JPH1135493A - New antiestrogen gene therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject gene therapeutic agent effective against estrogen- relatedly developing or progressing osteosarcoma, bone metastasis cancer, etc. by including a DNA encoding an intranuclear receptor TR4 or a mutant thereof as an active ingredient. SOLUTION: The objective antiestrogen gene therapeutic agent is obtained by screening cDNA library derived from human hypothalamus, prostate or testis by using a TR4-specific primer or probe to provide a DNA encoding a cloned intranuclear receptor TR4 or a mutant thereof, and allowing the obtained DNA encoding the cloned intranuclear receptor TR4 or the mutant thereof to be included as an active ingredient. The gene therapeutic agent inhibits the transcription-promoting activities of the estrogen target gene, and is useful for effective treatment, prevention, etc., of osteosarcoma, bone metastasis cancer derived from mammary cancer, etc., which are diseases estrogen- relatedly developed or progressed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel anti-estrogen gene therapy agent. More specifically, the present invention relates to a novel anti-estrogen gene therapeutic, which comprises a DNA encoding a nuclear receptor TR4 or a mutant thereof as an active ingredient and suppresses the activity of estrogen to promote the transcription of an estrogen target gene. . The anti-estrogen gene therapeutic agent of the present invention is a therapeutic or preventive agent for diseases that develop or progress in an estrogen-dependent manner. In particular, since the anti-estrogen gene therapeutic agent of the present invention exerts its effect on cells derived from bone, it is effective for the treatment or prevention of bone diseases progressing by the action of estrogen, ie, osteosarcoma or bone metastatic cancer derived from breast cancer. It is an agent.

[0002]

2. Description of the Related Art Ligands having a steroid skeleton such as estrogen androgen and highly lipophilic ligands such as retinoic acid / vitamin D are transcription factor-type receptors localized in the cell nucleus, that is, "intranuclear". Through the “receptor” to induce or suppress the expression of target genes for these ligands (protein, nucleic acid, enzyme
Vol.41, No.8, p1265 (1996). That is, the nuclear receptor functions as a ligand-dependent transcription factor, and the nuclear receptor activated by binding of a ligand such as the steroid or vitamin binds to a ligand-specific target gene, Induces or suppresses transcription of the gene (Shigeaki Kato, Nuclear Receptor and Signaling, Yodosha, 1994)
Year).

[0003] These nuclear receptors are highly homologous in the primary structure, and in particular, D is located at the center of the nuclear receptor protein.
The NA binding region (also called C region) has a characteristic structure called Zn finger structure, and its homology is
It is over 50% among all nuclear receptors. Based on such characteristic structures and homology that are commonly found in nuclear receptors, it is considered that these nuclear receptor gene groups form a gene superfamily (Shigeaki Kato) , Nuclear receptor and information transmission, Yodosha,
1994). As a result of gene cloning in recent years, a large number of genes of factors that are structurally judged to be "nuclear receptors" have been cloned by utilizing high homology with known nuclear receptors. Of these, the ligand is unknown,
Factors that are thought to be absent or absent are called "orphan receptors." Many of the functions of these orphan receptors in vivo are still unknown, but their functions were revealed by gene knockout (SF-1 (ste
roidgenic factor-1)), an example in which the function could be inferred because the ligand was identified in vivo (PPARα, PPAR
γ; LXR, FXR, RXR).

[0004] TR4, also called TAK1, is an orphan receptor that two groups have successfully cloned at the same time (MOL. ENDO., Vol. 8, No. 1).
2, p1667-1680 (1994), Proc. Natl. Acad. Sci. USA, Vol. 91,
p6040-6044 (1994), Gene, 163, p239-242 (1995)). At present, the ligand is unknown, and its function in vivo is also unknown. However, recently, it has become clear that TR4 has an effect of suppressing signal transmission of thyroid hormone, retinoic acid and vitamin D. That is, by binding to thyroid hormone, retinoic acid and vitamin D target genes, the TR4 antagonizes the binding of receptors for these ligands to target genes,
As a result, it has been revealed that the signaling of these ligands is suppressed (Biochem. Biophys. Res. Commun. 21
1, p83-91, 1995). On the other hand, the document indicates that the TR4 does not bind to an estrogen response element in an estrogen target gene. Therefore, the TR
No. 4, unlike the results for the above thyroid hormones, suggests that estrogen signaling is not suppressed.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel anti-estrogen gene therapeutic comprising a DNA encoding TR4 or a mutant thereof as an active ingredient. That is, the present invention comprises a DNA encoding TR4 or a mutant thereof as an active ingredient,
An object of the present invention is to provide a novel anti-estrogen gene therapeutic agent capable of treating or preventing a disease such as osteosarcoma or bone metastatic cancer derived from breast cancer.

[0006]

DISCLOSURE OF THE INVENTION To elucidate the function of the nuclear receptor TR4, the present inventors first studied rabbit heart cD.
When the NA library was screened using a DNA binding region having a very high homology between nuclear receptors as a probe, it was found that the homology with rat, mouse, and human TR4, which had been reported previously, indicates that rabbit TR4 CDN
A was successfully cloned and the protein was replaced with TR4-0
It was named. Furthermore, the present inventors also cloned the isoform of TR4-0 lacking the ligand binding region located on the C-terminal side, and named the protein TR4-1. These TR4-0 (TR-4) and TR4-1
When the effect on estrogen was examined using cDNA of (TR-4 mutant), unexpectedly,
-0 and TR4-1 inhibit estrogen signaling, that is, the activity of estrogen through the estrogen receptor to promote the transcription of a target gene.
The present inventors have found for the first time that R4 or a mutant thereof suppresses. The present inventors have further revealed that the inhibitory effect is observed in bone-derived cells (osteosarcoma-derived cells) but not in kidney-derived cells.

[0007] Estrogen plays a particularly important role in fat metabolism and bone metabolism in women.
It is a drug that has been frequently used as a treatment for osteoporosis since it was discovered that osteoporosis, which is particularly common in elderly women, is due to estrogen deficiency (Bilezikian, JP et al., P.
rinciples of Bone Biology, Academic Press, p507-520,
1996). However, on the other hand, the estrogen is also known as a causative factor that increases the risk of developing breast cancer and uterine cancer (Toshio Matsumoto, Toshitaka Nakamura, ed., Osteoporosis, Yodosha, p162-163, 1995). Studies using human breast cancer-derived cell lines have revealed that breast cancer has a large number of estrogen receptors and proliferates in an estrogen-dependent manner (Ann. New York Acad. Sci. 761, Edi).
ted by Henderson, Detal 1995). Furthermore, it is also known that breast cancer has a very high probability of metastasis to bone, and such metastatic bone cancer derived from breast cancer is also considered to estrogen-dependently grow (Yoneda, T. et al., Breast Cance).
r Res. Treat. 32, 73-84, 1994). In addition, since osteosarcoma growth tends to be promoted in women with high blood estrogen levels before menopause, such osteosarcoma growth is also expected to be estrogen-dependent (Yoneda , T.etal, Breast Cancer Res. Treat.32,73-
84,1994).

[0008] Therefore, by using the DNA encoding the above-mentioned TR4 or its mutant as an anti-estrogen gene therapy agent, it becomes possible to obtain estrogen-dependent diseases such as the above-mentioned breast cancer, uterine cancer, bone metastasis derived from breast cancer, and osteosarcoma. It is an effective therapeutic or prophylactic agent for a disease that develops or progresses. In particular, the present invention has revealed for the first time that TR4 and TR4 mutants suppress estrogen signaling in bone-derived cells. Therefore, the gene therapy agent of the present invention is an effective therapeutic or prophylactic agent for bone metastatic cancer derived from breast cancer or osteosarcoma, among the above diseases.

[0009] Incidentally, antiestrogens are currently used for the treatment of osteosarcoma, but since they cause systemic effects, there is a concern about adverse effects on other organs such as effects on blood hormone levels (Ann) .New York Acad.Sci.76
1, Edited by Henderson, Detal 1995). In this regard, since TR4 or its mutant of the present invention has high specificity to bone, it can be expected to treat osteosarcoma with little adverse effect on other organs. The present invention has been completed based on the above findings.

[0010] That is, the gist of the present invention is: 1) an anti-estrogen gene therapeutic agent containing a DNA encoding the nuclear receptor TR4 or a mutant thereof as an active ingredient; 2) suppressing the transcription promoting activity of an estrogen target gene. The anti-estrogen gene therapeutic agent according to the above 1), 3) the anti-estrogen gene therapeutic agent according to the above 1) or 2), which specifically acts on bone-derived cells, and 4) The anti-estrogen gene therapeutic agent according to 3), which is for treating or preventing osteosarcoma or breast cancer-derived bone metastatic cancer.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, TR4 is TA
It is also called K1, for example, rabbit, rat,
Mouse or human TR4 (MOL. ENDO., Vol. 8, No. 12, p1667-1680 (1994), Proc. Nat)
l.Acad.Sci.USA, Vol.91, p6040-6044 (1994), Gene, 163, p.
239-242 (1995). These TR4s of the present invention are prepared by preparing TR4-specific primers or probes from the nucleotide sequences disclosed in the above-mentioned documents and the like, and using the primers or probes, cDNA libraries derived from the human hypothalamus, prostate or testis, etc. Can be easily cloned. These cloning methods include, for example, Molecular Cloning
2nd Edt. Cold Spring Harbor Laboratory Press (198
9) and the like, and specifically include a method using hybridization, a method using PCR, and the like.

In the present invention, the term "TR4 mutant" refers to an artificially produced so-called modified protein, or a TR4 isoform present in vivo, which suppresses the estrogen signal transmission activity found in the present invention. That is, it refers to a substance having an inhibitory effect on the transcription promoting property of an estrogen target gene by estrogen. DNA encoding the mutant can be obtained, for example, by site-directed mutagenesis (Methods in Enzymology, 100 , 448- (1
983)) and PCR (Molecular Cloning: A Laboratory)
Manual, 2nd Edt., Chapter 15, Cold Spring Harbor Laborato
ry Press (1989), PCR A Practical Approach, IRL Pres
s 200-210 (1991)), and can be easily prepared by those skilled in the art. In the case of an isoform present in a living body, it can be cloned by screening a cDNA library of human testis or heart using a TR4-specific primer or probe as described above.

As a specific example of the TR4 mutant, for example, in the amino acid sequence of TR4 of SEQ ID NO: 2, positions 236 to 246 are substituted with different amino acids due to a frame shift, and positions 247 and after are deleted. Lost TR4 isoforms. Due to its structural features, nuclear receptors are composed of A to F regions, of which the A / B region promotes transcription, the C region promotes DNA binding, and the E region promotes ligand binding. It has been found to be involved. The above TR4 isoform has a part of the E / F region substituted with a different amino acid, and the amino acid after the substitution site is deleted. Since it has been found that the isoform has an estrogen signal transduction inhibitory action, it is possible to produce a more active TR4 mutant based on the isoform. In addition to this, for example, it is conceivable that the above-mentioned inhibitory action is present in, for example, a TR4 mutant retaining only the C region or a TR4 mutant retaining only the A / B region.

In the present invention, the term "estrogen target gene" means a known estrogen target gene, that is, PS2, cathepsin D, stremesin 3, IGF-1,
IGFBP3, creatine kinase, vitellogenin,
Genes such as IL-6, progesterone receptor or ovalbumin are mentioned, and more preferably, PS2,
Cathepsin D and stremesin 3. That is, PS2 is isolated from human breast cancer-derived MCF-7 cells, and its expression is estrogen-dependent, and is currently used as one marker gene for malignant cancer. Cathepsin D, a protease,
It has been suggested that it is important for cancer cell invasion. And it is known that stremesin 3 is expressed only in estrogen-dependent malignant breast cancer (Shigeaki Kato,
Nuclear receptors and signal transduction, Yodosha, 1994). Thus, these PS2, cathepsin D and stremesin 3
Are considered to act specifically on estrogen-dependent cancers, and the gene therapy agent of the present invention suppresses the transcription of these target genes. It becomes a prophylactic agent.

The action of TR4 or a mutant thereof in the present invention, ie, the action of estrogen to suppress the transcription promoting activity of an estrogen target gene, can be measured, for example, by the following method using CAT enzyme activity as an index.

First, 1) an estrogen receptor expression vector, 2) an estrogen response element-CAT reporter plasmid,
3) Prepare TR4 or TR4 mutant expression vector. Here, the estrogen receptor expression vector is a previously reported estrogen receptor cDNA sequence (Nature 320, 13).
4-139 1986; Proc. Natl. Acad. Sci. USA 93, 5925-5930 199
Based on 6), appropriate primers are prepared, the cDNA is cloned by a method such as PCR, and thereafter, it is possible to construct by connecting to an expression vector such as pSG5 (Stratagene). Examples of the estrogen response element-CAT reporter plasmid include a plasmid in which a CAT reporter gene is linked to an estrogen response element present in the Xenopus vitellogenin gene, which is one of estrogen target genes, and such a vitellogenin gene estrogen Response element-CAT reporter plasmid is a previously reported estrogen response element in the vitellogenin gene (Nucl.Aci.
ds.Res. 12 8611-8626 1984; Cell 46 1053-1061 1986)
Can be prepared upstream of a CAT reporter plasmid (Promega). The TR4 or TR4 expression vector, which is a test sample, is constructed by cloning or modifying the DNA encoding the TR4 or TR4 mutant as described above, for example, pSG.
5 (Stratagene) or the like. In addition to the DNA for introduction as described above, it is preferable to introduce a β-galactosidase expression vector (PCH110; manufactured by Pharmacia) for correcting the introduction efficiency.

Next, the above-mentioned DNA mixture for introduction is introduced into cells. Examples of the cells include osteosarcoma-derived cells, UMR-106 (Dainippon Pharmaceutical Co., Ltd.). Other examples include MCF-7 (Dainippon Pharmaceutical), which is a cell derived from breast cancer. As a method for introducing DNA into cells, for example, the calcium phosphate method (Takashi Yokota, Kenichi Arai,
Transfection and expression / analysis method, Yodosha, 1994). After an appropriate time, estrogen is added to the medium after the introduction. As the estrogen, for example, 17
β-estradiol (Sigma) is used. afterwards,
After a suitable time, the CAT enzyme activity is measured. The C
The AT enzyme activity is that the estrogen binds to the estrogen receptor in the cell, and the conjugate binds to the estrogen response element, whereby the transcription of the CAT reporter gene linked to the response element is promoted. It is based on the mechanism by which enzyme activity is detected.
Examples of a method for measuring the CAT enzyme activity include the method of Kato et al. (Cell, 68 , 731-743, 1992). The above-mentioned assay was performed using the D-encoding variously prepared TR4 mutants.
By providing NA, the TR4 mutant of the present invention, which suppresses the transcription promoting activity of an estrogen target gene, can be easily selected.

The TR4 of the present invention or a mutant thereof has an inhibitory effect on the transcription promoting activity of an estrogen target gene as described above. Therefore, a gene therapy agent having an anti-estrogen action can be prepared by using a DNA encoding the TR4 or a mutant thereof as an active ingredient of a medicine. When such an anti-estrogen gene therapeutic is administered, TR4 or a mutant thereof is highly expressed in cells, and it is considered that the action of estrogen in the cells is suppressed. Therefore, as described above, the anti-estrogen gene therapeutic agent of the present invention is used for treating or preventing estrogen-dependent diseases which develop or progress, such as breast cancer, uterine cancer, metastatic bone cancer derived from breast cancer, or osteosarcoma. Agent. In particular, TR4 of the present invention or a mutant thereof suppresses the action of estrogen in bone-derived cells, and thus is an effective therapeutic or preventive agent for the above-mentioned breast cancer-derived bone metastatic cancer or osteosarcoma.

As a method for introducing the gene therapy agent of the present invention into cells, a gene transfer method using a viral vector or a non-viral gene transfer method (Nikkei Science, April 1994, pp. 20-45). , Experimental Medicine Special Issue, 12 (15)
(1994), Experimental Medicine Supplement, “Basic Technologies for Gene Therapy”, Youtosha (1996)).

As a method for introducing a gene using a viral vector, for example, a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a pox virus, a polio virus, a simbis virus, etc.
4 or a mutant TR4. Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus, and a vaccinia virus is particularly preferable. Non-viral gene transfer methods include a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method,
Examples thereof include a calcium phosphate method and an electroporation method, and a DNA vaccine method and a liposome method are particularly preferable.

In order for the gene therapy agent of the present invention to actually act as a medicine, DNA is directly introduced into the body.
ex vivo method, ex vivo extracting certain cells from humans, introducing DNA into the cells outside the body, and returning the cells to the body
o There is a law (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Special Issue on Experimental Medicine, 12 (15) (19
94)). In vivo methods are more preferred.

When administered by the in vivo method, it can be administered by an appropriate route depending on the disease, symptom and the like. For example, it can be administered intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, and the like. When administering by the in vivo method,
Generally, it is an injection or the like, and a conventional carrier may be added as necessary. In the case of a liposome or a membrane-fused liposome (such as Sendai virus (HVJ) -liposome), a liposome preparation such as a suspending agent, a freezing agent, and a centrifugal concentrated frozen agent can be obtained.

[0023]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Reference Example 1 Cloning of Rabbit TR4 cDNA The full-length cDNA of TR4 was obtained by using a rabbit heart cDNA library (λZAPII) (Stratagene) as an oligonucleotide primer in the DNA binding region conserved in all nuclear receptors. 5-GAA (G / A) AA (G / A) CC (C /
T) TT (G / A) CA (G / A) CC (C / T) TC (G / CA-3) was obtained by plaque hybridization using TC (G / CA-3) as a probe. Hybridization was performed at 42 ° C. for 18 hours using 6 × SSC.
(1 × SSC = 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 1 × Denhardt's solution (=
0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.02% Ficoll 400), 0.1% SDS,
100 μg / ml denatured salmon sperm DNA in solution and 2 ×
The above probe labeled with 10 ⁶ cpm / ml of ³² P (T
(5 terminal labeling with 4 polynucleotide kinase). After hybridization, twice at room temperature,
After washing in 6 × SSC, 0.1% SDS for 10 minutes, further washing was performed once at room temperature in 4 × SSC, 0.1% SDS for 10 minutes. The positive clone was converted into a pBluescript vector (manufactured by Stratagene) using the in vivo excision method (edited by Hiroshi Nojima, method for preparing a gene library, Yodosha Co., Ltd., 1994), and the nucleotide sequence was determined using an ABI DNA sequencer.

As a result, one cDNA clone was 5
It encoded a novel protein consisting of 96 amino acids. Homology search revealed that the novel protein was rabbit TR-4,
4-0. The nucleotide sequence determined is described in SEQ ID NO: 1, and the amino acid sequence is described in SEQ ID NO: 2. Further, the amino acid sequence of SEQ ID NO: 2
6th to 246th are As for frame shift
Another cDNA clone substituted with p-Ala-Cys-Glu-His-Pro-Ala-Ala-Phe-Asp-Thr and deleted from position 247 onward was also isolated and named TR4-1. The TR4-1
Was determined to be a TR4 isoform at the DNA level, since only deletions at positions 705 to 727 of SEQ ID NO: 1 were observed, and the others were completely homologous.

Reference Example 2 Construction of Expression Vector The expression vectors of TR4-0 and TR4-1 cloned above were pSG5, an animal cell expression vector.
(Stratagene). That is, T
In order to insert the full-length cDNAs of R4-0 and TR4-1 into the EcoRI site of pSG5, EcoRI sites were added to the N-terminal side and the C-terminal side by PCR. At that time, a Kozak sequence was introduced immediately before the ATG of the start codon to increase the translation efficiency. After confirming the base sequence of the constructed plasmid with a DNA sequencer,
A large amount was prepared by ultracentrifugation using a cesium chloride density gradient method.

Example 1 Inhibition of estrogen action of TR4-0 and TR4-1
Rate Measurements gene (manufactured by Dainippon Pharmaceutical) introducing cells and a rat osteosarcoma-derived UMR-106 cells and monkey kidney-derived COS-1 cells (Dainippon Pharmaceutical Co., Ltd.) was coated dextran Chaco - Le The cells were cultured in a phenol red-free DMEM medium (COSMO BIO) containing 5% of treated fetal calf serum (GIBCO). UMR-
As a mixture of DNA to be introduced into 106 cells, 0.5 μg estrogen receptor α or β expression vector (see the embodiment of the invention), 1 μg Xenopus vitellogenin
Estrogen response element-CAT reporter plasmid (see the embodiment of the present invention), 4.5 μg β-galactosidase expression vector (PCH110; manufactured by Pharmacia), and 0.5 μg TR4-prepared in Reference Example 2 described above. 0 expression vector, 25 μg TR4-1 expression vector or both expression vectors were mixed. Also as a control
A DNA mixture not containing the TR4 expression vector was also prepared. To this DNA mixture, pBluscribeM13 + plasmid (manufactured by Stratagene) was added, and the amount of DNA per each dish was adjusted to 31.5 μg in total.
When the MR-106 cells reached 40-60% confluence in a 9 cm Petri dish, the D
An NA mixture was introduced.

On the other hand, in COS-1 cells, 0.2 μg estrogen receptor expression vector, 0.4 μg Xenopus vitellogenin estrogen response element-CAT reporter plasmid, 1.5 μg β-galactosidase expression vector, and 0.2 μg TR4- produced in Reference Example 2
0 expression vector, 10 μg TR4-1 expression vector or both expression vectors were mixed. As a control, a DNA mixture containing no TR4 expression vector was also prepared. In these DNA mixtures,
After adding the pBluscribeM13 + plasmid (manufactured by Stratagene) to adjust the amount of DNA per dish to 12.5 μg in total, when the COS-1 cells reached 40-60% confluence in a 6 cm Petri dish Then, the above DNA mixture was introduced by the calcium phosphate method.

Both cells were cultured for 24 hours after gene transfer, the medium was changed, and then the cells were cultured in a 17-well medium to a final concentration of 10 ^-9 M.
Tar-estradiol (Sigma) was added. Where 1
7-beta estradiol was dissolved in ethanol to a final concentration of 0.1% ethanol. A control containing no drug was also prepared so as to be 0.1% ethanol. After further culturing for 24 hours, the cells were scraped off, the β-galactosidase activity was measured by a conventional method to confirm the efficiency of DNA transfer into the cells, and then the activity of the CAT enzyme accompanying the activation of the estrogen receptor was examined. That is, cell extract 1 was prepared according to the method of Kato et al. (Cell, 68 731-743, 1992).
Unit (for UMR-106) or 30 units (for COS-1)
Was reacted with acetyl-CoA (Sigma) and ¹⁴ C-chloramphenicol (Amersham) to separate acetyl ^14A separated on silica gel thin-layer chromatography.
The radioactivity of spots of C-chloramphenicol and ¹⁴ C-chloramphenicol was quantified using an image analyzer (BAS-2000; manufactured by Fuji Photo Film Co., Ltd.), and the production rate of acetyl ¹⁴ C-chloramphenicol was determined. Was calculated. At that time, the result of an experiment using a medium containing only 0.1% ethanol as a ligand was used as a control value, and the estrogen action inhibition rate of TR4-0 and TR4-1 was measured based on the relative value.

The results are shown in Tables 1 and 2. In the table, ERα
(Β) is an estrogen receptor α (β) expression vector, ERE-CAT is a Xenopus vitellogenin estrogen response element-CAT reporter plasmid, and PCH110 is a β-galactosidase expression vector.
And BSM uses the pBluscribeM13 + plasmid for TR4-0.
Is the TR4-0 expression vector, TR4-1 is the TR4-1
Expression vectors are respectively referred to. According to Tables 1 and 2,
In COS-1 cells, TR4-0 or TR4
No estrogen action-suppressing effect by -1 was observed, but in UMR-106 cells, TR4-0 or TR4-1 was found to suppress the action of estrogen.

[0031]

[Table 1]

[0032]

[Table 2]

[0033]

Industrial Applicability According to the present invention, there is provided a novel anti-estrogen gene therapeutic containing a DNA encoding nuclear receptor TR4 or a mutant thereof as an active ingredient. The anti-estrogen gene therapeutic agent of the present invention is a therapeutic or preventive agent for diseases that develop or progress in an estrogen-dependent manner. Particularly, since the anti-estrogen gene therapeutic agent of the present invention exerts an effect on cells derived from bone, it is effective for osteosarcoma, which is a bone disease progressed by the action of estrogen, or bone metastatic cancer derived from breast cancer. It is a therapeutic or prophylactic agent.

[0034]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1791 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Sequence characteristics Characterized method: E sequence ATG ACC AGC CCC TCC CCG CGC ATC CAG ATC ATC TCC ACC GAC TCC ACG 48 Met Thr Ser Pro Ser Pro Arg Ile Gln Ile Ile Ser Thr Asp Ser Thr 5 10 15 GTA GCC TCT CCT CAG CGC ATT CAG ATT GTG ACT GAC CAG CAG ACA GGA 96 Val Ala Ser Pro Gln Arg Ile Gln Ile Val Thr Asp Gln Gln Thr Gly 20 25 30 CAG AAG ATA CAG ATA GTC ACA GCT GTG GAC GCC TCC GGA TCC CCC AAA 144 Gln Lys Ile Gln Ile Val Thr Ala Val Asp Ala Ser Gly Ser Pro Lys 35 40 45 CAG CAG TTC ATT CTG ACC GGT CCA GAT GGG GCT GGA ACC GGG AAG GTG 192 Gln Gln Phe Ile Leu Thr Gly Pro Asp Gly Ala Gly Thr Gly Lys Val 50 55 60 ATC CTG GCT TCT CCA GAG ACA ACC AGC GCC AAG CAG CTC ATA TTC ACC 240 Ile Leu Ala Ser Pro Glu Thr Thr Ser Ala Lys Gln Leu Ile Phe Thr 65 70 75 80 ACC TCA GAC AAC CTT GTC CCT GGC AGG ATC CAG ATC GTC ACG GAT TCT 288 Thr Ser Asp Asn L eu Val Pro Gly Arg Ile Gln Ile Val Thr Asp Ser 85 90 95 GCT TCT GTG GAG CGA TTG CTG GGC AAG GCG GAC GTG CAG CGG CCC CAG 336 Ala Ser Val Glu Arg Leu Leu Gly Lys Ala Asp Val Gln Arg Pro Gln 100 105 110 GTG GTG GAG TAC TGT GTG GTC TGC GGC GAC AAA GCC TCT GGC CGT CAC 384 Val Val Glu Tyr Cys Val Val Cys Gly Asp Lys Ala Ser Gly Arg His 115 120 125 TAT GGG GCT GTC AGT TGT GAA GGT TGC AAA GGT TTC TTC AAA AGG AGC 432 Tyr Gly Ala Val Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Ser 130 135 140 GTG AGG AAA AAT CTG ACC TAC AGC TGC CGG AGC AAC CAA GAC TGC ATC 480 Val Arg Lys Asn Leu Thr Tyr Ser Cys Arg Ser Asn Gln Asp Cys Ile 145 150 155 160 ATC AAT AAA CAT CAC CGC AAT CGC TGT CAG TTT TGC CGG CTG AAA AAA 528 Ile Asn Lys His His Arg Asn Arg Cys Gln Phe Cys Arg Leu Lys Lys 165 170 175 TGC TTG GAG ATG GGC ATG AAA ATG GAA TCT GTG CAG AGT GAA CGG AAG 576 Cys Leu Glu Met Gly Met Lys Met Glu Ser Val Gln Ser Glu Arg Lys 180 185 190 CCC TTT GAC GTG CAG CGG GAG AAA CCA AGC AAT TGT GCC GCA TCA ACT 624 Pro Phe Asp Val Gln Arg Glu Lys Pro Ser Asn Cys Ala Ala Ser Thr 195 200 205 GAG AAA ATC TAT ATC CGG AAG GAC CTG AGG AGT CCT CTG ATA GCC ACC 672 Glu Lys Ile Tyr Ile Arg Lys Asp Leu Arg Ser Pro Leu Ile Ala Thr 210 215 220 CCC ACC TTT GTG GCA GAC AAA GAT GGA GCA AGA CAA ACA GGT CTT CTT 720 Pro Thr Phe Val Ala Asp Lys Asp Gly Ala Arg Gln Thr Gly Leu Leu 225 230 235 240 GAT CCA GGG ATG CTT GTG AAC ATC CAG CAG CCT TTG ATA CGT GAG GAT 768 Asp Pro Gly Met Leu Val Asn Ile Gln Gln Pro Leu Ile Arg Glu Asp 245 250 255 GGT ACG GTT CTC CTG GCC ACG GAT TCC AAG GCA GAA ACA AGC CAG GGA 816 Gly Thr Val Leu Leu Ala Thr Asp Ser Lys Ala Glu Thr Ser Gln Gly 260 265 270 270 GCT CTG GGC ACA CTG GCA AAC GTA GTG ACC TCC CTT GCC AAC CTG AGT 864 Ala Leu Gly Thr Leu Ala Asn Val Val Thr Ser Leu Ala Asn Leu Ser 275 280 285 GAG TCC TTG AAC AAC GGT GAC ACT TCA GAG ATC CAG CCA GAG GAC CAA 912 Glu Ser Leu Asn Asn Gly Asp Thr Ser Glu Ile Gln Pro Glu Asp Gln 290 295 300 TCT GCA AGC GAG ATT ACA CGG GCA TTT GAT ACC TTA GCT AAA GCA CTT960 Ser Ala Ser Glu Ile Thr Arg Ala Phe Asp Thr Leu Ala Lys Ala Leu 305 310 315 320 AAT ACC ACA GAC AGC TCC TCG CCT CCA AGC CTG GCC GAC GGG ATA GAC 1008 Asn Thr Thr Asp Ser Ser Ser Pro Pro Ser Leu Ala Asp Gly Ile Asp 325 330 335 GCC AGT GGA GGG GGC AGC ATC CAC GTC ATC AGC AGA GAC CAG TCG ACA 1056 Ala Ser Gly Gly Gly Ser Ile His Val Ile Ser Arg Asp Gln Ser Thr 340 345 350 CCC ATC ATT GAG GTT GAA GGC CCT CTC CTT TCA GAC ACA CAC GTC ACA 1104 Pro Ile Ile Glu Val Glu Gly Pro Leu Leu Ser Asp Thr His Val Thr 355 360 365 TTT AAG CTG ACA ATG CCC AGC CCG ATG CCG GAA TAC CTC AAC GTG CAC 1152 Phe Lys Leu Thr Met Pro Ser Pro Met Pro Glu Tyr Leu Asn Val His 370 375 380 TAC ATC TGC GAG TCT GCG TCC CGC CTG CTC TTC CTC TCC ATG CAC TGG 1200 Tyr Ile Cys Glu Ser Ala Ser Arg Leu Leu Phe Leu Ser Met His Trp 385 390 395 400 GCC AGG TCC ATC CCA GCC TTT CAG GCA CTT GGG CAG GAC TGT AAC ACC 1248 Ala Arg Ser Ile Pro Ala Phe Gln Ala Leu Gly Gln Asp Cys Asn Thr 405 410 415 AGC CTA GTG CGG GCC TGC TGG AAC GAG CTC T TC ACC CTA GGC CTG GCC 1296 Ser Leu Val Arg Ala Cys Trp Asn Glu Leu Phe Thr Leu Gly Leu Ala 420 425 430 CAG TGT GCC CAG GTC ATG AGT CTC TCC ACC ATC CTG GCC GCC ATT GTC 1344 Gln Cys Ala Gln Val Met Ser Leu Ser Thr Ile Leu Ala Ala Ile Val 435 440 445 AAC CAC CTG CAG AAC AGC ATC CAG GAA GAT AAA CTT TCT GGC GAC CGG 1392 Asn His Leu Gln Asn Ser Ile Gln Glu Asp Lys Leu Ser Gly Asp Arg 450 455 460 ATC AAG CAG GTC ATG GAG CAC ATC TGG AAG CTG CAG GAG TTC TGC AAC 1440 Ile Lys Gln Val Met Glu His Ile Trp Lys Leu Gln Glu Phe Cys Asn 465 470 475 475 480 AGC ATG GCG AAG CTG GAT ATA GAC GGC TAT GAG TAC GCATAC CTAC AAA 1488 Ser Met Ala Lys Leu Asp Ile Asp Gly Tyr Glu Tyr Ala Tyr Leu Lys 485 490 495 GCT ATA GTT CTC TTT AGC CCC GAT CAT CCA GGT TTG AGT GGC ACA AGC 1536 Ala Ile Val Leu Phe Ser Pro Asp His Pro Gly Leu Ser Gly Thr Ser 500 505 510 CAA ATC GAA AAA TTC CAA GAA AAG GCA CAG ATG GAG TTG CAG GAC TAC 1584 Gln Ile Glu Lys Phe Gln Glu Lys Ala Gln Met Glu Leu Gln Asp Tyr 515 520 525 GTG CAG AAA ACC TAC TAC TC G GAA GAC ACG TAC CGA TTG GCC CGG ATT CTC 1632 Val Gln Lys Thr Tyr Ser Glu Asp Thr Tyr Arg Leu Ala Arg Ile Leu 530 535 540 GTC CGC CTG CCG GCA CTC AGG CTG ATG AGC GCC AAC ATA ACA GAA GAA 1680 Val Arg Leu Pro Ala Leu Arg Leu Met Ser Ala Asn Ile Thr Glu Glu 545 550 555 560 CTT TTT TTT ACT GGT CTC ATT GGC AAC GTT TCC ATA GAC AGC ATA ATC 1728 Leu Phe Phe Thrhr Gly Leu Ile Gly Asn Val Ser Ile Asp Ser Ile Ile 565 570 575 CCC TAT ATC CTC AAG ATG GAG ACC GCA GAA TAC AAC GGC CAG ATC ACA 1776 Pro Tyr Ile Leu Lys Met Glu Thr Ala Glu Tyr Asn Gly Gln Ile Thr 580 585 590 590 GGA GCC AGC CTA TAG 1791 Gly Ala Ser Leu 595

SEQ ID NO: 2 Sequence length: 596 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Met Thr Ser Pro Ser Pro Arg Ile Gln Ile Ile Ser Thr Asp Ser Thr 5 10 15 Val Ala Ser Pro Gln Arg Ile Gln Ile Val Thr Asp Gln Gln Thr Gly 20 25 30 Gln Lys Ile Gln Ile Val Thr Ala Val Asp Ala Ser Gly Ser Pro Lys 35 40 45 Gln Gln Phe Ile Leu Thr Gly Pro Asp Gly Ala Gly Thr Gly Lys Val 50 55 60 Ile Leu Ala Ser Pro Glu Thr Thr Ser Ala Lys Gln Leu Ile Phe Thr 65 70 75 80 Thr Ser Asp Asn Leu Val Pro Gly Arg Ile Gln Ile Val Thr Asp Ser 85 90 95 Ala Ser Val Glu Arg Leu Leu Gly Lys Ala Asp Val Gln Arg Pro Gln 100 105 110 Val Val Glu Tyr Cys Val Val Cys Gly Asp Lys Ala Ser Gly Arg His 115 120 125 Tyr Gly Ala Val Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Ser 130 135 140 Val Arg Lys Asn Leu Thr Tyr Ser Cys Arg Ser Asn Gln Asp Cys Ile 145 150 155 160 Ile Asn Lys His His Arg Asn Arg Cys Gln Phe Cys Arg Leu Lys Lys 165 170 175 Cys Leu Glu Met G ly Met Lys Met Glu Ser Val Gln Ser Glu Arg Lys 180 185 190 Pro Phe Asp Val Gln Arg Glu Lys Pro Ser Asn Cys Ala Ala Ser Thr 195 200 205 Glu Lys Ile Tyr Ile Arg Lys Asp Leu Arg Ser Pro Leu Ile Ala Thr 210 215 220 Pro Thr Phe Val Ala Asp Lys Asp Gly Ala Arg Gln Thr Gly Leu Leu 225 230 235 240 Asp Pro Gly Met Leu Val Asn Ile Gln Gln Pro Leu Ile Arg Glu Asp 245 250 255 Gly Thr Val Leu Leu Ala Thr Asp Ser Lys Ala Glu Thr Ser Gln Gly 260 265 270 Ala Leu Gly Thr Leu Ala Asn Val Val Thr Ser Leu Ala Asn Leu Ser 275 280 285 Glu Ser Leu Asn Asn Gly Asp Thr Ser Glu Ile Gln Pro Glu Asp Gln 290 295 300 Ser Ala Ser Glu Ile Thr Arg Ala Phe Asp Thr Leu Ala Lys Ala Leu 305 310 315 320 Asn Thr Thr Asp Ser Ser Ser Pro Pro Ser Leu Ala Asp Gly Ile Asp 325 330 335 Ala Ser Gly Gly Gly Ser Ile His Val Ile Ser Arg Asp Gln Ser Thr 340 345 350 Pro Ile Ile Glu Val Glu Gly Pro Leu Leu Ser Asp Thr His Val Thr 355 360 365 Phe Lys Leu Thr Met Pro Ser Pro Met Pro Glu Tyr Leu Asn Val His 370 375 380 380 Tyr Ile Cys Glu SerAla Ser Arg Leu Leu Phe Leu Ser Met His Trp 385 390 395 400 400 Ala Arg Ser Ile Pro Ala Phe Gln Ala Leu Gly Gln Asp Cys Asn Thr 405 410 415 Ser Leu Val Arg Ala Cys Trp Asn Glu Leu Phe Thr Leu Gly Leu Ala 420 425 430 Gln Cys Ala Gln Val Met Ser Leu Ser Thr Ile Leu Ala Ala Ile Val 435 440 445 Asn His Leu Gln Asn Ser Ile Gln Glu Asp Lys Leu Ser Gly Asp Arg 450 455 460 Ile Lys Gln Val Met Glu His Ile Trp Lys Leu Gln Glu Phe Cys Asn 465 470 475 480 480 Ser Met Ala Lys Leu Asp Ile Asp Gly Tyr Glu Tyr Ala Tyr Leu Lys 485 490 495 Ala Ile Val Leu Phe Ser Pro Asp His Pro Gly Leu Ser Gly Thr Ser 500 505 510 Gln Ile Glu Lys Phe Gln Glu Lys Ala Gln Met Glu Leu Gln Asp Tyr 515 520 525 Val Gln Lys Thr Tyr Ser Glu Asp Thr Tyr Arg Leu Ala Arg Ile Leu 530 535 540 Val Arg Leu Pro Ala Leu Arg Leu Met Ser Ala Asn Ile Thr Glu Glu 545 550 555 560 Leu Phe Phe Thr Gly Leu Ile Gly Asn Val Ser Ile Asp Ser Ile Ile 565 570 575 Pro Tyr Ile Leu Lys Met Glu Thr Ala Glu Tyr Asn Gly Gln Ile Thr 580 585 590 Gly Ala Ser Leu 595

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (72) Inventor Shigeaki Kato 1-1-1 Yayoi Bunyo-ku, Tokyo Inside the Institute for Molecular and Cell Biology, The University of Tokyo

Claims (4)

[Claims] 1. An anti-estrogen gene therapy agent comprising as an active ingredient DNA encoding the nuclear receptor TR4 or a mutant thereof. 2. The anti-estrogen gene therapeutic agent according to claim 1, which suppresses the transcription promoting activity of an estrogen target gene. 3. The anti-estrogen gene therapy agent according to claim 1, wherein the agent acts specifically on bone-derived cells. 4. The anti-estrogen gene therapeutic according to claim 3, which is used for treating or preventing bone metastasis derived from osteosarcoma or breast cancer.