JP2008094766A - Agent for preventing or treating hypertriglyceridemia, and method for screening the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for preventing or treating hypertriglyceridemia, having a new mechanism of action, and to provide a method for screening the agent. <P>SOLUTION: The agent for preventing or treating the hypertriglyceridemia contains a substance inhibiting the expression or activity of Stra13. The method for screening the substance preventing or treating the hypertriglyceridemia includes evaluating whether a test agent inhibits the expression or activity of the Stra13 or not. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a prophylactic / therapeutic agent for hypertriglyceridemia, a screening method thereof, and the like.

Hypertriglyceridemia is a type of hyperlipidemia in which a lot of triglycerides are present in blood (150 mg / dL or more). Hypertriglyceridemia is common in visceral fat obesity and is considered to be associated with the onset of cardiovascular diseases (eg, arteriosclerosis).
Since most of the etiology of hypertriglyceridemia is excessive intake of alcohol and sugar, diet therapy is generally effective for the treatment. However, dietary therapy prevents and treats hypertriglyceridemia without relying on diet alone, because it imposes mental stress on patients such as being unable to eat enough of their favorite food due to dietary restrictions. Development of new drugs that can be obtained is required.
On the other hand, Stra13 (also called Dec1, Clast5, or Sharp2) is a bHLH type transcription factor (Non-patent Document 1) isolated as a factor induced by retinoic acid. The transcription factor is induced by Hypoxia (hypoxic conditions) and has an action of suppressing differentiation of adipocytes (Non-patent Document 2, Patent Document 1), and is one of genes induced by leptin, (Patent Document 2) and expression induction of TGF-β, BMP2 or insulin in mouse EC cell line ATDC5 (Non-patent Document 3) have been reported.
Further, the present inventors have found that insulin-derived signal transduction in the liver, that is, insulin responsiveness can be detected by using the expression of the Stra13 gene or a protein encoded by the gene (Stra13) as an index ( Patent Document 3).
However, the relationship between Stra13 and fatty acid synthesis and hypertriglyceridemia in the liver was not known at all.

Special Table 2002-507405 Special Publication 2002-525067 Japanese Unexamined Patent Publication No. 2005-6645 Genes & Dev., 11, 2052-2065 (1997) Dev. Cell, 2, 331-341 (2002) J. Biol. Chem., 277, 50112-50120 (2002)

  The problem to be solved by the present invention is to identify transcription factors involved in the expression control of fatty acid synthesis genes in the liver, and to prevent and treat hypertriglyceridemia by regulating the expression and function of the transcription factors It is an object of the present invention to develop a drug and provide a screening method for a prophylactic / therapeutic agent for hypertriglyceridemia using the expression and function of the transcription factor as an index.

As a result of diligent studies to solve the above problems, the present inventors surprisingly suppress the expression of Stra13 using shRNA against Stra13, and surprisingly, the genes related to fatty acid synthesis such as SREBP1c and SCD1. It was found that expression was reduced and serum triglyceride levels were reduced. This result was in contrast to a report (Patent Document 3) that promotion of Stra13 expression in the liver can improve or treat insulin resistance and glucose metabolism.
From the above findings, it has been clarified that Stra13 has an action of promoting the expression of genes involved in fatty acid synthesis such as SREBP1c and SCD1 in the liver and increasing the serum triglyceride concentration. Therefore, a substance that inhibits the expression and function of Stra13 can be a prophylactic / therapeutic agent for hypertriglyceridemia.
The present invention has been completed based on the above findings. That is, the present invention relates to the following.

[1] A prophylactic / therapeutic agent for hypertriglyceridemia, comprising a substance that suppresses the expression or activity of Stra13.
[2] The agent according to [1] above, wherein the substance that suppresses the expression or activity of Stra13 is any one of (i), (ii), and (iii) below:
(I) a substance that suppresses the expression of Stra13 selected from the group consisting of Stra13 antisense nucleic acid, Stra13 ribozyme and Stra13 siRNA;
(Ii) a dominant negative mutant, a nucleic acid encoding the mutant, a substance that suppresses the activity of Stra13 selected from the group consisting of Stra13 antibody and a nucleic acid encoding Stra13 antibody; or (iii) (i), (ii) An expression vector capable of expressing any of the nucleic acids.
[3] The agent according to [1] above, wherein the substance suppresses the expression or activity of Stra13 in the liver.
[4] A screening method for a substance capable of preventing or treating hypertriglyceridemia, comprising evaluating whether a test substance suppresses the expression or activity of Stra13.
[5] The method described in [4] above, wherein the substance that suppresses the expression or activity of Stra13 is selected as a substance that can prevent or treat hypertriglyceridemia.
[6] The method according to [4] above, comprising the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of measuring the expression of Stra13;
(B) a step of measuring the expression level of Stra13 in the cell contacted with the test substance and comparing the expression level with the expression level of Stra13 in the control cell not contacted with the test substance;
(C) A step of selecting a test substance that suppresses the expression level of Stra13 based on the comparison result of (b).
[7] The method described in [6] above, wherein the cells capable of measuring the expression of Stra13 are hepatocytes.

  The agent of the present invention is useful for the prophylaxis or treatment of diseases / conditions in which a decrease in blood triglyceride concentration is desired by a novel mechanism of action of suppressing the expression or activity of Stra13. The screening method of the present invention is useful because it enables the development of a substance capable of preventing / treating hypertriglyceridemia.

  Hereinafter, the content of the present invention will be described more specifically, but the present invention is not limited to this description.

1. The present invention provides a prophylactic / therapeutic agent for hypertriglyceridemia comprising a substance that suppresses the expression or activity of Stra13. Preferably, the substance suppresses Stra13 expression or activity in the liver (hepatocytes). More preferably, the inhibitory effect is liver (hepatocyte) specific.

  As used herein, “hypertriglyceridemia” refers to a type of hyperlipidemia in which the blood triglyceride concentration is higher than normal. According to the diagnostic criteria established by the Japanese Society for Arteriosclerosis, in humans, a subject can usually be diagnosed with hypertriglyceridemia when the blood triglyceride concentration is 150 mg / dl or higher.

  As used herein, “expression” means that a protein is produced and functionally localized at its site of action. Therefore, a “substance that suppresses expression” acts at any stage of gene transcription, post-transcriptional regulation, translation into protein, post-translational modification, localization (ie, nuclear translocation) and protein folding. It may be. On the other hand, the “substance that suppresses activity” refers to a substance that acts on a protein in a cell and prevents the action of the protein.

  Stra13 used in the present invention is usually mammalian Stra13. As used herein, “mammal” includes, for example, primates, laboratory animals, domestic animals, pets and the like, and is not particularly limited. Rat, mouse, guinea pig, rabbit, horse, cow, goat, sheep, dog, cat and the like.

  Stra13 is a known gene, and its nucleotide sequence is also known. As the nucleotide sequence (cDNA sequence) of the human Stra13 gene, the nucleotide sequence represented by SEQ ID NO: 1 is known (GenBank Accession No. NM_003670), and the method for obtaining it is also known as Biochem. Biophys. Res. Commun. 236 ( 2), 294-298 (1997). The orthologs include mouse Stra13 gene (Genbank Accession No. NM_011498) having the nucleotide sequence (cDNA sequence) represented by SEQ ID NO: 3 and rat Stra13 gene having the nucleotide sequence (cDNA sequence) represented by SEQ ID NO: 5 ( Genbank Accession No. NM_053328) etc., and these acquisition methods are also described in Genes Dev. 11 (16), 2052-2065 (1997), Mol. Cell. Neurosci. 10 (3-4), 460-475 (1997). ).

  Examples of the activity of Stra13 include activity of promoting the expression of genes involved in fatty acid synthesis such as SREBP1c and SCD1 in the liver and the like, and binding activity to DNA containing class B E-box elements.

(Substance that suppresses the expression of Stra13)
Examples of substances that suppress the expression of Stra13 include transcriptional repressors, RNA polymerase inhibitors, RNA-degrading enzymes, protein synthesis inhibitors, nuclear translocation inhibitors, proteolytic enzymes, and protein denaturing agents. In order to minimize the adverse effects on other genes and proteins expressed in, it is important that the substance be able to act specifically on the target molecule.

  From such a viewpoint, one preferred embodiment of the substance that suppresses the expression of Stra13 is an antisense nucleic acid of Stra13 mRNA or initial transcription product. The “antisense nucleic acid” is a nucleotide sequence that can hybridize with the target mRNA (initial transcript) under physiological conditions of a cell that expresses the target mRNA (early transcript), and in the hybridized state, A nucleic acid capable of inhibiting translation of a polypeptide encoded by a target mRNA (early transcript). The type of the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. In addition, since the phosphodiester bond of a natural type antisense nucleic acid is easily degraded by a nucleolytic enzyme present in cells, the antisense nucleic acid of the present invention has a thiophosphate type (phosphorus) that is stable to the degrading enzyme. It is also possible to synthesize using modified nucleotides such as P′O in the acid bond and P═S) or 2′-O-methyl type. Other important factors for the design of antisense nucleic acid include enhancement of water solubility and cell membrane permeability. These can also be overcome by devising dosage forms such as the use of liposomes and microspheres.

  The length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the mRNA or initial transcription product of Stra13 and inhibit the translation of Stra13 polypeptide in the hybridized state, and is about 15 It may be a sequence that is as long as a base and includes a sequence complementary to the full-length sequence of mRNA (initial transcription product). Considering the specificity of hybridization, the length of the antisense nucleic acid is, for example, about 15 bases or more, preferably about 18 bases or more, more preferably about 20 bases or more. In consideration of easiness of synthesis, antigenicity problems, etc., the length of the antisense nucleic acid is, for example, about 200 bases or less, preferably about 50 bases or less, more preferably about 30 bases or less. That is, examples of the antisense nucleic acid include oligonucleotides composed of about 15 to about 200 bases, preferably about 18 to about 50 bases, more preferably about 20 to about 30 bases.

  The target nucleotide sequence of the antisense nucleic acid is not particularly limited as long as the antisense nucleic acid hybridizes to inhibit the translation of Stra13 or a functional fragment thereof. The full length of Stra13 mRNA (initial transcription product) It may be a sequence or a partial sequence (for example, about 15 bases or more, preferably about 18 bases or more, more preferably about 20 bases or more), or an intron part of an initial transcription product, When an oligonucleotide is used as an antisense nucleic acid, the target sequence is preferably located from the 5 ′ end of Stra13 mRNA to the C terminus of the coding region.

  The nucleotide sequence of the antisense nucleic acid usually has at least 70%, preferably 80%, more preferably 90%, most preferably 95% or more (eg 100%) homology with the complementary sequence of the target nucleotide sequence. Nucleotide sequence homology can be calculated under default conditions using the homology calculation algorithm NCBI BLAST or the like.

  Furthermore, antisense nucleic acids not only hybridize with Stra13 mRNA or initial transcripts to inhibit translation, but also bind to the Stra13 gene, a double-stranded RNA, to form a triplex. It may be one that can inhibit transcription into the.

  Another preferred embodiment of the substance that suppresses the expression of Stra13 is a ribozyme that can specifically cleave Stra13 mRNA or an initial transcript within the coding region (including an intron in the case of the initial transcript). . “Ribozyme” refers to RNA having an enzyme activity for cleaving nucleic acids. Recently, it has been clarified that oligoDNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. Then, as long as it has sequence-specific nucleic acid cleavage activity, it shall be used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. Further, when the ribozyme is used in the form of an expression vector containing RNA encoding the ribozyme, it can be a hybrid ribozyme in which a sequence modified with tRNA is further linked in order to promote the transfer to the cytoplasm [ Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

  Yet another embodiment of the substance that suppresses the expression of Stra13 is homologous to the nucleotide sequence of Stra13 mRNA or the initial transcript or a partial sequence thereof (preferably within the coding region) (including an intron in the case of the initial transcript). It is a double-stranded oligo RNA containing a unique nucleotide sequence, so-called siRNA. When a short double-stranded RNA is introduced into a cell, the mRNA complementary to the RNA is degraded. So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Recently, it has been confirmed that this phenomenon also occurs in animal cells [Nature, 411 (6836): 494-498 (2001)], and has attracted attention as an alternative to ribozyme. As siRNA, one synthesized by itself can be used as described later, but a commercially available one may be used.

  siRNA is typically a double-stranded oligo RNA consisting of RNA having a sequence homologous to the nucleotide sequence of the mRNA or initial transcription product of the target gene or a partial sequence thereof (hereinafter referred to as target nucleotide sequence) and its complementary strand. . A single-stranded RNA in which a sequence homologous to a target nucleotide sequence (first sequence) and its complementary sequence (second sequence) are linked via a hairpin loop portion, RNA (small hairpin RNA: shRNA) in which the first sequence forms a double-stranded structure with the second sequence by taking the structure is also one of the preferred embodiments of siRNA.

  The length of the portion homologous to the target nucleotide sequence contained in siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases) in length, but RNA interference Is not particularly limited as long as it can cause If the siRNA is longer than 23 bases, the siRNA is degraded in the cell to produce siRNA of about 20 bases. Therefore, theoretically, the upper limit of the length of the portion homologous to the target nucleotide sequence is the target. It is the full length of the nucleotide sequence of the gene mRNA or early transcript. However, considering the ease of synthesis, antigenicity problems, etc., the length of the homologous portion is, for example, about 200 bases or less, preferably about 50 bases or less, more preferably about 30 bases or less. That is, the length of the homologous portion is, for example, about 18 bases or more, preferably about 18 to about 200 bases, more preferably about 20 to about 50 bases, and further preferably about 20 to about 30 bases.

  Further, the total length of siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases) in length, but is particularly limited as long as it can cause RNA interference. In theory, there is no upper limit on the length of siRNA. However, considering the ease of synthesis, antigenic problems, etc., the length of the siRNA is, for example, about 200 bases or less, preferably about 50 bases or less, more preferably about 30 bases or less. That is, the length of siRNA is, for example, about 18 bases or more, preferably about 18 to about 200 bases, more preferably about 20 to about 50 bases, and further preferably about 20 to about 30 bases. In addition, the length of the nucleic acid of shRNA shall be shown as the length of the double stranded part at the time of taking a double stranded structure.

  The relationship between the target nucleotide sequence and the sequence homologous to that contained in the siRNA is preferably 100% identical. However, for mutations of bases at positions off the center of the siRNA (which may be within a homology range of at least 70%, preferably 80%, more preferably 90%, most preferably 95% or more) However, the cleavage activity due to RNA interference is not lost, but partial activity may remain. On the other hand, the mutation of the base at the center of siRNA has a large effect, and the mRNA cleavage activity due to RNA interference can be extremely reduced.

  siRNA may have an additional base consisting of 5 bases or less, preferably 2 bases, not forming a base pair, at the 5 'or 3' end. The additional base may be DNA or RNA, but the use of DNA can improve the stability of siRNA. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples include, but are not limited to, ', uuuuu-3'.

  The length of the loop part of the hairpin loop of shRNA is not particularly limited as long as it can cause RNA interference, but is usually about 5 to 25 bases. The nucleotide sequence of the loop portion is not particularly limited as long as it can form a loop and shRNA can cause RNA interference.

  Antisense oligonucleotides and ribozymes determine mRNA or initial transcript target sequences based on Stra13 cDNA sequences or genomic DNA sequences, and are commercially available DNA / RNA automatic synthesizers (Applied Biosystems, Beckman, etc.) Can be prepared by synthesizing a complementary sequence thereto. For siRNA, the sense strand and the antisense strand were respectively synthesized by a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, and then about 30 to about 70 ° C. For about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to overlap each other, annealing them, and ligating with ligase.

  When the substance that suppresses the expression of Stra13 is a nucleic acid molecule such as an antisense nucleic acid or siRNA (hereinafter also referred to as an effective nucleic acid molecule if necessary), the preventive / therapeutic agent of the present invention expresses the effective nucleic acid molecule. A possible (encoding) expression vector can also be used as an active ingredient. In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule is usually operably linked to a promoter capable of exhibiting promoter activity in mammalian cells (eg, hepatocytes) to be administered. Has been.

  The promoter to be used is not particularly limited as long as it can function in mammalian cells to be administered. As the promoter, a pol I promoter, pol II promoter, pol III promoter, or the like can be used. Specifically, SV40-derived early promoter, viral promoter such as cytomegalovirus LTR, mammalian constituent protein gene promoter such as β-actin gene promoter, and RNA promoter such as tRNA promoter are used.

  When the effective nucleic acid molecule is RNA such as ribozyme or siRNA, it is preferable to use a pol III promoter as the promoter. Examples of the polIII promoter include U6 promoter, H1 promoter, tRNA promoter and the like.

  The expression vector of the present invention preferably contains a transcription termination signal, that is, a terminator region, downstream of an oligo (poly) nucleotide encoding an effective nucleic acid molecule. Furthermore, a selection marker gene for selecting transformed cells (a gene that imparts resistance to drugs such as tetracycline, ampicillin, and kanamycin, a gene that complements an auxotrophic mutation, and the like) can be further contained.

  The vector used as the expression vector in the present invention is not particularly limited, and examples of suitable vectors for administration to mammals such as humans include viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses. Among these, adenovirus has advantages such as extremely high gene transfer efficiency and can be introduced into non-dividing cells. However, since integration of the transgene into the host chromosome is extremely rare, gene expression is transient and usually lasts only about 4 weeks. Considering the persistence of the therapeutic effect, use of an adeno-associated virus that has relatively high gene transfer efficiency, can be introduced into non-dividing cells, and can be integrated into the chromosome via an inverted terminal repeat (ITR) Also preferred.

  Effective nucleic acid molecules such as antisense nucleic acids and siRNA are inherently foreign, and their constitutive and excessive expression is highly toxic to host animals into which the gene has been introduced, and may cause side effects. Therefore, in a preferred embodiment of the present invention, the expression vector can specifically express an effective nucleic acid molecule in a tissue (eg, liver) in order to prevent adverse effects due to overexpression of the effective nucleic acid molecule at an unnecessary site. As shown in the Examples, the blood triglyceride concentration can be strongly suppressed by suppressing the expression or activity of liver Stra13.

  As a first embodiment of such a vector, an oligo (poly) encoding an effective nucleic acid molecule operably linked to a promoter derived from a gene specifically expressed in the liver (hepatocyte) of an animal to be administered. Examples include vectors containing nucleotides. Examples of promoters derived from genes that are specifically expressed in hepatocytes include promoters of hepatocyte-specific genes such as albumin promoters.

  In addition, a second embodiment of the vector includes a vector capable of delivering an oligo (poly) nucleotide encoding an effective nucleic acid molecule specifically to the liver (hepatocyte) of an animal to be administered. Examples of such vectors include adenovirus vectors. Since adenovirus infects the liver (hepatocytes) with extremely high efficiency, the use of adenovirus makes it possible to efficiently / specifically suppress the expression of Stra13 in the liver (hepatocytes).

  Furthermore, in another preferred embodiment of the present invention, the expression vector can express the effective nucleic acid molecule in a time-specific manner in order to prevent adverse effects due to overexpression of the effective nucleic acid molecule at an unnecessary time. Such an embodiment includes a vector comprising an oligo (poly) nucleotide encoding an effective nucleic acid molecule operably linked to an inducible promoter whose expression is controlled in trans by an exogenous substance. If this vector is used, the expression of an effective nucleic acid molecule can be induced at any time by administering the inducer at a desired time. In addition, by administering the inducer locally to a desired tissue (eg, liver), the expression of an effective nucleic acid molecule can be induced specifically in the tissue.

  Administration of the prophylactic / therapeutic agent of the present invention comprising an expression vector capable of expressing an effective nucleic acid molecule as an active ingredient is carried out by an in vivo method in which the vector is directly introduced into the body of the administration subject. In this case, the viral vector is administered intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, intraperitoneally, etc. in the form of an injection or the like. When the production of neutralizing antibodies against viral vectors becomes a problem, if the vectors are injected locally in the vicinity of the liver (in situ method), adverse effects due to the presence of antibodies can be reduced.

(Substance that suppresses Stra13 activity)
The substance that suppresses the activity of Stra13 is not particularly limited as long as it is a substance that can reduce the activity of Stra13, but in order to minimize the adverse effect on other genes and proteins, it is specific to the target molecule. It is important that the substance be able to act. Examples of the substance that specifically suppresses the activity of Stra13 include a Stra13 dominant negative mutant, a nucleic acid encoding the same, an expression vector containing the nucleic acid, a low molecular weight organic compound, and the like. Here, the expression vector is the same as described above.

  The dominant negative mutant of Stra13 refers to a substance whose activity is reduced by introducing a mutation into Stra13. Stra13 dominant negative mutants can inhibit its activity indirectly by competing with natural Stra13. A dominant negative mutant of Stra13 can be produced by introducing a mutation into Stra13. Examples of the mutation include an amino acid mutation (for example, deletion, substitution or addition of one or more amino acids) that causes a decrease in activity at a site such as a helix-loop-helix domain or a DNA binding site. It is done. The amino acid mutation can be introduced by a method known per se using PCR or a known kit.

  Another preferred embodiment of the substance that suppresses the activity of Stra13 is an antibody that specifically recognizes Stra13 (Stra13 antibody). The Stra13 antibody may be a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. The Stra13 antibody may be an antibody fragment (eg, Fab, F (ab ′) 2) or a recombinant antibody (eg, a single chain antibody). Furthermore, a nucleic acid encoding a Stra13 antibody and an expression vector containing the nucleic acid are also preferable as substances that suppress the activity of Stra13. Here, the expression vector is the same as described above.

  The prophylactic / therapeutic agent of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that suppresses the expression or activity of Stra13 as described above.

  Examples of pharmaceutically acceptable carriers include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, citric acid, Fragrances such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspensions such as methylcellulose and polyvinylpyrrolide, dispersants such as surfactants, water, Although diluents, such as physiological saline, base wax, etc. are mentioned, it is not limited to them.

  Preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like.

  Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  The dosage of the preparation of the present invention varies depending on the activity and type of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc. The amount of active ingredient per day for an adult is about 0.001 to about 500 mg / kg.

  The prophylactic / therapeutic agent of the present invention is preferably administered to a mammal so that a substance that suppresses the expression or activity of Stra13, which is its active ingredient, is delivered to the liver (hepatocytes).

  The prophylactic / therapeutic agent of the present invention is useful for the prophylaxis / treatment of diseases / conditions in which a reduction in blood triglyceride concentration is desired, such as hypertriglyceridemia, hyperlipidemia, obesity / obesity, metabolic syndrome and the like. is there.

2. Method for screening substance capable of preventing / treating hypertriglyceridemia The present invention also provides a method for preventing / treating hypertriglyceridemia, comprising evaluating whether a test substance suppresses the expression or activity of Stra13. A method for screening a substance to be obtained, and a substance obtainable by the method are provided. In the screening method of the present invention, a substance that suppresses the expression or activity of Stra13 is selected as a substance that can prevent or treat hypertriglyceridemia.

  The test substance used for the screening method of the present invention may be any known compound and novel compound, for example, using nucleic acid, carbohydrate, lipid, protein, peptide, low-molecular-weight organic compound, combinatorial chemistry technique. The prepared compound library, random peptide library, natural components derived from microorganisms, animals and plants, marine organisms, and the like can be mentioned.

The screening method for evaluating whether the test substance suppresses the expression of Stra13 includes, for example, the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of measuring the expression of Stra13;
(B) a step of measuring the expression level of Stra13 in the cell contacted with the test substance and comparing the expression level with the expression level of Stra13 in the control cell not contacted with the test substance;
(C) A step of selecting a test substance that suppresses the expression level of Stra13 based on the comparison result of (b).

  “A cell capable of measuring expression” refers to a cell capable of directly or indirectly evaluating the expression level of mRNA or protein to be measured. Cells that can directly evaluate the expression level of the mRNA or protein to be measured include cells that can naturally express the measurement target, while the expression level of the mRNA or protein to be measured can be indirectly evaluated. Examples of such cells include cells that enable a reporter assay for the transcriptional regulatory region to be measured.

  The measurement target, for example, a cell capable of naturally expressing Stra13 is not particularly limited as long as it can potentially express Stra13 mRNA. Examples of the cell include primary cultured cells, cell lines derived from the primary cultured cells, and the like. Can be used. Examples of cells that can naturally express Stra13 include hepatocytes.

  A cell that enables reporter assay for a transcriptional regulatory region of Stra13, such as a transcriptional regulatory region of Stra13, is operably linked to the transcriptional regulatory region of Stra13 (eg, DNA consisting of a base sequence of about 2 kbp upstream from the transcription start point). Cells containing a reporter gene (eg, GFP gene). Hepatocytes are preferred as the cells to be measured because they express a physiological transcriptional regulatory factor for Stra13 and are considered to be more appropriate for evaluation of the expression regulation of Stra13.

  Contact between the test substance and cells capable of measuring the expression of Stra13 is performed in a culture medium. The culture medium is appropriately selected according to the cells capable of measuring the expression of Stra13. For example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM), etc. is there. The culture conditions are appropriately determined in the same manner. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is about 12 to about 72 hours.

  When cells capable of expressing Stra13 are used, the expression level is measured for mRNA or protein. The expression level of mRNA is measured, for example, by preparing total RNA from cells and performing RT-PCR, Northern blotting or the like. The amount of protein expression can be measured, for example, by preparing an extract from cells and immunologically. As an immunological technique, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. When cells containing a reporter gene are used, the expression level is measured based on the signal intensity of the reporter gene.

  The comparison of expression levels can be preferably performed based on the presence or absence of a significant difference. It should be noted that the expression level of Stra13 in the control cells not contacted with the test substance is the expression level measured at the same time even if the expression level was measured in advance compared to the measurement of the expression level of Stra13 in the cells contacted with the test substance. Although it may be an amount, it is preferably an expression amount measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

A screening method for evaluating whether a test substance suppresses the activity of Stra13 includes, for example, the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of measuring the activity of Stra13;
(B) measuring the Stra13 activity in a cell contacted with a test substance and comparing the activity with the Stra13 activity in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that suppresses the Stra13 activity based on the comparison result of (b).

  The activity of Stra13 is not particularly limited as long as it can be measured or evaluated. For example, the activity of promoting the expression of genes involved in fatty acid synthesis such as SREBP1c and SCD1 in the liver (hepatocytes), class B E Binding activity to DNA containing -box elements can be evaluated. The activity of Stra13 preferably includes SCD1 expression promoting activity.

  Here, when evaluating the activity that promotes gene expression related to fatty acid synthesis as Stra13 activity, “cell capable of measuring Stra13 activity” means the level of gene expression related to fatty acid synthesis promoted by intracellular Stra13 expression. Refers to cells that can be evaluated. Examples of the cells include cells that express Stra13 and express genes (SREBP1c, SCD1, etc.) involved in fatty acid synthesis induced thereby (for example, hepatocytes). In the cells, the Stra13 activity can be indirectly measured as the expression level of a gene involved in fatty acid synthesis.

  Contact between the test substance and cells capable of measuring the activity of Stra13 is performed in a culture medium. The culture conditions are the same as those in the screening method for evaluating whether the test substance suppresses the expression of Stra13.

  The measurement of Stra13 activity is performed on mRNA or protein of a gene involved in fatty acid synthesis. The method for measuring the expression levels of mRNA and protein is the same as that in the screening method for evaluating whether or not the test substance suppresses the expression of Stra13.

  The comparison of Stra13 activity can be preferably made based on the presence or absence of a significant difference. The Stra13 activity in the case where the test substance is not contacted may be the activity measured in advance or the activity measured simultaneously with respect to the measurement of the Stra13 activity in the case where the test substance is contacted. The activity measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment is preferable.

  Then, a substance that suppresses the expression or activity of Stra13 obtained as a result of the comparison is selected as a substance that can prevent or treat hypertriglyceridemia.

  The compound obtained by the screening method of the present invention can be used for diseases / conditions in which a reduction in blood triglyceride concentration is desired, such as hypertriglyceridemia, hyperlipidemia, obesity / obesity, metabolic syndrome, etc. It is useful as an active ingredient of prophylactic / therapeutic agents for diseases.

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

Example 1: Construction of shStra13 adenovirus vector
A DNA encoding shRNA against Stra13 having GTGAAAGCATTGACAAATC (SEQ ID NO: 7) as a target sequence was synthesized. The nucleotide sequence of the DNA is
gtttGTGGAAGTATTGATAAATAACGTGTGCTGTCCGTTATTTGTCAATGCTTTCACTTTTT (SEQ ID NO: 8)
The sequence includes a loop sequence consisting of 15 bases.
A fragment obtained by connecting the above synthesized DNA fragment encoding mouse Stra13 gene shRNA downstream of the mouse U6 promoter was inserted into the SwaI site of cosmid vector pAxCAwt (Takara Bio Inc.) to obtain pAxCAwt-shSTRA13. Using the λ packaging kit GigapackXL (Stratagene), in vitro packaging of the obtained pAxCAwt-shSTRA13 was performed to obtain λ phage, and the λ phage was infected with E. coli DH5α. Infected E. coli was cultured in LB medium containing 50 ml 50 μg / ml ampicillin, and a large amount of cosmid DNA was prepared by Qiagen plasmid midi kit (Qiagen).
293 cells (Takara Bio Inc.) were cultured in D-MEM medium supplemented with 10% FCS (Nissui Pharmaceutical) at 37 ° C. and 5% carbon dioxide. 8 μg of pAxCAwt-shSTRA13 prepared as described above and 5 μl of restriction enzyme-treated DNA-TPC (Takara Bio) were mixed, and co-transfection into 293 cells was performed using the mixture by the calcium phosphate method. The cells were cultured as they were for 18 hours, and then the medium was replaced with 10% FCS-added D-MEM medium (Nissui Pharmaceutical), and further cultured for 12 hours. After completion of the culture, the cells were detached from the dish, and the recovered cell suspension and 293 cells were mixed and further cultured in 10% FCS-added D-MEM medium (Nissui Pharmaceutical). When the 293 cells were completely killed, the culture was rapidly frozen with dry ice. After subjecting the cells to ultrasonic disruption, the disrupted solution was centrifuged at 5000 rpm for 5 minutes, and the resulting supernatant was stored as a primary recombinant adenovirus solution (pAxCAwt-shmSTRA13).
10 μl of the primary recombinant virus solution prepared by the above method was added to 293 cells (Takara Bio Inc.), 5% FCS-added D-MEM medium (Nissui Pharmaceutical) was added thereto, and then the mixture was incubated at 37 ° C. The cells were cultured for 1 hour under 5% carbon dioxide. Further, 5% FCS-added D-MEM medium (Takara Bio Inc.) was added to the mixture, and the mixture was cultured at 37 ° C. under 5% carbon dioxide. After culturing for 3 days, the culture solution of virus-infected 293 cells (Takara Bio Inc.) was collected and rapidly frozen with dry ice. After subjecting the cells to ultrasonic disruption, the disrupted solution was centrifuged at 5000 rpm for 5 minutes, and the resulting supernatant was stored as a secondary recombinant adenovirus solution. On the other hand, the precipitate (cells) obtained at the same time by the above operation was also collected and snap frozen with dry ice. 10 × TNE (500 mM Tris-hydrochloric acid (pH = 7.5), 1 M NaCl, 100 mM EDTA) 40 μl, proteinase K (20 mg / ml) 4 μl and sterilized distilled water 356 μl were added to the collected cells, and the mixture was well suspended in Voltex. . To the obtained suspension, 4 μl of 10% SDS was added, and this was incubated at 50 ° C. for 1 hour. After treatment with phenol / chloroform / isoamyl alcohol (Nippon Gene), ethanol precipitation is performed, and the resulting DNA pellet is air-dried and dissolved in 50 μl of sterile distilled water containing 20 μg / ml RNaseA (Nippon Gene). A viral DNA solution was obtained. The obtained recombinant adenovirus DNA solution was cleaved with EcoRI 5U (Takara Bio Inc.), and the target fragment (about 1 Kbp) was confirmed. Subsequently, by repeating the above operation using this recombinant adenovirus DNA, the recombinant virus was subcultured to produce a high-titer recombinant virus, which was used in the following experiments. The titer of the virus was measured by an existing method (Japanese Patent Laid-Open No. 7-298877).

Example 2: Effect of suppressing liver-specific Stra13 expression in KKAy mice
From the tail vein of 8-9 week old male KKAy mice, adenoviral vector encoding shRNA against Stra13 (shStra13) or adenoviral vector encoding only U6 promoter (Cont) was obtained at 9 × 10 ⁹ pfu per 30 g mouse body weight. Administered in doses. 0.2 ml of virus suspension was injected per mouse. On day 4 after virus administration, the amount of liver Stra13 protein, the expression level of various genes in the liver, and the serum triglyceride concentration in the fed state were measured as needed. Significant difference test was performed by Student t-test.
As a result, the expression of genes related to fatty acid synthesis such as SREBP1c and SCD1 was significantly decreased by suppressing the expression of Stra13 in the liver (FIGS. 1A and 1B), and the serum triglyceride concentration was significantly decreased (FIG. 1C).

Example 3: Effect of suppressing liver-specific Stra13 expression in C57 / BL6 mice
From the tail vein of 8-9 week old male C57 / BL6 mice, an adenoviral vector encoding shRNA against Stra13 (shStra13) or an adenoviral vector encoding only the U6 promoter (Cont) was obtained at 9 × 10 ⁹ per 30 g of mouse body weight. Administered at a pfu dose. 0.2 ml of virus suspension was injected per mouse. On day 4 after virus administration, the amount of liver Stra13 protein, the expression level of various genes in the liver, and the serum triglyceride concentration in the fed state were measured as needed. Significant difference test was performed by Student t-test.
As a result, suppression of the expression of Stra13 in the liver significantly reduced the expression of the SCD1 gene involved in fatty acid synthesis (FIGS. 2A and 2B), and the serum triglyceride concentration significantly decreased (FIG. 2C). There was no effect on the expression of SREBP1c.

Example 4: Effect of suppressing liver-specific Stra13 expression in db / db mice
From the tail vein of 8-9 week old male db / db mice, adenoviral vector encoding shRNA against Stra13 (shStra13) or adenoviral vector encoding only U6 promoter (Cont) was obtained at 9 × 10 ⁹ per 30 g of mouse body weight. Administered at a pfu dose. 0.2 ml of virus suspension was injected per mouse. On day 4 after virus administration, the amount of liver Stra13 protein, the expression level of various genes in the liver, and the serum triglyceride concentration in the fed state were measured as needed. Significant difference test was performed by Student t-test.
As a result, the serum triglyceride concentration was significantly reduced by suppressing the expression of Stra13 in the liver (FIG. 3C). Expression of genes involved in fatty acid synthesis such as SREBP1c and SCD1 showed a decreasing tendency (FIG. 3B).
based on the above results,
(1) Stra13 has the effect of promoting the expression of genes involved in fatty acid synthesis such as SREBP1c and SCD1 in the liver and increasing the serum triglyceride concentration, and (2) using shRNA against Stra13 These results suggest that hypertriglyceridemia can be prevented and treated by suppressing the expression and activity of Stra13.

  The agent of the present invention is useful for the prophylaxis or treatment of diseases / conditions in which a reduction in blood triglyceride concentration is desired by a novel mechanism of action that suppresses the expression of Stra13. The screening method of the present invention is useful because it enables the development of a substance capable of preventing / treating hypertriglyceridemia.

Fig. 3 shows the effects of suppressing liver-specific Stra13 expression in KKAy mice on liver Stra13 protein levels (A), various gene expression levels (B) in liver, and serum triglyceride concentrations (C). B, C) shStra13, n = 15; Cont, n = 9. Each value is expressed as mean ± sem. ** P <0.01. Fig. 6 shows the effects of suppressing liver-specific Stra13 expression in C57 / BL6 mice on liver Stra13 protein levels (A), various gene expression levels (B) in liver, and serum triglyceride concentrations (C). B) shStra13, n = 7; Cont, n = 6. C) shStra13, n = 7; Cont, n = 8. Each value is expressed as mean ± sem. ** P <0.01, * P <0.05. The effect of suppressing liver-specific Stra13 expression in db / db mice on liver Stra13 protein levels (A), liver gene expression levels (B), and serum triglyceride concentrations (C) is shown. B, C) shStra13, n = 11; Cont, n = 6. Each value is expressed as mean ± sem. ** P <0.01.

Claims (7)

  A prophylactic / therapeutic agent for hypertriglyceridemia, comprising a substance that suppresses the expression or activity of Stra13. The agent according to claim 1, wherein the substance that suppresses the expression or activity of Stra13 is any of the following (i), (ii), and (iii):
(I) a substance that suppresses the expression of Stra13 selected from the group consisting of Stra13 antisense nucleic acid, Stra13 ribozyme and Stra13 siRNA;
(Ii) a dominant negative mutant, a nucleic acid encoding the mutant, a substance that suppresses the activity of Stra13 selected from the group consisting of Stra13 antibody and a nucleic acid encoding Stra13 antibody; or (iii) (i), (ii) An expression vector capable of expressing any of the nucleic acids.   The agent according to claim 1, wherein the substance suppresses the expression or activity of Stra13 in the liver.   A screening method for a substance capable of preventing or treating hypertriglyceridemia, comprising evaluating whether a test substance suppresses the expression or activity of Stra13.   The method according to claim 4, wherein the substance that suppresses the expression or activity of Stra13 is selected as a substance that can prevent or treat hypertriglyceridemia. The method of claim 4 comprising the following steps (a), (b) and (c):
(A) contacting a test substance with a cell capable of measuring the expression or activity of Stra13;
(B) a step of measuring the expression level or activity of Stra13 in a cell contacted with a test substance and comparing the expression level or activity with the expression level or activity of Stra13 in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that suppresses the expression level or activity of Stra13 based on the comparison result of (b).   The method according to claim 6, wherein the cell capable of measuring the expression or activity of Stra13 is a hepatocyte.