtained in step 2):
4) a step of extracting a total RNA fraction from each of the animal to which the oligonucleotide having the nucleotide sequence shown in step 1) has been administered and the animal to which the oligonucleotide has not been administered;
5) analyzing the difference in the expression level of each gene output in step 3) between the total RNA fractions obtained in step 4);
6) a step of modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each gene obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and having no effect on the expression level of each gene output in step 3) The process of selecting. A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence data and the base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting all polypeptide fractions from cells incubated with the oligonucleotide having the nucleotide sequence shown in step 1) and cells incubated without the oligonucleotide;
5) analyzing the difference in the expression level of the polypeptide encoded by each gene output in step 3) among the total polypeptide fractions obtained in step 4);
6) modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each polypeptide obtained in step 5);
7) Using the oligonucleotide having the base sequence obtained in step 6), an oligonucleotide having an antisense effect on the target gene and having no effect on the expression level of each gene output in step 3) The process of selecting. A method for designing an antisense compound comprising the following steps 1) to 7):
1) inputting nucleotide sequence data of an oligonucleotide having an antisense effect on a target gene;
2) a step of determining homology between the input base sequence and base sequence data from the base sequence database of the gene whose base sequence has been analyzed;
3) outputting data of the highly homologous gene obtained in step 2);
4) a step of extracting all polypeptide fractions from animals to which the oligonucleotide having the nucleotide sequence shown in step 1) has been administered and animals to which the oligonucleotide has not been administered, respectively;
5) analyzing the difference in the expression level of the polypeptide encoded by each gene output in step 3) among the total polypeptide fractions obtained in step 4);
6) modifying the nucleotide sequence of the oligonucleotide shown in step 1) based on the difference in the expression level of each polypeptide obtained in step 5);
7) Step 6) Using the oligonucleotide having the base sequence obtained, select an oligonucleotide that has an antisense effect on the target gene and does not affect the expression level of each gene output in Step 3) Process. Modification of the base sequence, i) reducing the number of bases constituting the oligonucleotide, ii) moving the nucleotide sequence of the oligonucleotide to the 3 ′ or 5 ′ side in the target gene or iii) constituting the oligonucleotide The number of bases is reduced, and the nucleotide sequence of the oligonucleotide is moved to the 3 ′ or 5 ′ side in the target gene, which is performed by the method according to any one of i) to iii). A method according to any one of claims 1 to 4. The method according to claim 1 or 2, wherein the expression level of the gene is measured by Northern blotting, dot blotting, slot blotting, or RT-PCR. The method according to claim 3 or 4, wherein the expression level of the polypeptide is measured by Western blotting, dot blotting, slot blotting or enzyme-linked immunosorbent assay (ELISA). The method according to any one of claims 1, 3, and 5 to 7, wherein the cells are cultured cells. The method according to claim 8, wherein the cultured cells are animal cells. The method according to any one of claims 2, 4 and 5 to 7, wherein the animal is a mammal. The method according to claim 10, wherein the mammal is a rodent. The method according to claim 11, wherein the mammal is a mouse or a rat. The method according to any one of claims 1 to 12, wherein the target gene is a VEGF gene. The following general formula (I)
HO-B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-H (I)
(Where B1 to B3, B8 and B11 are the same or different
(Hereinafter referred to as “C ^p ”), a formula
(Hereinafter referred to as “C ^s ”) or a general formula C ^e
(In the formula, l represents an integer of 1 to 5.)
B4, B5, B7, B9, B12, B14 to B16 are the same or different
(Hereinafter referred to as “A ^p ”), a formula
(Hereinafter referred to as “A ^s ”) or a general formula A ^e
(In the formula, m represents an integer of 1 to 5.)
B6, B10, B13 and B17 are the same or different
(Hereinafter referred to as “G ^p ”), a formula
(Hereinafter referred to as “G ^s ”), a formula
(Hereinafter referred to as “G ^m ”) or a general formula G ^e
(In the formula, n represents an integer of 1 to 5.)
B18 and B19 are the same or different
(Hereinafter referred to as “T ^p ”), a formula
(Hereinafter referred to as “T ^s ”) or a general formula T ^e
(In the formula, o represents an integer of 1 to 5.)
B20 is the formula
(Hereinafter referred to as “E”)
And / or a salt thereof. According to claim 14, B1 to B3, B8 and B11 are the same or different C ^p or C ^e,
B4, B5, B7, B9, B12, B14 to B16 are the same or different and A ^p or A ^e,
B6, B10, B13 and B17 are the same or different and G ^p, a G ^m or G ^e, and,
Compound is a T ^p or T ^e B18 and B19 are identical or different, and / or its salts. In any one of claims 14 or 15, the compounds B1 to B3 is C ^e, and / or its salts. 17. The compound according to claim 16, wherein B4 is ^Ae , and / or a salt thereof. The compound according to claim 17, wherein B5 is ^Ae , and / or a salt thereof. In claim 18, the compound B6 is G ^m or G ^e, and / or its salts. 20. The compound according to claim 19, wherein B7 is ^Ae , and / or a salt thereof. In claim 20, the compound B8 is C ^e, and / or its salts. According to claim 21, compound B19 is T ^e, and / or its salts. According to claim 22, B18 is a T ^e compounds, and / or its salts. According to claim 23, B17 is G ^m or G ^e compound, and / or its salts. 25. The compound according to claim 24, wherein B16 is ^Ae , and / or a salt thereof. 26. The compound according to claim 25, wherein B15 is ^Ae , and / or a salt thereof. 27. The compound according to claim 26, wherein B14 is ^Ae , and / or a salt thereof. 28. The compound according to any one of claims 14 to 27, wherein l, m, n and o are the same or different and are 1 or 2, and / or a salt thereof. 29. The compound according to claim 28, wherein l, m, n and o are the same and are 1 or 2, and / or a salt thereof. 30. The compound according to claim 29, wherein l, m, n and o are 2, and / or a salt thereof.