JP4412734B2 - A novel gene Brachyury Expression Nuclear Inhibitor, BENI that governs mesoderm formation in early vertebrate development - Google Patents

Description

The present invention relates to a cell differentiation regulator. More specifically, it relates to a differentiation regulator of mesodermal cells.

  In the early development period after fertilization, the process of determining the fate of undifferentiated cells starts in order to become various organs and tissues in the future. As a differentiation inducing factor having a crucially important function in determining the initial fate of cells at this time, there is maternal factor activin A identified in 1990 by Asashima et al. (Non-patent Document 1). That is, activin A, which is already present in the embryo as a maternal factor, switches on the induction of cell differentiation in the embryo immediately after fertilization, and many other factors are activated or suppressed depending on the switch. Then, various differentiation-inducing factors work stepwise and continuously to form various organs and tissues such as pancreas, muscle, heart, liver, blood vessels. However, if there is any abnormality in the factor that activates or suppresses the response to activin A in this way, the embryo cannot perform normal development (early development) after fertilization. Serious death and in some cases death. Such an abnormality is a genetic disease caused by a genetic abnormality. Therefore, by identifying a factor that responds to the action of activin A and identifying its action, it is possible not only to elucidate the mechanism of differentiation induction in the embryo in detail, but also to utilize this knowledge to investigate various genetic diseases. Early diagnosis and further clues for treatment can be expected.

  An exhaustive determination of the genomic sequence for several organisms, including humans, has been made and the existence of many genes has been revealed, but many of these genes remain unknown. There are many unknown genes that control the early development of the embryo.

The induction phenomenon in which a cell group having a new property is born by the interaction of cell groups having different properties has been attracting attention for a long time in understanding the mechanism of development. In particular, mesoderm induction is the earliest induction phenomenon in the embryo in which cells on the animal hemisphere side receive a signal from the plant hemisphere side to form a planned mesoderm region in the middle blastocyst stage. Nerve induction occurs following mesoderm induction, and is a phenomenon in which the ectoderm lining the mesoderm invades into the inside of the embryo by morphogenic movement is induced into the nerve. These induction phenomena have a great influence on the body plan in the subsequent vertebrate development, and molecular biological analysis is underway. In the early development of multicellular organisms, the identity of individual cells is very important. Individual cells establish their individuality by detecting the presence of extracellular ligand proteins with receptor proteins on the surface of the cell membrane, transmitting the signal into the cell, and controlling the expression of the target gene via proteins in the cytoplasm. To go. A series of these systems is called an intracellular signal transduction system.

Asashima, M .; Nakano, H .; , Shimada, K .; Kinoshita, K .; , Ishii, K .; Shiba, H .; & Ueno, N .; (1990) Roux's Arch. Dev. Biol. 198, 330-335.

  An object of the present invention is to provide a gene that responds early to activin A.

In order to identify such genes, the present inventors have first comprehensively captured genes that respond early to activin A, and then has a method for identifying the function of each gene. One suitable method for achieving the first objective is an exhaustive microarray method. The present inventors also examined Xenopus laevis embryos using an exhaustive microarray method. As a gene whose expression greatly decreases early in response to activin A, Unigene code Xl. 7756 (BENI, named Brachyury Expression N uclear Inhibitor) was discovered. This gene has a homologous gene in mammals such as humans and mice, but its function is completely unknown (FIG. 1).

  As shown in FIG. 1, Xenopus tropicalis, human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegirus), green fowl (Tetradon nigro), and the like. The homology with them is as high as 57% -93% in the N-terminal 1-58 amino acid sequence. On the other hand, homology on the C-terminal side is low.

  Accordingly, the present invention provides the following.

    (Item 1)
A composition comprising a nucleic acid encoding Brachyury Expression Nuclear Inhibitor (BENI) or a vector containing the nucleic acid for inhibiting differentiation into mesoderm,
    (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
    (B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity A polypeptide; or
    (C) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
  A composition.
    (Item 2)
The composition according to item 1, wherein the nucleic acid encodes the sequence represented by SEQ ID NO: 2.
    (Item 3)
The composition according to item 1, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 1.
    (Item 4)
A composition comprising a Brachyury Expression Nuclear Inhibitor (BENI) polypeptide for inhibiting differentiation into mesoderm, wherein the BENI comprises:
  (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
  (B) In the amino acid sequence set forth in SEQ ID NO: 2, one or more or one or several amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and suppression of Brachyury expression A polypeptide having activity; or
  (C) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
A composition.
    (Item 5)
Item 5. The composition according to Item 4, wherein the BENI has the sequence shown in SEQ ID NO: 2.
    (Item 6)
Item 5. The composition according to Item 4, wherein the BENI comprises a sequence represented by SEQ ID NO: 1.
    (Item 7)
  A composition for inducing mesoderm differentiation, the composition comprising:
  (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
  (B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity A polypeptide; or
  (C) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
A composition comprising an antisense nucleic acid molecule or RNAi molecule of a nucleic acid molecule encoding or a vector comprising said antisense nucleic acid molecule or RNAi molecule.
    (Item 8)
  8. A composition according to item 7, further comprising means for delivering the antisense nucleic acid molecule or RNAi molecule to a specific site.
    (Item 9)
  Item 9. The composition according to Item 8, wherein the means expresses the antisense nucleic acid molecule or RNAi molecule specifically at the specific site.
    (Item 10)
  Item 10. The composition according to Item 9, wherein the means is a promoter.
    (Item 11)
  Item 9. The composition according to Item 8, wherein the means is a directional liposome.
    (Item 12)
A method for screening a regulator of mesoderm differentiation induction,
  A) providing a candidate substance,
  B) subjecting it to an assay system to determine whether the candidate substance modulates the activity of BENI;
  C) A step of identifying a factor that regulates BENI among the candidate substances, and determining that the factor that regulates BENI is a regulator of BENI
And the BENI is
    (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
    (B) In the amino acid sequence set forth in SEQ ID NO: 2, one or more or one or several amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and suppression of Brachyury expression A polypeptide having activity; or
    (C) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1
Is that way.
    (Item 13)
13. The method according to item 12, wherein the candidate substance is selected from the group consisting of a nucleic acid molecule, polypeptide, lipid, sugar chain, small organic molecule and complex molecule thereof, and a combinatorial chemistry library.
    (Item 14)
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity A polypeptide; or
(C) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1
Or a nucleic acid molecule encoding the polypeptide of (a), (b) or (c)
A mesodermal gastrulation inhibitor, comprising:
    (Item 15)
    (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
    (B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity A polypeptide; or
    (C) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
  A mesodermal gastrulation promoter, comprising an antisense nucleic acid molecule or an RNAi molecule of a nucleic acid molecule encoding

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  Accordingly, it is understood that these and other advantages of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description, with reference where appropriate to the accompanying drawings and the like.

  A gene that responds early to activin A is provided.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles or adjectives (eg, “a”, “an”, “the”, etc., in the English language) also include the plural concept unless otherwise stated. is there. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition)
Listed below are definitions of terms particularly used in the present specification.

The "BENI" or "Brachyury Expression N uclear Inhibitor" as used herein, is a factor found by the present inventors, while the expression of a mesoderm-specific agent Brachyury suppressed in the nucleus, medium Regulates the induction of germ layer differentiation. Homologous genes BENI the other Xenopus l a e vis (SEQ ID NO: 1-2), as well as related species of Toripikarisu (Xenopus tropicalis) (SEQ ID NO: 3-4), human (SEQ ID NO: 5-10) rats (R.norvegirus) (SEQ ID NO: 15 - 16), mouse (M.musculus) (SEQ ID NO: 11 -1 4), are present a wide range Tetraodon (T.nigroviridis) (SEQ ID NO: 1 7 20). The present inventors have found that all of these homologous genes regulate the differentiation induction of mesoderm while suppressing the expression of this mesoderm-specific factor Brachyury in the nucleus.

This BENI is
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(C) a variant polypeptide or fragment thereof in which one or more or one or several amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2 A polynucleotide encoding a variant polypeptide having biological activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or a fragment thereof;
(E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to It may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or its complementary sequence, and encodes a polypeptide having biological activity.

BENI, when it comes to amino acid sequence,
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(B) in the amino acid sequence of SEQ ID NO: 2 , one or more or one or several amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and biological activity Having a polypeptide;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 ;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2 ; or (e) an amino acid sequence that is at least 70% identical to any one polypeptide of (a)-(d) And a polypeptide having biological activity,
It can be.

  As the biological activity of BENI, for example, the activity that regulates differentiation induction of mesoderm while suppressing the expression of Brachyury, which is a mesoderm-specific factor in the nucleus, is significant when activin A is added. In particular, the activity of increasing expression can be mentioned.

  As used herein, the term “mesoderm” refers to a germ layer that appears in the middle of the ectoderm and endoderm during the early development of most metazoans. Leaves) to fill the gaps between germ layers. In animals showing somite system, the mesoderm is segmented to show a repetitive structure in the anterior-posterior direction. Among adult organ systems and tissues, those whose main parts are derived from mesoderm are somewhat different depending on the group of animals, but there are muscle systems, connective tissues, skeletal systems, circulatory systems, excretory systems, reproductive systems, etc. is there. Scheduled mesoderm cells are present in specific parts of the embryo prior to gastrulation.

  As used herein, “differentiation” or “cell differentiation” refers to two or more morphologically and / or functionally qualitative differences among daughter cell populations derived from the division of one cell. A phenomenon in which types of cells are generated. Therefore, the process in which a cell population (cell lineage) derived from cells that cannot originally detect special characteristics exhibits distinct characteristics such as production of a specific protein is also included in differentiation. At present, it is common to consider cell differentiation as a state in which a specific gene group in the genome is expressed, and cell differentiation by searching for intracellular or extracellular factors or conditions that cause such gene expression state. Can be identified. The result of cell differentiation is in principle stable, especially in animal cells, which differentiates into other types of cells only in exceptional cases. Inducing differentiation is referred to as “differentiation induction”.

  The cells used in the present invention may be cells derived from any organism (for example, any kind of multicellular organisms (eg, animals (eg, vertebrates, invertebrates), plants (eg, monocotyledonous plants, dicotyledonous plants, etc.)). ) Etc.)). Preferably, cells derived from vertebrates (eg, eelfish, lamprey, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) are used, more preferably mammals (eg, single pores, Marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed, rodent, scale, sea cattle, cetacean, primate , Rodents, rabbit eyes, etc.). More preferably, mammals (eg, rodents (mouse, rat, etc.), primates (monkey, human, etc.)) are used.

  As used herein, “tissue” refers to a population of cells having substantially the same function and / or morphology in a multicellular organism. A “tissue” usually has the same origin, but even cell populations with different origins can be referred to as a tissue if they have the same function and / or morphology. Therefore, when a tissue is regenerated using the stem cells of the present invention, a cell population having two or more different origins can constitute one tissue. Usually, tissue constitutes part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis.

In the present specification, “activin” refers to a protein hormone that is secreted from ovarian granulosa cells and the like and promotes secretion of follicle stimulating hormone (FSH) in the anterior pituitary gland. Representative activins include, for example, activin-A, activin-B, activin-C, and inhibin, which are well conserved among animals, and humans act on Xenopus and vice versa. Is also expected to be true. SEQ ID NO: 21 and SEQ ID NO: 23 (respectively accession number: NM _ 002192 (SEQ ID NO: and accession numbers of the human inhibin βA): X68250 (Xenopus activator SEQ ID = nucleic acid sequence of bins A)) and SEQ ID NO: 22 and SEQ ID NO: 24 (respectively human inhibin βA SEQ ID NO: accession number X68250 (Xenopus activin A) SEQ ID NO = amino acid sequence) and a corresponding factor (ortholog) in other species of animals. But not limited to. Xenopus has a registration of Inhibin βB (accession number: S61773). Molecular weight 25000. Inhibin β-chain subunits are S—S-linked dimers, and three types of activin A (βAβA), activin AB (βAβB), and activin B (βBβB) are known. Activin A is also referred to as an erythroblast differentiation factor (EDF). Since inhibin β chain has about 40% homology with transforming growth factor TGF-β, and the position of cysteine residues in the primary structure is well conserved, activin may be included in the TGF-β family. . Activin is a protein hormone that is secreted from granulosa cells of the ovary and promotes secretion of follicle stimulating hormone (FSH) in the anterior pituitary gland. Isolated from follicular fluid in the course of purifying inhibin (1986). Molecular weight 25000. Inhibin β-chain subunits are S—S-linked dimers, and three types of activin A (βAβA), activin AB (βAβB), and activin B (βBβB) are known. Activin A is also referred to as an erythroblast differentiation factor (EDF). Since inhibin β chain has about 40% homology with transforming growth factor TGF-β, and the position of cysteine residues in the primary structure is well conserved, activin may be included in the TGF-β family. . Activin gene expression has been reported in various vertebrate organs, promotes the synthesis of FSH receptors in ovarian granulosa cells, suppresses proliferation of erythroid progenitor cells in friend cells and bone marrow, and induces hemoglobin synthesis, It has many physiological activities such as promotion of insulin secretion from the pancreas and mesoderm induction in amphibian embryos. For activin, reference can be made to: Nakamura et al. , Isolation and charactarization of native activin B. J Biol. Chem. 1992, 267, 16385-9; Uchiyama and Asashima, Specific erythrophilic differentiation of mouse erythroleukemia cells by activities and its sensitization activity. Biochem Biophys. Res. Commun. 1992, 187, 347-52; Fukui et al. , Isolation and characterization of Xenopus follistatin and activins. Dev. Biol. 1993, 159, 131-9; Fukui et al. , Identification of activins A, AB, and B and follistatin proteins in Xenopus embryos. Dev. Biol. 1994, 163, 279-81; Nakano et al. , Comparison of mesoderm-inducing activity with monometic and dimeric inhibin alpha and beta-A subunits on Xenopus ectoderm. Horm. Res. 1995, 44, Suppl. 2, 15-22.

Activins are known to have homologues in mammals including humans, rats, mice and Xenopus, as well as in Drosophila. Therefore, in this specification, activin usually refers to activin which exists not only in mammals but also in living organisms in general. Activin -A is a dimer of inhibin [beta] A (accession human inhibin [beta] A session number NM _ 002192; SEQ ID NO: 21 and 22 (nucleic acid and amino acid)). Activin-AB is a dimer of inhibin βA and inhibin βB (human inhibin βB accession number NM002193). Activin-B is a dimer of inhibin βB. Activin-C is a dimer of inhibin βC (human inhibin βC accession number NM005538). Inhibin is a dimer of inhibin α (accession number NM002191 for human inhibin α).

  In the polypeptide such as activin used in the present invention, as a moiety to which a sugar chain can be added, a moiety capable of N-glucoside binding to which N-acetyl-D-glucosamine and the like, and N-acetyl-D-galactosamine are bound. Examples include a moiety that forms an O-glycoside bond (a part where serine or threonine residues frequently appear). The activin used in the present specification is not particularly affected by the presence or absence of a sugar chain, but proteins with these sugar chains are usually stable against degradation in vivo. , May have strong physiological activity. Therefore, polypeptides to which these sugar chains are added are also within the scope of the present invention.

  As used herein, “ligand” refers to a substance that specifically binds to a protein. Examples of the ligand include lectins, antigens, antibodies, hormones, neurotransmitters, and the like that specifically bind to various receptor protein molecules present on the cell membrane.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Is done. The gene product of the present invention usually takes the form of a polypeptide. The gene product of the present invention in such a polypeptide form is useful as a composition for diagnosis, prevention, treatment or prognosis of the present invention.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”.

  As used herein, the term “oligonucleotide derivative” or “polynucleotide derivative” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides, and is used interchangeably. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C− Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, cytosine in oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is replaced with 2'-O-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98). (1994)). The gene of the present invention usually takes this polynucleotide form.

  As used herein, “nucleic acid molecule” is also used interchangeably with nucleic acids, oligonucleotides and polynucleotides and includes cDNA, mRNA, genomic DNA, and the like. As used herein, nucleic acids and nucleic acid molecules can be included within the concept of the term “gene”. Nucleic acid molecules encoding certain gene sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition. Therefore, in the present specification, for example, a gene (activin gene) that can be used in the present invention may include a splice variant thereof.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that influence the expression are called regulatory genes (for example, promoters). In the present specification, genes include structural genes and regulatory genes unless otherwise specified. Therefore, a gene such as activin usually includes both a structural gene of the gene of the present invention and a transcriptional and / or translational regulatory sequence such as its promoter. In the present invention, it is understood that in addition to structural genes, regulatory sequences such as transcription and / or translation are also useful for nerve regeneration, diagnosis, treatment, prevention, prognosis, etc. of nerve diseases. As used herein, “gene” refers to “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide”. Sometimes. Also herein, a “gene product” is a “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein”, “polypeptide”, “oligo” expressed by a gene. Includes "peptide" and "peptide". A person skilled in the art can understand what a gene product is, depending on the situation.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. In this specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of two or more comparable sequences that are identical to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct comparison of the sequences or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.

  In the present specification, the similarity, identity and homology of amino acid sequences and nucleotide sequences are compared, and the search for identity is performed using, for example, NCBI BLAST 2.2.9 (issued 2004.12). be able to. In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  In the present specification, the “amino acid” may be natural or non-natural as long as it satisfies the object of the present invention. “Amino acid derivative” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such amino acid derivatives and amino acid analogs are well known in the art.

  The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.

  As used herein, the term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine.

  As used herein, “amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. As activin in this specification, in addition to those containing natural amino acids, those containing such amino acid analogs or amino acid derivatives can be used.

  In the present specification, the “nucleotide” may be natural or non-natural. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). .

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

  As used herein, a “corresponding” amino acid or nucleic acid has the same action as a predetermined amino acid or nucleotide in a polypeptide or polynucleotide to be compared in a certain polypeptide molecule or polynucleotide molecule, or An amino acid or nucleotide that is predicted to have, especially in an enzyme molecule, refers to an amino acid that is present at a similar position in the active site and contributes similarly to catalytic activity. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Therefore, in this specification, a specific amino acid sequence in mouse activin can be associated with a specific amino acid in human activin by analysis such as alignment. Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.

  As used herein, a “corresponding” gene (for example, a polypeptide molecule or a polynucleotide molecule) has or has the same action as a given gene in a species to be compared in a certain species. It refers to a predicted gene (for example, a polypeptide molecule or a polynucleotide molecule), and when there are a plurality of genes having such an action, it refers to one having the same evolutionary origin. Thus, a gene corresponding to a gene can be an ortholog of that gene. Therefore, genes corresponding to genes such as Xenopus activin can also be found in other animals (human, rat, pig, cow, etc.). Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in an animal is a sequence database of that animal (eg, human, rat) using the sequence of the gene that serves as a reference for the corresponding gene (eg, a gene such as Xenopus activin) as a query sequence. Can be found by searching.

  As used herein, “fragment” (or fragment) refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length n). . The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. Whether the length of a fragment useful in the present specification retains at least one of the functions of a full-length protein that serves as a reference for the fragment (for example, differentiation-regulating action, specific interaction with other molecules) It can be decided by how.

  By the term “region” as used herein is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of a protein, a region is defined by a contiguous portion of the amino acid sequence of the protein. The term “domain” is defined herein to refer to the structural portion of a biomolecule that contributes to the known or suspected function of the biomolecule. A domain can have the same extent as a region or portion thereof; a domain can also incorporate a portion of a biomolecule that is distinct from a particular region in addition to all or a portion of the region. Examples of activin or BENI domains of the present invention include, but are not limited to, signal peptides, extracellular (ie, N-terminal) domains, leucine-rich repeat domains. The BENI of the present invention was found to have a nuclear translocation signal as a domain.

  As used herein, a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor). Specific interactions for a substance or factor include, for example, when both nucleic acid and protein are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites. Thus, when both a substance or factor is a nucleic acid, the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor. Further, for example, when both substances or factors are proteins, the fact that the first substance or factor “specifically interacts” with the second substance or factor includes, for example, interaction by antigen-antibody reaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction. When two substances or factors include proteins and nucleic acids, the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included.

  Therefore, in the present specification, a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide, The affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.

  As used herein, an “agent” can be any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) that can achieve the intended purpose. Good. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to. As used herein, activin can be used for stem cell differentiation, but it is understood that any factor having the same biological activity as activin can be used interchangeably with activin in the present invention. Such factors can be identified by screening using technical common sense in the field based on the disclosure in the present specification, and it is understood that these are within the scope of well-known and conventional techniques. .

  As used herein, “compound” means any identifiable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids, including: Without limitation, such compounds can be natural or synthetic.

  In the present specification, the “small organic molecule” means an organic molecule having a relatively small molecular weight. Usually, a low molecular weight organic material has a molecular weight of about 1000 or less, but may have a higher molecular weight. The organic small molecule can be synthesized usually by using a method known in the art or a combination thereof. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, small molecule ligands). It is not limited.

  As used herein, “contacting” refers to physically bringing a compound, either directly or indirectly, into an activin or the like (eg, a polypeptide or polynucleotide) of the present invention. It means close proximity. The polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like. Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof. Typically, the contact can be achieved by placing a contact object (for example, a cell) in a solution (for example, a medium) containing a factor (for example, activin) to be contacted.

  As used herein, “complex molecule” refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules. Examples of such complex molecules include, but are not limited to, glycolipids and glycopeptides. In this specification, activin can also be used in the form of such a complex molecule.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, When it is a nucleic acid, it is substantially separated from a non-nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The terms “purified” and “isolated” as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably Means that at least 98% by weight of the same type of biological agent is present.

  As used herein, “biological activity” refers to the activity that a factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). Activity is included. For example, when two factors interact (eg, activin and its specific factor bind), the biological activity is the binding between the two molecules and the resulting biological change, eg, When one molecule is precipitated using an antibody, the other molecule is also co-precipitated and the two molecules are considered to be bound. Therefore, seeing such coprecipitation is one method of judgment.

  Thus, “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus); Refers to various measurable indicators, such as the affinity of a compound that binds directly to the polypeptide or polynucleotide used in the present invention, or the amount of protein upstream or downstream, eg, after some stimulation or event Alternatively, other similar measures of function can be mentioned. Such activity can be measured by assays such as competitive inhibition of binding of specific factors to activin.

  In this specification, the term “interaction” refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state.

  The term “binding” as used herein means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.

  The term “modulate” or “modify” as used herein means an increase or decrease or maintenance in the amount, quality or effect of a particular activity or protein.

  As used herein, “polynucleotide that hybridizes under stringent conditions” refers to a polynucleotide obtained under well-known conditions commonly used in the art. Obtaining such a polynucleotide by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides used in the present invention as a probe. Can do. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the polynucleotide capable of hybridizing is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide (for example, activin) having the amino acid sequence specifically shown in the present invention. Preferably, a polynucleotide having a homology of 80% or more, more preferably a polynucleotide having a homology of 95% or more.

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al. Nucleic Acid Hybridization: a Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
Tm (° C.) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + in the hybrid Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  As used herein, “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can be generated under “highly stringent conditions”. . Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate, 50-65 ° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20 % Formamide, 37-50 ° C. By way of example, “moderately stringent” conditions at 50 ° C. in 0.015 M sodium ion tolerate a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there may not be a complete distinction between “highly” stringent conditions and “moderate” stringent conditions herein. For example, at 0.015M sodium ion (no formamide), the melting temperature of perfectly matched long DNA is about 71 ° C. In washing at 65 ° C. (same ionic strength), this allows about 6% mismatch. In order to capture more distant related sequences, one of ordinary skill in the art can simply decrease the temperature or increase the ionic strength.

For oligonucleotide probes up to about 20 nucleotides, a reasonable estimate of the melting temperature in 1M NaCl is
Tm = (2 ° C. for one AT base) + (4 ° C. for one GC base pair)
Provided by. The sodium ion concentration in 6 × sodium citrate (SSC) is 1 M (see Suggs et al., Developmental Biology Using Purified Genes, page 683, Brown and Fox (ed.) (1981)).

  Natural nucleic acids encoding proteins such as activin or variants or fragments thereof can be readily obtained from a cDNA library having PCR primers and hybridization probes comprising, for example, a portion of a nucleic acid sequence such as SEQ ID NO: 1 or a variant thereof. To be separated. A nucleic acid encoding a preferred activin or variant or fragment thereof essentially comprises 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaPO 4); 1 mM EDTA; hybridization comprising 7% SDS at a temperature of 42 ° C. Under low stringent conditions, more preferably essentially 50, defined by a wash buffer containing buffer and essentially 2 × SSC (600 mM NaCl; 60 mM sodium citrate); 0.1% SDS at 50 ° C. Hybridization buffer containing 1% bovine serum albumin (BSA) at a temperature of ° C; 500 mM sodium phosphate (NaPO4); 15% formamide; 1 mM EDTA; 7% SDS, and 1 × SSC essentially at 50 ° C. 300 mM NaCl; 3 mM sodium citrate); 1% bovine serum albumin (BSA) under low stringent conditions defined by a wash buffer containing 1% SDS, most preferably essentially at a temperature of 50 ° C .; 200 mM sodium phosphate ( NaPO4); 15% formamide; 1 mM EDTA; hybridization buffer containing 7% SDS and essentially 0.5 × SSC at 65 ° C. (150 mM NaCl; 15 mM sodium citrate); wash containing 0.1% SDS It can hybridize to one or a portion of the sequence shown in SEQ ID NO: 1 or 3 under low stringency conditions as defined by the buffer.

  In the present specification, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. As an electronic search, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:44:44). -453 (1970)) and the like. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, it is intended that activin and the like used in the present invention should include corresponding genes identified by such electronic search and biological search.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, the reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequences contain additions, in the portion of the polynucleotide or polypeptide sequence within the comparison window, Reference sequences herein shall include no additions or deletions (ie, gaps) as compared to the reference). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol.Biol.215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res.22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol.266: 383-402. Altschul et al., 1990, J. Mol.Biol.215 (3): 403-410, Altschul et al. 1993, Nature Genetics 3: 266-272), but is not limited thereto. In particularly preferred embodiments, the Basic Local Alignment Tool (BLAST) (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990, well known in the prior art. J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

(1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) Comparison of a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX in six reading frames with a protein sequence database;
(4) Compare protein query sequence with TBLASTN to nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.

  The BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although less preferred than this matrix, a PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrixes for Detection Distance Relationships: Atlas of Protein Sequence and Strategur Strateg. thing). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). thing).

(Modification of genes, protein molecules, nucleic acid molecules, etc.)
In certain protein molecules (eg, BENI of the present invention, activin, etc.), certain amino acids included in the sequence can be expressed, for example, as a cationic region or a binding site of a substrate molecule, with no apparent reduction or loss of interaction binding capacity. In other protein structures can be substituted with other amino acids. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility. It will be understood that variants thus modified are also within the scope of the present invention.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity). It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

  The hydrophilicity index is also useful for modifying the amino acid sequence of the present invention. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present specification, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid are similar as described above. Examples of conservative substitution include those having a hydrophilicity index or hydrophobicity index within ± 2, preferably within ± 1, and more preferably within ± 0.5. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and Examples include, but are not limited to, isoleucine.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Such variants include one or several substitutions, additions and / or deletions, or one or more substitutions, additions and / or deletions relative to a reference nucleic acid molecule or polypeptide. But are not limited thereto. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs can usually perform the same function as the original species in another species, the orthologs of the present invention can also be useful in the present invention.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide. Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, ligation treatment by treatment with DNA ligase, etc., site-specific base substitution using synthetic oligonucleotides (specification) Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)), but other modifications may also be made by methods usually used in the field of molecular biology. it can.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include amidation, carboxylation, sulfation, halogenation, shortening, lipidation, phosphorylation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg acetylation), etc. Including, but not limited to. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the term “peptide analog” or “peptide derivative” refers to a compound that is different from a peptide but is equivalent to at least one chemical or biological function of the peptide. Thus, peptide analogs include those in which one or more amino acid analogs or amino acid derivatives are added or substituted to the original peptide. Peptide analogs have the same function as the original peptide (for example, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similarity in sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog.

  Chemically modified polypeptide compositions in which the polypeptide used in the present invention is conjugated to a polymer are included within the scope of the present invention. The polymer can be water soluble and can prevent precipitation of the protein in a water soluble environment (eg, a physiological environment). Suitable aqueous polymers can be selected, for example, from the group consisting of: polyethylene glycol (PEG), monomethoxy polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymer, poly (N-vinyl pyrrolidone) polyethylene glycol, Polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohol. The selected polymer is usually modified and has a single reactive group (eg, an active ester for acylation or an aldehyde for alkylation) so that the degree of polymerization can be controlled. The polymer can be of any molecular weight, and the polymer can be branched or unbranched, and mixtures of such polymers can also be used. The chemically modified polymer used in the present invention is selected for use by a pharmaceutically acceptable polymer as determined for therapeutic use.

  If the polymer is modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, if the polymer is modified by reductive alkylation, the polymer should have a single reactive aldehyde group. Preferred reactive aldehydes are polyethylene glycol, propionaldehyde (which is water-soluble) or mono-C1-C10 alkoxy or aryloxy derivatives thereof (eg, US Pat. No. 5,252,714). No., which is hereby incorporated by reference in its entirety).

  Pegylation of polypeptides used in the present invention can be performed by any pegylation reaction known in the art, for example as described in the following references: Focus on Growth Factors 3 , 4-10 (1992); EP 0 154 316; and EP 0 401 384, each of which is hereby incorporated by reference in its entirety. Preferably, this pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or similar reactive water-soluble polymer). A preferred water-soluble polymer for pegylation of polypeptides (eg, activin, Mel-18, M33, Mph-1 / Rae28, etc.) used in the present invention is polyethylene glycol (PEG). As used herein, “polyethylene glycol” is meant to encompass any form of PEG, where the PEG is another protein (eg, a mono (C1-C10) alkoxy polyethylene. Glycol or mono (C1-C10) aryloxypolyethylene glycol).

  Chemical derivatization of the polypeptides used in the present invention can be carried out under suitable conditions used to react biologically active substances with activated polymer molecules. A method for preparing a pegylated polypeptide used in the present invention generally comprises the following steps: (a) polyethylene glycol (eg, under conditions such that activin is attached to one or more PEG groups. Reacting the polypeptide with a reactive ester or aldehyde derivative of PEG) and (b) obtaining the reaction product. It is easy for those skilled in the art to select the optimal reaction conditions or acylation reaction based on known parameters and the desired result.

  Pegylated polypeptides used in the present invention can generally be used to treat conditions that can be alleviated or modulated by administering the polypeptides described herein, but the present The polypeptide molecules used in the present invention that are chemically derivatized as disclosed herein have additional activity, increased or decreased biological activity, or others compared to their non-derivative molecules. Characteristics (eg, increased or decreased half-life). The polypeptides, their fragments, variants and derivatives used in the present invention may be used alone, in combination, or in combination with other pharmaceutical compositions. These cytokines, growth factors, antigens, anti-inflammatory agents and / or chemotherapeutic agents are suitable for treating symptoms.

  Similarly, “polynucleotide analog” or “nucleic acid analog” refers to a compound that is different from a polynucleotide or nucleic acid but is equivalent to at least one chemical or biological function of the polynucleotide or nucleic acid. . Thus, polynucleotide analogs or nucleic acid analogs include those in which one or more nucleotide analogs or nucleotide derivatives are added or substituted to the original peptide.

  As used herein, a nucleic acid molecule may have a portion of its nucleic acid sequence deleted or otherwise as long as the expressed polypeptide has substantially the same activity as the native polypeptide. It may be substituted with a base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

(Genetic engineering)
Activins and the like and fragments and variants thereof used in the present invention can be produced using genetic engineering techniques.

  In the present specification, when referring to a gene, “vector” or “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Such vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or can be integrated into chromosomes and used in the present invention. Examples include those containing a promoter at a position suitable for transcription of the polynucleotide to be produced. Among vectors, a vector suitable for cloning is called “cloning vector”. Such cloning vectors usually contain multiple cloning sites that contain multiple restriction enzyme sites. Such restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra). Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses and integratable DNA fragments (ie, fragments that can be integrated into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenovirus, baculovirus, parvovirus, herpes virus, pox virus, adeno-associated virus, Semliki Forest virus, vaccinia virus and retrovirus.

  One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) can replicate autonomously in the host cell into which they are introduced. Other vectors (eg, non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell, and are thereby replicated along with the host genome. Furthermore, certain vectors may direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “expression vectors”.

  Therefore, in the present specification, the “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory elements used can vary depending on the host cell.

“Recombinant vectors” for prokaryotic cells that can be used in the present invention include pcDNA3 (+), pBluescript-SK (+/−), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DEST ^™ 42GATEWAY ( Invitrogen) and the like.

  Examples of “recombinant vectors” for animal cells that can be used in the present invention include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 [JP-A-3-229 (Invitrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem. , 268, 22782-2787 (1993)], a retroviral expression vector based on murine stem cell virus (MSCV), pEF-BOS, pEGFP, and the like.

  As used herein, “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA, and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.

  In this specification, “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is usually a base sequence that initiates transcription upon binding of RNA polymerase. . Therefore, in this specification, a part having a promoter function of a gene is referred to as a “promoter part”. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic base sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.

  As used herein, “replication origin” refers to a specific region on a chromosome where DNA replication begins. The origin of replication can either be provided by constructing the vector to include an endogenous origin, or it can be provided by the host cell's chromosomal replication machinery. The latter may be sufficient if the vector is integrated into the host cell chromosome. Alternatively, rather than using a vector containing a viral origin of replication, one of skill in the art can transform mammalian cells by a method that co-transforms a selectable marker and the DNA of the present invention. Examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase (see US Pat. No. 4,399,216).

  For example, recombinant mammalian expression vectors can preferentially direct expression of a nucleic acid in a particular cell type by expressing the nucleic acid using tissue-specific regulatory elements. Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include developmentally regulated promoters (eg, mouse hox promoter (Kessel and Gruss (1990) Science 249, 374-379) and α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537-546)), albumin promoter (liver specific; Pinkert et al. (1987) Genes Dev. 1, 268-277), lymphoid specific promoter (Calame and Eaton (1988) Adv. Immunol.43, 235-275), in particular T cell receptors (Winoto and Baltimore (1989) EMBO J. 8, 729-733) and immunoglobulins. (Banerji et al. (1983) Cell 33, 729-740; Queen and Baltimore (1983) Cell 33, 741-748), neuron-specific promoters (eg, nerve fiber promoter; Byrne and Ruddle (1989) Proc. Natl. Acad.Sci.USA 86, 5473-5477), pancreas specific promoter (Edrund et al. (1985) Science 230, 912-916), and mammary gland specific promoter (eg, whey promoter; US Pat. No. 4,873, 316 and European Application Publication No. 264,166), but are not limited thereto.

  As used herein, “enhancer” refers to a sequence used to increase the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject. The cell used in the present invention may be a transformant.

  When prokaryotic cells are used in genetic manipulation or the like in the present invention, prokaryotic cells include, for example, prokaryotic cells belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified.

  As used herein, animal cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, and human leukemia cells. HBT5637 (Japanese Patent Laid-Open No. 63-299), human colon cancer cell line, and the like. Mouse myeloma cells such as ps20 and NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1, and Africa COS-1, COS-7 as the kidney monkey cell, HCT-15 as the human colon cancer cell line, human neuroblastoma SK-N-SH, SK-N-SH-5Y, mouse neuroblastoma Neuro2A, etc. Is exemplified.

  As used herein, any method for introducing a DNA can be used as a method for introducing a recombinant vector. For example, a calcium chloride method, an electroporation method [Methods. Enzymol. , 194, 182 (1990)], lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

(Method for producing polypeptide)
Transformants derived from microorganisms, animal cells, etc. that possess a recombinant vector incorporating a DNA encoding a polypeptide (for example, activin, LIF or a variant or fragment thereof) used in the present invention are usually used. The polypeptide used in the present invention is produced by accumulating the polypeptide used in the present invention, collecting the polypeptide used in the present invention from the culture of the present invention, and producing the polypeptide according to the present invention. Can do.

  The method of culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host. A medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism of the present invention. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.

  Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol can be used.

  Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing substances, and peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.

  As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

  The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin or tetracycline, to a culture medium as needed during culture | cultivation.

  When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter, and indole acrylic is used when a microorganism transformed with an expression vector using the trp promoter is cultured. An acid or the like may be added to the medium. Cells or organs into which a gene has been introduced can be cultured in large quantities using a jar fermenter.

  For example, when animal cells are used, the culture medium for culturing the cells of the present invention includes RPMI1640 medium (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM medium (Science, 122). , 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, 1 (1950)), or fetal bovine serum or the like was added to these media A medium or the like is used.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  In order to isolate or purify a polypeptide used in the present invention from a culture of a transformant transformed with a nucleic acid sequence encoding the polypeptide of the invention used in the present invention, it is well known in the art. Conventional conventional enzyme isolation or purification methods can be used. For example, when the polypeptide of the present invention is secreted outside the transformant for producing the polypeptide of the present invention, the culture is treated with a technique such as centrifugation to obtain a soluble fraction. Get minutes. From the soluble fraction, anion exchange chromatography using a resin such as a solvent extraction method, a salting out method using ammonium sulfate, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), etc. Chromatography method, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography A purified sample can be obtained by using a technique such as a chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide used in the present invention (for example, activin, LIF or a variant or fragment thereof) accumulates in a dissolved state in the cells of the transformant for producing the polypeptide used in the present invention, culture is performed. The cells in the culture are collected by centrifuging the product, and after washing the cells, the cells are disrupted by an ultrasonic disrupter, a French press, a Manton Gaurin homogenizer, a dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical) anion exchange chromatography using a resin, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose and phenyl sepharose A purified sample can be obtained by using a technique such as a chromatography method, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide used in the present invention is expressed by forming an insoluble substance in the cells, similarly, the cells are collected and then disrupted and centrifuged from the precipitate fraction obtained by a conventional method. After recovering the polypeptide used in the present invention, an insoluble substance of the polypeptide is solubilized with a polypeptide denaturant. This solubilized solution is diluted or dialyzed into a solution that does not contain a polypeptide denaturant or is so thin that the concentration of the polypeptide denaturant does not denature the polypeptide, and then converts the polypeptide used in the present invention into a normal three-dimensional solution. After making it into a structure, a purified sample can be obtained by the same isolation and purification method as described above.

  Further, a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458]. In addition, the polypeptide used in the present invention can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having affinity for the fused protein [Akio Yamakawa, Experimental Medicine, 13, 469-474 (1995)]. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227-8231 (1989), Genes Develop. , 4, 1288 (1990)], the polypeptide used in the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

  In addition, the polypeptide used in the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)]. In such fusion proteins, in the expression vector, the proteolytic cleavage site joins the fusion moiety to the recombinant protein to allow separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Introduced into the department. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase. Representative fusion expression vectors include pGEX (Pharmacia Biotech; Smith and Johnson (1988) Gene 67, which fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A to the target recombinant protein, respectively. , 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ).

  Furthermore, it can also be purified by affinity chromatography using an antibody against the polypeptide itself used in the present invention. Polypeptides used in the present invention can be prepared by known methods [J. Biomolecular NMR, 6,129-134, Science, 242, 1162-1164, J. Biol. Biochem. , 110, 166-168 (1991)], can be produced using an in vitro transcription / translation system.

  Polypeptides used in the present invention can be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method) based on the amino acid information. it can. Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Applied Biosystems, Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

  The structural analysis of the purified polypeptide used in the present invention is performed by a method usually used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hirano Hisashi, Tokyo Chemical Dojin, 1993). It can be implemented. The physiological activity of the polypeptide used in the present invention can be measured according to a known measurement method.

  Deletion, substitution or addition (including fusion) of amino acids of a polypeptide (for example, activin, LIF, etc.) used in the present invention can be carried out by site-directed mutagenesis which is a well-known technique. Such deletion, substitution or addition of one or several amino acids is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratories Press (1989), Current Protocols in BioJ. 1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci. USA, 81, 5562 (1984), Science, 224, 1431 (1984), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985) and the like.

(Cell culture)
The cell culture properties of mesodermal cell differentiation of a wide variety of media formulations known in the art can be dramatically altered by adjusting the amount of BENI, activin or equivalents disclosed herein. Was found.

  Many media formulations have been developed over the years to maximize cell growth, cell viability, and / or biologic production for a given cultured cell or cell line. These media compositions differ, for example, in the number, type, and concentration of growth factors, antibiotics, and amino acid supplements added to the media, and components such as protease inhibitors, and particularly cells in suspension. When grown, it contains one or more anti-foaming agents. Other personalized ingredients include additives that enhance recombinant protein production. For example, if the host cell was developed for gene amplification of a recombinant protein, the selectable marker DHFR (dihydrofolate reductase) typically constitutes part of the host transfected as a selectable marker, And methotrexate is contained in the culture medium. Insulin or other growth factors, such as factors that play a role in the energy source cascade, such as IGF, are also often included to enhance cell proliferation.

  The BENI, activin or equivalents disclosed herein are expected to have an effect on various media formulations as well as in standard formulations.

For example, all serum-free media formulations contain essential ingredients that allow cell growth, and (1) an energy source, typically glucose or glutamine, or other sugars such as fructose, galactose, mannose, etc .; (2) contains a nitrogen source (typically obtained by inclusion of one or more amino acids); and (3) vitamins (cofactors in enzymatic reactions). Also includes a wide range of inorganic salts including Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cl ⁻ , HPO ₃ ^{2− and the} like, as well as fatty acids (preferably conjugates), cholesterol, phospholipids, and their precursors. A wide range of fat and fat-soluble ingredients are also essential. The components of a standard serum-free medium formulation are listed in Table 1. This includes GrandIsland Biological Co. (GIBCO), Grand Island, N .; Y. Ingredients found in “DMEM / F-12” obtained from media manufacturers such as For a review of animal cell culture media, see Mizrahi and Lazar, Cytotechnology, 1: 199-214 (1988). Media considerations for insect cell culture are described in Goodwin, R .; H. (1990) Nature 347: 209-210. An example of a defined serum-free medium that is individualized for hybridoma culture is described in Kovar, J. et al. (1987) Folia Biology 33: 377-384. Imagawa et al. (1989) PNAS 86: 4122-4126 describe a medium developed to maximize cell proliferation in mouse mammary epithelial cells, and Miyazaki et al. (1991) Res. Exp. Med. 191: 77-83 describes a medium to enhance the viability of rat hepatocytes in vitro.

  As used herein, “nutrient medium” includes natural medium, semi-synthetic medium, synthetic medium, solid medium, semi-solid medium, liquid medium, etc., but undifferentiated cells are allowed to grow, differentiate and mature, including self. Alternatively, any medium may be used as long as it is used for storage and is usually used for cell culture. Examples include Steinberg medium, α-MEM medium, RPMI-1640 medium, or MEM basic medium. As basic components, sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, hormones, antibiotics, fatty acids, sugars, or other chemical components or biological components such as serum may be contained depending on the purpose.

  The media components can typically be added in any order, and the final combination can be sterilized by standard methods, such as filter sterilization.

  Utilizing the media of the present invention, a wide range of cell stem cells (both vertebrates and invertebrates) can be differentiated in a desired direction. The culture medium of the present invention can be used to improve cell culture properties in many different cell culture systems.

  In the case of animals, serum-free media such as Iskov media, RPMI media, Dulbecco MEM media, MEM media, and F12 media that are usually used for culturing animal cells can be used. In addition, factors other than serum known to be effective for cell growth and maintenance, such as lipid and fatty acid sources, cholesterol, pyruvate, glucocorticoid, DNA and RNA synthetic nucleosides, etc. are added according to known literature. May be.

  As used herein, the term “culture vessel” refers to a vessel used when proliferating desired cells, for example, undifferentiated cells. Feeder cells such as stromal cells can be maintained and survived as needed, and undifferentiated. Any material or shape may be used as long as it does not inhibit the maintenance, survival, differentiation, maturation, or self-replication of cells. Specific examples of the culture vessel material include glass, synthetic resin, natural resin, metal, and plastic. Specific examples of the shape of the culture vessel include a triangular prism, a cube, a rectangular parallelepiped, a triangular pyramid, a quadrangular pyramid, and the like. Polygonal pyramids, arbitrary shapes such as gourds, spheres, hemispheres, cylinders (including bottom, circular, elliptical, semicircular, etc.) can be mentioned, and during culture, for example from hemispheres to spheres The shape may be changed as necessary. Cultivation may be under open conditions or under closed (sealed) conditions.

  The cell culture method in the present invention is a culture dish having a different matrix coating, a two-dimensional culture method using a culture dish having a different presence or absence of matrix coating, a three-dimensional culture method using a soft gel such as Matrigel, a collagen sponge, etc. Alternatively, a method using them in combination may be mentioned, but preferably a two-dimensional culture method using a culture dish having a different matrix coating or a culture dish having a different presence or absence of a matrix coating, such as a gelatin-coated culture dish, type I collagen. It is a two-dimensional culture method using two of a coated culture dish or a laminin coated culture dish. In addition, when human-derived cells are used as cells having multipotency, it is preferable to use type I collagen-coated culture dishes.

The culture conditions of the method of the present invention are not limited to specific conditions such as Examples, and generally acceptable conditions can be taken. For example, as the number of cells at the start of differentiation induction, a range of 5.0 × 10 ^{3 to} 5.0 × 10 ⁶ cells / culture dish can be exemplified. The differentiation induction period is, for example, 2 to 10 days (preferably 5 days), or 1 to 4 days (preferably 2 days), and 2 to 5 days (preferably 3 days) in the preculture step. In the case of human mesenchymal cells, the differentiation induction period is 12 to 21 days (preferably 14 days).

  In culturing, physical environmental conditions such as temperature, osmotic pressure, and light, and chemical environmental conditions such as oxygen, carbon dioxide, pH, and oxidation-reduction potential are maintained and maintained by undifferentiated cells before and after differentiation. Any environmental condition may be used as long as it does not inhibit the maintenance, survival, differentiation, maturation, and self-replication of undifferentiated cells. Preferred conditions are shown below.

  Specifically, the temperature is 30 ° C. to 40 ° C., preferably 37 ° C. The osmotic pressure is specifically an osmotic pressure under physiological conditions, and preferably an osmotic pressure equal to that of physiological saline.

  The light may be as dark as a dark room, or as bright as the brightness outside in sunny weather. Specifically, as for the oxygen concentration, the culture system is in contact with the gas phase having the oxygen concentration in the gas phase in contact with the gas phase having the oxygen concentration in the gas phase of 10% to 30% in the gas phase. The oxygen concentration may be the oxygen concentration in a state of being in contact with the gas phase, preferably in the state of being in contact with the gas phase having a concentration of oxygen in the gas phase of 20%. Concentration.

  In general, the pH for controlling the pH in the culture system is specifically pH 6.0 to pH 8.0, preferably the pH equivalent to physiological conditions. Carbon dioxide may be used to control the pH, or any other buffer may be used. Specifically, the concentration of carbon dioxide is the concentration of dissolved carbon dioxide in a state where the culture system is in contact with the 5% gas phase.

  As used herein, the term “colony” refers to a visible clump formed from a single cell in a solid medium.

  As used herein, the term “kit” refers to a unit in which parts to be provided (eg, reagents, particles, etc.) are usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such kits preferably include instructions that describe how to treat the provided parts (eg, reagents, particles, etc.). Such a manual may be any medium. Examples of such a medium include, but are not limited to, a paper medium, a transmission medium, and a recording medium. Examples of the transmission medium include, but are not limited to, the Internet, an intranet, an extranet, and a LAN. Examples of the recording medium include, but are not limited to, CD-ROM, CD-R, flexible disk, DVD-ROM, MD, mini disk, MO, and memory stick.

(screening)
As used herein, “screening” refers to selecting a target such as a target organism or substance having a specific property from a large number of populations by a specific operation / evaluation method. For screening, an agent (eg, antibody), polypeptide or nucleic acid molecule of the invention can be used. For the screening, a system using an actual substance such as in vitro or in vivo may be used, or a library generated using an in silico (computer system) system may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also encompassed within the scope of the present invention. The present invention also contemplates providing a computer modeling drug based on the disclosure of the present invention.

  In one embodiment, the invention is for screening candidate or test compounds that bind to or modulate the activity of the protein of the invention (BENI or activin), or a biologically active portion thereof. An assay is provided. Test compounds of the invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries; spatially Accessible parallel solid phase or solution phase library; synthetic library method requiring deconvolution; “one bead one compound” library method; and synthetic library method using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Des. 12). 145).

  Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. MoI. Med. Chem 37: 2678; Cho et al. (1993) Science 2611: 1303; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. MoI. Med. Chem 37: 1233.

  The library of compounds can be in solution (eg, Houghten (1992) BioTechniques 13: 412-421), or on beads (Lam (1991) Nature 354: 82-84), on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner, supra), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwilla et al. (1990) Proc. Natl. Acad. ci.U.S.A.87: 6378~6382; Felici (1991) J Mol Biol 222: 301~310; Ladner above) can be shown in.

(disease)
In one aspect, the present invention relates to any disease, disorder or abnormal state related to a disorder of mesoderm abnormality, for example a related disease such as a muscular system, connective tissue, skeletal system, circulatory system, excretory system, reproductive system disease, A method for treating a fault or abnormal condition is provided. More particularly, any disease, disorder or condition associated with a disorder of mesoderm abnormality is any disease, disorder or (abnormal or normal) condition associated with mesoderm differentiation.

  In the present specification, “state related to mesoderm” refers to a state (disorder, disease, etc.) related to the state of mesoderm (muscle system, connective tissue, skeletal system, circulatory system, excretory system, reproductive system, etc.) Point to. In the present specification, when referring specifically to a disorder or disease associated therewith, it may be referred to as a “mesoderm-related disorder” or “mesoderm-related disease”, and when related to mesoderm differentiation, It may be referred to as “a state associated with mesoderm differentiation”.

In the present specification, the “mesoderm-related disorder” or “mesoderm-related disease” includes, for example, abnormal states such as muscular system, connective tissue, skeletal system, circulatory system, excretory system, and reproductive system (for example, Inflammation, cancer, etc.), and more specifically, myosphere, musculoskeletal, myoma, myopathy, connective tissue inflammation, connective tissue tumor, connective tissue disease, skeletal dysplasia , Circulatory collapse, hypercirculatory blood volume, myocarditis, myocardial ischemia, myocardial infarction, epicarditis, ventricular septal defect, cardiomyocyte lysis, cardiomyopathy, myocardial fibrosis, vascular dystrophy, vascular hyaline, vascular myelin Disorder, angiolymphangioma, vasomotor paralysis, angina pectoris, vasculitis, hemangioblastoma, angiomyolipoma, angiomyoma, angiomyopathy, angiogenesis abnormality, angiogenesis dysfunction, vascular stone, vascular sclerosis Disease, renal amyloidosis, nephritis, nephroblastoma, Dysfunction, renal dysplasia, renal fistula, renal stone, nephrosclerosis, nephroma, cystocele, bladder hematuria, urinary tract infection, urolithiasis, urethritis, periurethritis, urethral neuropathy, urethral disorder, Ovitis, hypoovarian function, ovarian hyperfunction, ovarian goiter, ovarian varices, ovarian male germinoma, ovarian abscess, ovarian parahistitis, uterine myositis, cervical lymphatic swelling, cervicitis, cervical endocarditis, cervical dysplasia, testicular Yin嚢癒deposition, orchitis, testicular hyperkinesia, testicular cancer, testicular loss disease, testicular sheath meningitis, epididymitis, testicular hypoplasia, testis - epididymis Inflammation, prostatitis, prostatic calculus, prostatic disease, prostatic hypertrophy, prostatic adenoma and the like. It is understood that “a state associated with mesoderm differentiation” is also associated with the differentiation state of cells, tissues, etc. in the specific disorders and diseases.

  Representative diseases associated with mesoderm abnormalities include mesoderm type, obesity type, mesoderm nephroma, and the like.

  As used herein, “preventing” refers to reducing the likelihood that an organism will contract or develop an abnormal condition.

  As used herein, “treating” refers to having a therapeutic effect and at least partially alleviating or suppressing an abnormal condition in an organism.

  As used herein, “therapeutic effect” refers to an inhibitor or activator that causes or contributes to an abnormal condition. The therapeutic effect alleviates one or more of the symptoms of the abnormal condition to some extent. With respect to the treatment of abnormal conditions, a therapeutic effect may refer to one or more of: (a) an increase in cell proliferation, growth, and / or differentiation; (b) inhibition of cell death. (I.e. delay or stop); (c) inhibition of degeneration; (d) some relief of one or more symptoms associated with an abnormal condition; and (e) enhance the function of the affected cell population. thing. Compounds that exhibit efficacy against abnormal conditions can be identified as described herein.

  As used herein, an “abnormal condition” refers to a function in a cell or tissue of an organism that deviates from its normal function in the organism. The abnormal condition can be associated with cell proliferation, cell differentiation, cell signaling, or cell survival. Abnormal conditions may also include hematopoietic disorders, obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and irregularities in fatty acid uptake and metabolism.

  Examples of abnormal cell growth include cancer, neoplasm, tumor and inflammation.

  Examples of abnormal differentiation include malformation and cancer.

  An example of an abnormal cell signaling state is abnormal cell differentiation.

  Abnormal cell viability is also associated with conditions in which the apoptotic (programmed cell death) pathway is activated or arrested. A number of protein kinases are associated with the apoptotic pathway. Abnormalities in the function of any one of the protein kinases can result in cell immortality or premature cell death.

  In another aspect, the present invention relates to a prophylactic method and a therapeutic method for treating a subject having (or possibly suffering from) a mesoderm abnormality-related disease, disorder or abnormality, or a subject having the disorder. Provide both.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the medicament of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product may optionally be attached to such containers, which may be made, used or sold for administration to humans. Represents approval by government agencies.

(Gene therapy)
In certain embodiments, a nucleic acid comprising a normal gene nucleic acid sequence of the invention, a sequence encoding an antibody or functional derivative thereof, is associated with aberrant expression and / or activity of a polypeptide used in the invention. Administered for gene therapy purposes to treat, inhibit or prevent a disease or disorder. Gene therapy refers to a therapy performed by administering to a subject a nucleic acid that is expressed or expressible. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

  Any method for gene therapy available in the art can be used in accordance with the present invention. An exemplary method is as follows.

  For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene TransferMand Transfer and ExplorMorL. , Stockton Press, NY (1990).

  Therefore, in the present invention, gene therapy using a nucleic acid molecule encoding activin or a variant or fragment thereof may be useful.

  As used herein and as understood in the art, the terms “synthesis” or “synthesize” are purely chemical as opposed to enzymatic methods. Refers to chemical substances (eg, polynucleotides, polypeptides, etc.) produced in Thus, “globally” synthesized chemicals (eg, polynucleotides, polypeptides, etc.) are generated entirely by chemical means, and “partially” synthesized chemicals (eg, Polynucleotide, polypeptide, etc. includes chemicals (eg, polynucleotides, polypeptides, etc.) in which only a portion of the resulting chemicals (eg, polynucleotides, polypeptides, etc.) are produced by chemical means. To do.

(Antisense / RNAi)
As used herein, “antisense” is referred to as “antisense molecule” or the like, and refers to RNA having a base sequence complementary to mRNA or the like. RNA having a complementary base sequence to the RNA molecule is thought to inhibit the functional expression of RNA through intermolecular bonds. Since antisense RNA can be artificially and easily produced for any target gene, it is also used as a method for artificially controlling gene expression.

  In the present specification, “RNAi” is an abbreviation for RNA interference, and by introducing a factor causing RNAi such as double-stranded RNA (also referred to as dsRNA) into cells, homologous mRNA is specifically degraded, It refers to a phenomenon in which the synthesis of a gene product is suppressed and the technology used for it. As used herein, RNAi can also be used interchangeably with an agent that causes RNAi in some cases.

  As used herein, “factor causing RNAi” refers to any factor capable of causing RNAi. In the present specification, the “factor causing RNAi” with respect to “gene” means that RNAi related to the gene is caused and an effect brought about by RNAi (for example, suppression of expression of the gene) is achieved. Factors that cause such RNAi include, for example, at least 10 nucleotides, including sequences having at least about 70% homology to a portion of the nucleic acid sequence of the target gene or sequences that hybridize under stringent conditions Examples include, but are not limited to, RNAs containing long double-stranded portions or variants thereof. Here, the factor preferably comprises a 3 'overhang, more preferably the 3' overhang can be 2 or more nucleotides in length (eg, 2 to 4 nucleotides in length).

  Without being bound by theory, one of the possible mechanisms for RNAi to work is that when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, a relatively long (eg, 40 base pairs or more) RNA, helicase In the presence of ATP, an RNaseIII-like nuclease called Dicer having a domain excises the molecule from the 3 ′ end by about 20 base pairs to produce a short dsRNA (also called siRNA). As used herein, “siRNA” is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, synthesized in an organism, or about 40 This refers to short double-stranded RNA of 10 base pairs or more formed by decomposing double-stranded RNA of bases or more in the body, and usually has a 5′-phosphate, 3′-OH structure. 'The end protrudes about 2 bases. A protein specific to the siRNA binds to form RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity. The relationship between the siRNA sequence and the mRNA sequence cleaved as a target is preferably 100% identical. However, with respect to the base mutation at a position off the center of siRNA, the cleavage activity by RNAi is not completely lost, but a partial activity remains. On the other hand, the mutation of the base at the center of siRNA has a large effect, and the cleavage activity of mRNA by RNAi is extremely reduced. Utilizing such properties, for mRNA having a mutation, only siRNA having the mutation can be specifically decomposed by synthesizing siRNA having the mutation in the center and introducing it into the cell. Therefore, in the present invention, siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA typically having about 40 bases or more) can be used as such a factor. it can.

  Moreover, although not wishing to be bound by theory, siRNA is synthesized separately from the above pathway, and the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP). It is also contemplated that this dsRNA becomes Dicer's substrate again, generating new siRNA and amplifying the action. Thus, in the present invention, siRNA itself and factors that produce siRNA are also useful. In fact, in insects and the like, for example, 35 dsRNA molecules almost completely degrade the mRNA in a cell having 1,000 copies or more, so it is understood that siRNA itself and factors that generate siRNA are useful. Is done.

  In the present invention, double-stranded RNA having a length of about 20 bases (for example, typically about 21 to 23 bases in length) or less than that called siRNA can be used. Such siRNA can be used for treatment, prevention, prognosis, etc. of a disease because it suppresses gene expression by expressing in a cell and suppresses expression of a pathogenic gene targeted by the siRNA.

  The siRNA used in the present invention may take any form as long as it can cause RNAi.

  In another embodiment, the factor causing RNAi of the present invention may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3 'end. As used herein, “shRNA” is a single-stranded RNA that includes a partially palindromic base sequence, and thus has a double-stranded structure within the molecule, resulting in a hairpin-like structure. The above molecules. Such shRNA is artificially chemically synthesized. Alternatively, such shRNA can be generated by synthesizing RNA in vitro using T7 RNA polymerase with hairpin structure DNA in which the DNA sequences of the sense strand and antisense strand are ligated in the opposite direction. Although not wishing to be bound by theory, such shRNAs are approximately 20 bases in length (typically 21 bases, 22 bases, 23 bases, etc.) in length after being introduced into the cell. It should be understood that it is degraded to cause RNAi as well as siRNA and has the therapeutic effect of the present invention. It should be understood that such effects are exerted in a wide range of organisms such as insects, plants, animals (including mammals). Thus, since shRNA causes RNAi similarly to siRNA, it can be used as an active ingredient of the present invention. The shRNA can also preferably have a 3 'overhang. The length of the double-stranded part is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3 'protruding end may be preferably DNA, more preferably DNA having a length of at least 2 nucleotides, and further preferably DNA having a length of 2 to 4 nucleotides.

  The factor causing RNAi used in the present invention can be either artificially synthesized (for example, chemical or biochemical) or naturally occurring, and the effect of the present invention can be achieved between the two. There is no essential difference. Those chemically synthesized are preferably purified by liquid chromatography or the like.

  The factor that causes RNAi used in the present invention can also be synthesized in vitro. In this synthesis system, antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is caused through the mechanism described above, and the effects of the present invention are achieved. Here, for example, such RNA can be introduced into cells by the calcium phosphate method.

  Factors causing RNAi of the present invention also include factors such as single strands that can hybridize to mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the treatment methods and compositions of the invention.

  A more detailed description of the RNAi design method is described below.

As RNAi used in the present invention, a double-stranded polynucleotide comprising DNA and RNA having substantially the same sequence as at least a part of the base sequence of a target gene is introduced into a cell, tissue or individual. A featured target gene expression inhibition method can be used. Here, a double-stranded polynucleotide comprising a single strand having self-complementarity can be used. Alternatively, the double-stranded polynucleotide may be a hybrid of a DNA strand and an RNA strand. Preferably, in the hybrid of DNA strand and RNA strand used, the sense strand can be DNA and the antisense strand can be RNA. Alternatively, the double-stranded polynucleotide used may be a chimera of DNA and RNA. In one embodiment, in the double-stranded polynucleotide used, at least a part of the upstream side of the polynucleotide may be RNA. In one embodiment, the upstream portion of the nucleotides used can consist of 9-13 nucleotides.

  In one embodiment, the double-stranded polynucleotide used consists of 19-25 nucleotides, of which at least about 1/2 upstream can be RNA.

  In RNAi technology, the target gene may be plural. As a result of inhibition of target gene expression by RNAi technology, the effect can be confirmed by analyzing phenotypic changes that appear in the cells, tissues, or individuals.

  Using RNAi technology, specific properties can be imparted to cells, tissues, or individuals by inhibiting the expression of target genes.

  The functional site of RNA in the RNAi method can be identified by a method including the following steps: (i) a double-stranded polynucleotide having a sequence substantially identical to at least a part of the base sequence of the target gene. (Ii) introducing the double-stranded polynucleotide into a cell, tissue, or individual, and (iii) expressing a target gene in the cell, tissue, or individual. Measure the degree of inhibition and (iv) identify the sequences that are required to be RNA for inhibition of target gene expression. In this method, in the double-stranded polynucleotide used, either one of the double strands may be an RNA strand.

  As used herein, the “target gene” used in RNAi technology refers to mRNA, and optionally protein, in a cell, tissue, or individual into which the gene is to be introduced (hereinafter sometimes referred to as “the recipient”). Anything can be used as long as it can be translated to yield. Specifically, the target to be introduced may be endogenous or exogenous. Further, it may be a gene present on the chromosome or an extrachromosomal one. Examples of exogenous substances include those derived from pathogens such as viruses, bacteria, fungi, or protozoa that can infect the recipient. The function may be known or unknown, and the function may be known in a cell of another organism, but the function may be unknown in the introduced body.

  A double-stranded polynucleotide comprising DNA and RNA having substantially the same sequence as the base sequence of at least a part of these genes (hereinafter sometimes referred to as “double-stranded polynucleotide”) Of the base sequence of the target gene, it consists of a sequence that is substantially identical to a sequence of 20 nucleotides or more, which may be any part. Here, “substantially identical” means having 50% or more, preferably 70% or more, more preferably 80% or more homology with the target gene sequence. The chain length of the nucleotide may be any length from 19 nucleotides to the full length of the open reading frame (ORF) of the target gene, but those having a chain length of 19 to 500 nucleotides are preferably used. However, in cells derived from mammals, the existence of a signal transduction system that is activated in response to a double-stranded RNA having a chain length of 30 nucleotides or more is known. This is called an interferon reaction (Mareus, PI, et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters the cell, PKR (dsRNA-responses) protein Kinase: Bass, BL, Nature, 411, 428-429 (2001)), the initiation of translation of many genes is non-specifically inhibited, and at the same time, 2 ′, 5 ′ oligoadenylate synthetase (Bass, LB, Nature, 411, 428-429 (2001)), activation of RNase L occurs, and nonspecific degradation of RNA in the cell is induced. These non-specific reactions mask the specific reaction of the target gene. Therefore, when a mammal, or a cell or tissue derived from the animal is used as an introducer, a double-stranded polynucleotide comprising 19 to 25, preferably 19 to 23, more preferably 19 to 21 nucleotides is used. The double-stranded polynucleotide of the present invention does not need to be double-stranded as a whole, and includes those in which the 5 ′ or 3 ′ end partially protrudes. The protruding end is 1 to 5 nucleotides, preferably 1 -3 nucleotides, more preferably 2 nucleotides. The most preferred example includes one having a structure in which the 3 'end of each polynucleotide chain protrudes by 2 nucleotides. A double-stranded polynucleotide means a polynucleotide in which the complementary portion is double-stranded, but a single-stranded polynucleotide having self-phase correction may be self-annealed. Examples of the single-stranded polynucleotide having self-phase correction include those having an inverted repeat sequence.

  Furthermore, for mixing DNA and RNA, a hybrid type of DNA chain and RNA chain, a chimeric type of DNA and RNA, or the like is used. The hybrid of the DNA strand and the RNA strand may be any as long as it has an activity that inhibits the expression of the target gene when it is introduced into the recipient, but preferably the sense strand is A DNA whose antisense strand is RNA is used. Moreover, as long as it has the activity which inhibits the expression of a target gene, when it introduce | transduces it into a introduce | transduced body, what kind of thing may be sufficient as the chimera type of DNA and RNA. In order to enhance the stability of the double-stranded polynucleotide, it is preferable to contain as much DNA as possible. However, among the chimeric double-stranded polynucleotides of the present invention, a sequence that is necessary for inhibition of target gene expression is RNA. It is desirable to appropriately determine within the range where expression inhibition occurs while analyzing the expression inhibition degree of the target gene described later. Thereby, the functional site of RNA in RNAi method can also be identified. Preferable examples of the chimeric type thus determined include those in which a part of the upstream side of the double-stranded polynucleotide is RNA. Here, the upstream side means the 5 'side of the sense strand and the 3' side of the antisense strand. The portion on the upstream side is preferably a portion of 9 to 13 nucleotides from the upstream end of the double-stranded polynucleotide. Further, as a preferred example of such a chimeric double-stranded polynucleotide, each polynucleotide has a chain length of 19 to 21 polynucleotides, and at least the upstream half of the polynucleotide is RNA, and A double-stranded polynucleotide in which the others are DNA. Of such double-stranded polynucleotides, those in which the antisense strand is entirely RNA have a higher target gene expression inhibitory effect.

  Although there is no restriction | limiting in particular as a preparation method of double stranded polynucleotide, It is preferable to use the chemical synthesis method known per se. In the chemical synthesis, single-stranded polynucleotides having complementarity can be synthesized separately and associated with each other by an appropriate method to be double-stranded. Specifically, as a method of association, for example, the synthesized single-stranded polynucleotide is preferably at a molar ratio of at least about 3: 7, more preferably at a molar ratio of about 4: 6, and most preferably essentially. Examples of the method include mixing in the same molar amount (that is, a molar ratio of about 5: 5), heating to a temperature at which the double strand is dissociated, and then gradually cooling. The associated double-stranded polynucleotide is purified, if necessary, by a commonly used method known per se. As a purification method, for example, a method of confirming by using an agarose gel or the like and arbitrarily removing the remaining single-stranded polynucleotide by decomposing with an appropriate enzyme can be used. In addition, when preparing a single-stranded polynucleotide having a self-phase correction having an inverted repeat sequence, the polynucleotide is prepared by a method such as chemical synthesis and then self-complementarity by the same method as described above. It is prepared by associating sequences having

  The introduction of a double-stranded polynucleotide into a cell, tissue, or individual and inhibition of target gene expression are described. The recipient to which the double-stranded polynucleotide thus prepared is introduced may be any one as long as the target gene can be transcribed into RNA or translated into protein in the cell. Specifically, the introducer used in the present invention means a cell, tissue, or individual. Examples of the cells used in the present invention include germline cells, somatic cells, differentiated totipotent cells, multipotent cells, divided cells, non-divided cells, parenchymal tissue cells, epithelial cells, immortalized cells, and transformed cells. It may be. Specific examples include undifferentiated cells such as stem cells, cells derived from organs or tissues, or differentiated cells thereof. Tissues include single-cell embryos or constitutive cells, multi-cell embryos, fetal tissues and the like. Examples of the differentiated cells include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, Examples include basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and endocrine or exocrine gland cells. Specific examples of such cells include CHO-KI cells (RIKEN Cell bank), Drosophila S2 cells (Schneider, I., et al., J. Embryol. Exp. Morph., 27, 353-365 (1972). ), Human HeLa cells (ATCC: CCL-2), human HEK293 cells (ATCC: CRL-1573), and the like are preferably used. Furthermore, specific examples of the individual used as the recipient in the present invention include those belonging to plant, animal, protozoan, virus, bacteria, or fungal species. The plant may be monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate. Preferred microorganisms as recipients of the present invention are those used in agriculture or by industry and are pathogenic to plants or animals. Fungi include organisms in both mold and yeast forms. Examples of vertebrates include mammals including fish, cattle, goats, pigs, sheep, hamsters, mice, rats, monkeys and humans, and invertebrates include nematodes and other reptiles, gyro Drosophila, and other insects are included. Specific examples of the cultured cells include CHO-KI cells (RIKEN Cell bank), Drosophila S2 cells (Schneider, I., et al., J. Embryol. Exp. Morph., 27, 353-365 ( 1972)), human HeLa cells (ATCC: CCL-2), human HEK293 cells (ATCC: CRL-1573), and the like are preferably used.

  As a method for introducing a double-stranded polynucleotide into a recipient, when the recipient is a cell or tissue, a calcium phosphate method, an electroporation method, a lipofection method, a virus infection, a double-stranded polynucleotide solution Soaking or transformation method is used. Examples of the method for introduction into the embryo include microinjection, electroporation, and virus infection. When the recipient is a plant, a method by injection or perfusion into the body cavity or stromal cells of the plant or spraying is used. In the case of an animal individual, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ophthalmic, intraperitoneal administration, etc. The method, electroporation method, virus infection or the like is used. For methods for oral introduction, double-stranded polynucleotides can be mixed directly with the food of the organism. Furthermore, when introduced into an individual, it can be administered, for example, as an implanted long-term release preparation or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced.

  The amount of double-stranded polynucleotide to be introduced can be appropriately selected depending on the introduced body and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell. Specifically, for example, when the recipient is a cultured human cell and a double-stranded polynucleotide is introduced by the calcium phosphate method, 0.1 to 1000 nM is preferable.

  Here, two or more types of double-stranded polynucleotides can be introduced simultaneously. In this case, inhibition of expression of two or more types of target genes is expected in cells, tissues, or individuals (hereinafter sometimes referred to as “introducers”) to which the polynucleotide has been introduced. In the present invention, the expression inhibition of the target gene not only completely inhibits its expression, but also means inhibition of 20% or more as the expression level of m-RNA or protein. The degree of target gene expression inhibition can be measured by comparing the accumulation of RNA of the target gene, or the amount of protein encoded by the target gene, between a double-stranded polynucleotide introduced product and a non-introduced product. . The amount of mRNA can be measured by a commonly used method known per se. Specifically, for example, Northern hybridization, quantitative reverse transfer PCR, or in situ hybridization can be used. In addition, the amount of protein produced can be measured by Western blotting using an antibody whose antigen is the protein encoded by the target gene, or by measuring the enzyme activity of the protein encoded by the target gene.

  As a result of inhibition of gene expression in the introduced body by the double-stranded polynucleotide of the present invention, the function of the gene targeted by the introduced double-stranded polynucleotide is identified by analyzing phenotypic changes that appear in the introduced body. be able to.

Here, even if the function of the target gene is known, the function of the target gene may be unknown. Double-stranded polynucleotide corresponding to the target gene is prepared as above SL, it is introduced in a manner similar to the introduction body. The phenotype whose change should be analyzed in the introducer is not particularly limited. For example, the form of the introducer, the amount of substance introduced into the introducer, the amount of substance secreted by the introducer, the dynamics of the substance in the introducer, the adhesion between the introducer, and the introducer Movement, or life-form behavior such as the life of an introducer. When the function of the target gene is known in other recipients, it is preferable to analyze the phenotype associated with that function. As a means for analyzing a change in phenotype, when analyzing a change in the form of an introduced body, a microscope or a method of detecting it visually can be used. In addition, for example, in the case of mRNA as a substance in the transduced body, Northern hybridization, quantitative reverse transfer PCR, in situ hybridization, etc. can be mentioned as an analysis method of the amount. In the case of a protein, examples of the amount analysis method include Western blotting using an antibody whose antigen is a protein encoded by the target gene, and a method of measuring the enzyme activity of the protein encoded by the target gene. Since the phenotypic change that appears only in the introduced body analyzed as described above is a result of inhibition of expression of the target gene, it can be identified as a function of the target gene.

  By inhibiting the expression of a target gene using the double-stranded polynucleotide of the present invention, a specific property can be imparted to a cell, tissue, or individual. The specific property refers to what appears in the introduced body as a result of inhibition of target gene expression. The target gene here may be a gene whose inhibition of expression gives the introduced body already known, or a function or an unknown function in the introduced body. For a target gene whose function is unknown, after introducing a double-stranded polynucleotide for the target gene, by selecting a desired phenotype of the introducer, the desired property is imparted to the introducer. Can do.

  Specific examples of desired properties to be imparted to the recipient include, for example, an intracellular production function, a function of inhibiting extracellular secretion, a function of repairing damage to cells and DNA, a function of resistance to specific diseases, and the like. . Specifically, when the recipient is a plant individual or the like, examples of target genes include enzymes related to fruit ripening, plant structural proteins, genes related to pathogenicity, and the like. When the target gene expression inhibition has a resistance function against a specific disease, the increase in the expression of a specific protein causes a specific disease, and the target gene is the gene encoding the protein or the above Examples thereof include a gene encoding a protein having a function of controlling protein expression. As a specific example, the target gene is a gene necessary for maintaining a canceration / tumorization phenotype, and the recipient is a cancerous cell or a tumor tissue.

  Such a double-stranded polynucleotide against the target gene inhibits the expression of the protein encoded by the target gene, and thus can be used as a therapeutic or prophylactic agent for a disease associated with the target gene. When a double-stranded polynucleotide is used as the active ingredient of the drug, the polynucleotide can be used alone, but can also be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. . The ratio of the active ingredient to the carrier at this time can vary between 1 and 90% by weight. In addition, such drugs can be administered in various forms, such as tablets, capsules, granules, powders, syrups or the like, or injections, drops, liposomes, Parenteral administration with a suppository or the like can be mentioned. Further, the dose can be appropriately selected depending on symptoms, age, body weight and the like.

  An introducer into which a double-stranded polynucleotide targeting such a gene has been introduced is selected depending on the phenotype predicted to accompany inhibition of the gene expression. In addition, if the double-stranded nucleotide to be introduced is linked to a specific genetic marker, for example, a sequence encoding a fluorescent protein, the degree of inhibition of the expression of the fluorescent protein introduced into the recipient with the double-stranded polynucleotide is increased. It is also possible to select based on. Among these, for example, when a gene that functions in cancer suppression is used as a target gene, the cell traits to be selected include malignant diseases such as increased proliferation ability, decreased cell adhesion ability, or increased exercise (metastasis) ability. Examples include tumor traits. In addition, when a gene that adjusts a biological rhythm is a target gene, examples of cell traits to be selected include loss of a cell-specific circadian rhythm. Furthermore, when a gene involved in repair of DNA damage caused by an environmental mutagen is used as a target gene, the cell trait to be selected includes sensitivity to the mutagen.

  The selected introducer can be established and obtained as a system by a known cloning technique suitable for each. Specifically, when the recipient is a cell, the recipient should be established and acquired as a cell line by the method of establishing a cell line in a normal cultured cell, such as the limiting dilution method or the drug resistance marker method. Can do. Introduced with a specific function obtained in the present invention is a cell line with increased production or secretion efficiency of useful substances, a cell line showing high sensitivity to environmental factors that cause damage to cells or DNA, It shows traits associated with diseases and can be used as a model for disease treatment.

  Among these, a method for obtaining a cell line as a model for disease treatment will be described as a further specific application example of the present invention. Examples of target genes include genes whose decrease in expression level or absence causes disease. Specifically, a factor (BENI or a variant thereof) gene of TSLC1 molecular pathway in small cell lung cancer and the like can be mentioned.

  By introducing a double-stranded polynucleotide consisting of DNA and RNA having substantially the same sequence as at least a part of the base sequences of these human genes, for example, into human-derived culture cells, human-type diseases Model cells can be obtained. Furthermore, by contacting a test substance with a cell, tissue, or individual that has been given this specific property, and analyzing whether or not there is a change in the symptom or trait of the disease involving the gene, It is also possible to screen for therapeutic and / or prophylactic agents for the above diseases.

  When a substance selected by such screening is used as an active ingredient of the drug, the substance can be used alone, but it is used as a pharmaceutical composition in combination with a pharmaceutically acceptable carrier. You can also. The ratio of the active ingredient to the carrier at this time can vary between 1 and 90% by weight. In addition, such drugs can be administered in various forms, such as tablets, capsules, granules, powders, syrups or the like, or injections, drops, liposomes, Parenteral administration with a suppository or the like can be mentioned. Further, the dose can be appropriately selected depending on symptoms, age, body weight and the like.

  The RNAi technique described above in the present specification is a method for introducing a double-stranded polynucleotide comprising DNA and RNA having substantially the same sequence as the base sequence of at least a part of a target gene into a recipient, In the method of the present invention, (a) an expression vector containing a DNA encoding an indicator protein, (b) a DNA having a sequence substantially the same as at least a part of the base sequence encoding the indicator protein; Analyzing only the introduced body in which the expression of the gene in the introduced body has been inhibited by introducing a double-stranded polynucleotide consisting of RNA and conducting primary screening of the introduced body using the signal amount emitted from the indicator protein as an index. And efficient analysis can be performed.

  As a more specific example of the present invention, a case will be described in which the recipient is a vertebrate-derived cultured cell and the indicator protein is a fluorescent protein. An expression vector containing DNA encoding a fluorescent protein is introduced into cultured cells derived from vertebrates and cultured, and cells with a fluorescence amount emitted from the indicator protein of a specific intensity or higher are selected. The cells selected here are further introduced with a double-stranded polynucleotide consisting of DNA and RNA having a sequence substantially the same as the base sequence of at least a part of the DNA encoding the indicator protein, and cultured. The degree of inhibition of gene expression is analyzed by the degree of attenuation of the amount of fluorescence emitted from the indicator protein.

Such primary screening is to confirm that the introduction of the double-stranded polynucleotide into the recipient, and that the target gene expression inhibition has occurred in the recipient, indicator protein, and the protein mass and it Hassu that signal amount needs is to correlate. Specific examples of such proteins include luciferase protein.

  Furthermore, when measuring the degree of inhibition of target gene expression, the amount of protein encoded by the target gene can also be calculated based on the expression level of the indicator protein.

  The kit for carrying out the RNAi technique described in this specification includes a double-stranded polynucleotide, a vector containing DNA encoding a marker protein, and a sequence substantially the same as at least a part of the nucleotide sequence of the marker gene. Examples include double-stranded polynucleotides consisting of DNA and RNA, reagents such as enzymes and buffers, reagents for introducing polynucleotides, and the like. The kit of the present invention does not need to contain all these reagents and the like, and any combination of reagents or the like may be used as long as it is a kit used in the above-described method of the present invention.

  When RNAi technology is used, even cells that have a weak expression of the RNAi effect may be subjected to primary screening for cells that express a particularly high RNAi effect and use stronger ones.

  Stability as a substance of a polynucleotide to be introduced by using a polynucleotide consisting of the exemplified DNA and RNA as described above, specifically a hybrid polynucleotide consisting of a DNA strand and an RNA strand, or a DNA-RNA chimera polynucleotide And manufacturing costs can be reduced. From this, the polynucleotide itself to be introduced can be developed as a preparation for cancer treatment using RNAi technology. In addition, DNA can be easily subjected to various modifications such as fluorescent labeling, biotin labeling, amination, phosphorylation, and thiolation compared to RNA. Therefore, when used as a medicine or a reagent, a function according to the purpose can be added by performing such chemical modification.

(Administration and composition for regeneration / treatment / prevention)
The present invention provides methods for the treatment, inhibition and prevention of mesoderm abnormality-related diseases, disorders or conditions by the administration of an effective amount of a compound or pharmaceutical composition of the invention to a subject. In a preferred aspect, the compound can be substantially purified (eg, in a state substantially free of substances that limit its effect or cause undesirable side effects).

  In the present specification, the “effective amount for diagnosis, prevention, treatment or prognosis” refers to an amount which is recognized as being medically effective in diagnosis, prevention, treatment (or treatment) or prognosis, respectively. Such amounts can be determined by one skilled in the art using techniques well known in the art, taking into account various parameters.

  The animal targeted by the present invention may be any organism (eg, animal (eg, vertebrate, invertebrate)) as long as it has a nervous system or a similar system. Preferably, it is a vertebrate (for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably mammals (for example, single holes, marsupials, Rodents, crustaceans, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents , Rabbit eyes, etc.). Exemplary subjects include, but are not limited to, animals such as cows, pigs, horses, chickens, cats, dogs and the like. More preferably, cells derived from primates (eg, chimpanzee, Japanese monkey, human) are used. Most preferably, human-derived cells are used.

  When the nucleic acid molecule or polypeptide of the present invention is used as a medicament, such a composition may further comprise a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. Examples include, but are not limited to, agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, a medicament of the invention comprises a polypeptide or polynucleotide, such as activin or a variant or fragment thereof, or a variant or derivative thereof, one or more physiologically acceptable carriers, excipients or It is administered in the form of a composition containing with a diluent. For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used, for example Phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, polyvinylpyrrolidone); amino acid (Eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol) Salt formation vs. Io (E.g., sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)) but the like are not limited thereto.

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  The general preparation method of the pharmaceutical composition of this invention is shown below. In addition, veterinary drug compositions, quasi drugs, marine drug compositions, food compositions, cosmetic compositions and the like can also be produced by known preparation methods.

  Polypeptides, polynucleotides and the like used in the present invention are blended with a pharmaceutically acceptable carrier, solid preparations such as tablets, capsules, granules, powders, powders, suppositories, or syrups, injections , And can be administered orally or parenterally as liquid preparations such as suspensions, solutions, sprays and the like. As described above, the pharmaceutically acceptable carrier includes an excipient, a lubricant, a binder, a disintegrant, a disintegration inhibitor, an absorption enhancer, an adsorbent, a humectant, a solubilizing agent in a solid preparation, Stabilizers, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like can be mentioned. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. Moreover, it is also possible to mix | blend substances other than the polynucleotide used in this invention, a polypeptide, etc. with the composition of this invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.

  Examples of the excipient in the solid preparation include glucose, lactose, sucrose, D-mannitol, crystalline cellulose, starch, calcium carbonate, light anhydrous silicic acid, sodium chloride, kaolin and urea.

  Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, boric acid powder, colloidal silicic acid, talc, and polyethylene glycol.

  Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mannitol, crystalline cellulose, dextrin, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, starch solution, gelatin solution, polyvinylpyrrolidone, calcium phosphate , Potassium phosphate, shellac and the like.

  Examples of disintegrants in solid preparations include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid Examples include, but are not limited to, esters, sodium lauryl sulfate, starch, stearic acid monoglyceride, lactose, and calcium calcium glycolate.

  Preferable examples of the disintegration inhibitor in the solid preparation include, but are not limited to, hydrogenated oil, sucrose, stearin, cocoa butter and hydrogenated oil.

  Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.

  Examples of the adsorbent in the solid preparation include, but are not limited to, starch, lactose, kaolin, bentonite and colloidal silicic acid.

  Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.

  Examples of the solubilizer in the solid preparation include, but are not limited to, arginine, glutamic acid, aspartic acid and the like.

  Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin and lactose.

  When preparing tablets, pills and the like as solid preparations, they may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) as necessary. Tablets include tablets with ordinary coatings as necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets. Capsules include hard capsules and soft capsules. When forming into the form of a suppository, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides and the like can be added in addition to the additives listed above, but are not limited thereto.

  Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil.

  Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. It is not limited to them.

  Suitable examples of the suspending agent in the liquid preparation include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, Examples include, but are not limited to, hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

  Preferable examples of the isotonic agent in the liquid preparation include, but are not limited to, sodium chloride, glycerin, D-mannitol and the like.

  Preferable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate and citrate.

  Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, procaine hydrochloride and the like.

  Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, α-tocopherol and cysteine.

  When preparing as an injection, it is preferable that the solution and suspension are sterilized and isotonic with blood. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericidal agent, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol, or a mixed solution thereof) just before use. May be.

  Furthermore, if necessary, the pharmaceutical composition may contain coloring agents, preservatives, flavors, flavoring agents, sweeteners, and the like, as well as other agents.

  The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. Such a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability and the like. In this specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.). Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent.

  The medicament of the present invention may be a physiologically acceptable carrier, excipient or stabilizer as required (Japanese Pharmacopoeia 15th edition, supplement or update as necessary, Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990, etc.) and a glycan composition having a desired degree of purity, by mixing with a lyophilized cake or aqueous solution form Can be prepared and stored.

  Various delivery systems are known and can be used to administer the compounds of the invention (eg, liposomes, microparticles, microcapsules, etc.). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route (eg, by infusion or bolus injection, by absorption through the epithelium or mucosal lining (eg, oral mucosa, rectal mucosa, intestinal mucosa, etc.), and others. It can be administered with a biologically active agent. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention may be administered by any suitable route (including intraventricular and intrathecal injection; intraventricular injection, for example, intraventricular attached to a reservoir, such as an Ommaya reservoir. It may be desirable to introduce into the central nervous system by a catheter). Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

  In certain embodiments, it may be desirable to administer the polypeptide, polynucleotide or composition of the invention locally to the area in need of treatment (eg, central nervous system, brain, etc.); this limits Although not intended, for example, local injection during surgery, topical application (eg in combination with a post-surgical wound dressing), by injection, by catheter, by suppository, or an implant (this implant is shearable ( a porous, non-porous, or glue-like material), including membranes or fibers such as (sialastic) membranes. Preferably, when administering a protein of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.

  In another embodiment, the compound or composition can be delivered encapsulated in vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious). Disesease and Cancer, Lopez-Berstein and Fidler (eds.), Liss, New York, pages 353-365 (1989); see Lopez-Berstein, pages 317-327; see broadly the same book).

  In yet another embodiment, the compound or composition can be delivered in a controlled sustained release system. In one embodiment, a pump can be used (Langer (supra); Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Florida (1974); Controlled DrugDrivable Drug Bioavailability. And Ball (eds.), Wiley, New York (1984); see Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 1985); During et al., Ann. l.25: 351 (1989); see also Howard et al., J. Neurosurg. 71: 105 (1989)).

  In yet another embodiment, the controlled sustained release system can be placed near the therapeutic target, i.e., the brain, and therefore requires only a portion of the systemic dose (e.g., Goodson, Medical Applications of Controlled Release). , (Supra), Vol. 2, pages 115-138 (1984)).

  Other controlled controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

  The amount of the composition used in the treatment method of the present invention takes into consideration the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. Can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  The dose of the polypeptide, polynucleotide and the like of the present invention varies depending on the age, weight, symptom or administration method of the subject, and is not particularly limited, but is usually 0.01 mg to 10 g in the case of oral administration per day for an adult. Preferably, it may be 0.1 mg to 1 g, 1 mg to 100 mg, 0.1 mg to 10 mg, and the like. In the case of parenteral administration, it is 0.01 mg to 1 g, preferably 0.01 mg to 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 0.1 mg to 10 mg, and the like.

  As used herein, “administering” refers to a polypeptide, polynucleotide, factor or the like of the present invention or a pharmaceutical composition containing the same, alone or in combination with other therapeutic agents, to cells or tissues of an organism. It means capturing. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This also includes a procedure in which the combined drugs are administered together as a therapeutic mixture, and the combined drugs are administered separately but simultaneously (eg, through separate intravenous lines to the same individual). Also includes. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

  An abnormal condition can also be prevented or treated by administering a compound (such as an agent identified by the present invention) to a group of cells having an abnormality in a signal transduction pathway to an organism. The effect of administering the compound on the biological function can then be monitored. This organism is preferably a laboratory animal such as a mouse, rat, rabbit, guinea pig, goat or monkey (monkey or ape), and most preferably a human.

  In the present specification, the “instruction” describes a method for administering or diagnosing the pharmaceutical of the present invention to a doctor, a patient or other person who performs administration, or a person to be diagnosed (which may be the patient). It is. This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the US) in which the present invention is implemented, and is approved by the regulatory authority It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page (web site) or e-mail provided on the Internet) It can also be provided in such a form.

  The end of treatment according to the method of the present invention may be determined by standard clinical laboratory results from commercially available assays or instrumentation or diseases associated with BENI, activin, etc. (eg, mesoderm abnormal disease, blood system disease) ) Can be supported by the disappearance of clinical symptoms characteristic of. Treatment can be resumed by recurrence of a disease associated with BENI, activin, etc. (eg, mesoderm abnormal disease, blood system disease).

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product may optionally be attached to such containers, which may be made, used or sold for administration to humans. Represents approval by government agencies.

  The plasma half-life and biodistribution of drugs and metabolites in organs that cause mesoderm abnormalities can also be determined to facilitate selection of the most appropriate drug to inhibit the disorder. Such a measurement can be performed. For example, HPLC analysis can be performed on the plasma of a drug-treated animal, and the location of the radiolabeled compound can be determined using detection methods such as X-ray, CAT scan and MRI. Compounds that exhibit potent inhibitory activity in screening assays but lack pharmacokinetic characteristics can be optimized by chemical structure alteration and retesting. In this regard, compounds that exhibit good pharmacokinetic characteristics can be used as models.

  Toxicity studies can also be performed by testing the compositions of the present invention. For example, toxicity studies can be performed in appropriate animal models such as: (1) a compound is administered to mice (untreated control mice should also be used); (2) each Blood samples are periodically obtained from one mouse in the treatment group via the tail vein; and (3) the sample is obtained from the number of red blood cells and white blood cells, the composition and the ratio of lymphocytes to polymorphonuclear cells Analyze about. Comparison of the results for each dosing regimen with controls shows whether there is toxicity.

  Further studies can be performed by sacrificing the animals at the end of each toxicology study (preferably, the American Veterinary Medical Guidelines Report of the American Veterinary Medical Assoc. Vet.Med.Assoc.202: 229-249). A representative animal from each treatment group can then be tested by global examination for direct evidence of metastasis, abnormal disease or toxicity. Overall abnormalities in the tissue are described and the tissue is examined histologically. Compounds that cause weight loss or blood component loss are not preferred, as are compounds that have adverse effects on major organs. In general, the greater the adverse effect, the less preferred the compound.

(Best Mode for Carrying Out the Invention)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(BENI)
In one aspect, the present invention provides a nucleic acid sequence or a variant or fragment thereof encoding the Brachyury Expression N uclear Inhibitor (BENI) . The BENI whose function was found in the present invention is typically (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (b) in the amino acid sequence set forth in SEQ ID NO: 2 A polypeptide having one or several amino acids having at least one mutation selected from the group consisting of substitutions, additions and deletions and having biological activity; (c) according to SEQ ID NO: 1 A polypeptide encoded by an allelic variant or a splice variant of the base sequence; (d) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2 ; or (e) (a)-(d A polypeptide having an amino acid sequence that is at least 70% identical to any one polypeptide and having biological activity It is a nucleic acid molecule or variant or fragment thereof.

In a preferred embodiment, BENI is a nucleic acid molecule encoding the sequence shown in SEQ ID NO: 2 or a variant or fragment thereof. Alternatively, in another preferred embodiment, BENI comprises the sequence shown in SEQ ID NO: 1 .

  In one embodiment, the biological activity that BENI may have is regulatory activity of mesoderm differentiation induction, typically organizer formation activity, gastrulation activity, nerve induction activity, pattern Examples include formation activity.

  In one exemplary embodiment, the biological activity possessed by the BENI of the present invention is modulating the mesoderm differentiation inducing factor Brachyury. The regulatory activity of the mesodermal differentiation inducing factor Brachyury can be measured using methods known to those skilled in the art.

In another aspect, the present invention provides a Brachyury Expression N uclear Inhibitor (BENI) polypeptide or variant or fragment thereof. A BENI polypeptide typically comprises (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (b) one or more or 1 or a number in the amino acid sequence set forth in SEQ ID NO: 2 A polypeptide having one mutation selected from the group consisting of substitutions, additions and deletions and having biological activity; (c) a splice mutation of the base sequence described in SEQ ID NO: 1 A polypeptide encoded by a body or allelic variant; (d) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2 ; or (e) any one of (a)-(d) A polypeptide or a variant or fragment thereof having an amino acid sequence with at least 70% identity to one polypeptide and having biological activity It is an instrument.

In one embodiment, the BENI of the invention is a polypeptide having the sequence shown in SEQ ID NO: 2 , or a variant or fragment thereof.

In another embodiment, the BENI of the invention is a polypeptide encoded by the sequence shown in SEQ ID NO: 1 , or a variant or fragment thereof.

  In another embodiment, the biological activity possessed by the BENI of the present invention is a regulatory activity for inducing differentiation of mesoderm.

  In another embodiment, the biological activity possessed by the BENI of the present invention may be an activity of regulating the mesodermal differentiation inducing factor Brachyury.

  In one specific embodiment, the present invention provides an agent capable of modulating the activity of modulating the mesodermal differentiation inducing factor Brachyury, wherein the factor comprises a nucleic acid molecule of the present invention or a variant or It may be a fragment or an agent having a function of inhibiting the function of the polypeptide of the present invention or a variant or fragment thereof.

  The agent of the present invention can be selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof.

  In one specific embodiment, an agent of the invention is characterized by interacting with a nucleic acid molecule of the invention or a variant or fragment thereof, or a polypeptide of the invention or a variant or fragment thereof.

  In another embodiment, an agent of the invention is an antisense nucleic acid molecule or RNAi molecule of a nucleic acid molecule of the invention or variant or fragment thereof, or an antibody against a polypeptide of the invention or variant or fragment thereof. It is characterized by being.

  In certain embodiments, the agent of the present invention further comprises means for delivering the agent to a specific site. Such delivery systems are well known and can be used as appropriate.

  In another embodiment, the means may express the nucleic acid molecule specifically at the specific site. As such an expression system, any one used in molecular biology can be used.

  In another embodiment, the means included in the factor of the present invention may be a promoter. This means can be a directional liposome.

  In another aspect, the invention provides a vector comprising a nucleic acid molecule of the invention (eg, encoding a BENI gene). It will be appreciated that any such vector can be used. The nucleic acid molecule contained in the vector may be an inhibitory nucleic acid molecule that inhibits the function of the nucleic acid molecule (BENI) of the present invention instead of the BENI gene.

  It will be appreciated that as a more preferred embodiment, any embodiment described in (BENI) can be used.

(Screening method)
In another aspect, the present invention provides a method of screening for a regulator of mesoderm differentiation induction. In this method, A) providing a candidate substance, B) subjecting the assay system to determine whether the candidate substance modulates the activity of BENI; C) regulating BENI among the candidate substances. Identifying a factor and determining that the modulatory factor is a modulator of BENI.

  In one embodiment, the candidate substance is selected from, but not limited to, the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof.

  In another embodiment, the present invention provides a modulator of BENI identified by the method of the present invention. Specific examples of the regulatory factor include, for example, an antibody, a variant thereof, an antisense molecule, and an RNAi molecule.

  Such a screening comprises at least one molecule selected from the group consisting of factors that specifically interact with the nucleic acid molecule or polypeptide of interest, and variants and fragments thereof, and molecules that interact with them. It can be identified by determining whether a test factor significantly affects the interaction (decrease, enhancement, disappearance, etc.). Such test factor determination methods are well known in the art, and the results can be calculated using any statistical technique. Such screening or identification methods are well known in the art, and for example, such screening or identification can be performed using a biomolecule array or chip such as a microtiter plate, DNA or protein. Examples of the subject containing the screening test factor include, but are not limited to, a gene library and a compound library synthesized with a combinatorial library.

  Therefore, in the present invention, it is also intended that a drug by computer modeling is provided based on the disclosure of the present invention.

  It will be appreciated that as a more preferred embodiment, any embodiment described in (BENI) can be used.

(Regulatory factor production method)
In another aspect, the present invention provides a method for producing a regulator of mesoderm differentiation induction. The method comprises: A) providing a candidate substance; B) subjecting it to an assay system for determining whether the candidate substance modulates the activity of BENI; C) regulating BENI among the candidate substances. Identifying a factor and determining that the modulating factor is a BENI modulator; D) producing the BENI modulator. Here, any production method can be used, and candidate substances can be selected from a combinatorial chemistry library.

  Accordingly, the present invention provides a BENI modulator produced by such a method of the present invention.

The present invention, in other embodiments, is obtained using computational quantitative structure activity relationship (QSAR) modeling techniques as a tool for screening efficacy for modulatory activity for compounds of the present invention. The compound is included. Here, computer technology encompasses several substrate templates prepared by a computer, pharmacophore, and produce such as homology model of the active site of the present invention. In general, from the data obtained in vitro, the normal characteristic group of an interacting substance for a substance is modeled by the CATALYST ^™ pharmacophore method (Ekins et al., Pharmacogenetics, 9: 477-489, 1999; Ekins). et al., J. Pharmacol. & Exp.Ther., 288: 21-29, 1999; Ekins et al., J. Pharmacol. & Exp.Ther., 290: 429-438, 1999; Ekins et al., J. Pharmacol. & Exp. Ther., 291: 424-433, 1999) and comparative molecular field analysis (CoMFA) (Jones et al. Drug Metabolism & Disposition, 24: 1~6,1996) are shown using, for example. In the present invention, computer modeling may be performed using molecular modeling software such as CATALYST ^™ version 4 (Molecular Simulations, Inc., San Diego, Calif.).

Fitting of the compound to the active site can row Ukoto using any of various known computer modeling techniques in the art. Visual inspection and manual manipulation of compounds to the active site are described in QUANTA (Molecular Simulations, Burlington, MA, 1992), SYBYL (Molecular Modeling Software, Tripos Associates, Inc., St. AM, St. Louis, MO, 1992). et al., J. Am. Chem. Soc., 106: 765-784, 1984), CHARMM (Brooks et al., J. Comp. Chem., 4: 187-217, 1983), etc. Can be done using. In addition, energy can be minimized using standard force fields such as CHARMM, AMBER, and the like. Other more specialized computer modeling are GRID (Goodford et al., J. Med. Chem., 28: 849-857, 1985), MCSS (Miranker and Karplus, Function and Genetics, 11: 29-34, 1991), AUTODOCK (Goodsell and Olsen, Proteins: Structure, Function and Genetics, 8: 195-202, 1990), DOCK (Kuntz et al., J. Mol. Biol., 161: 269-288, (1982) Etc. Further structural compounds include LUDI (Bohm, J. Comp. Aid. Molec. Design, 6: 61-78, 1992), LEGEND (Nishibata and Itai), such as blank active sites, active sites in known low molecular weight compounds, and the like. , Tetrahedron, 47: 8985,1991), can LeapFrog (Tripos Associates, St.Louis, also be constructed de novo using computer Tup programs such as MO). Such modeling is well known and commonly used in the art, and those skilled in the art can appropriately design compounds that fall within the scope of the present invention according to the disclosure of the present specification.

  In another aspect, the present invention provides an agent identified by the above identification or modeling method of the present invention.

  It will be appreciated that as a more preferred embodiment, any embodiment described in (BENI) can be used.

(Diagnosis)
In one aspect, the present invention provides a method for diagnosing a condition associated with mesoderm differentiation. The method includes A) measuring the level of BENI. This method preferably further includes the step of B) determining the state of mesoderm differentiation using the change in the level of BENI as an indicator.

  In another aspect, the present invention provides a diagnostic reagent for a condition associated with mesoderm differentiation, comprising a factor specific for BENI.

  It will be appreciated that as a more preferred embodiment, any embodiment described in (BENI) can be used.

(Prevention / treatment)
In one aspect, the present invention provides a method for the treatment or prevention of a condition associated with mesoderm differentiation comprising A) modulating the level of BENI. In this way, the modulating step can be achieved using factors specific for BENI. The factor specific to BENI may include a nucleic acid sequence encoding BENI or a variant thereof.

  It will be appreciated that as a more preferred embodiment, any embodiment described in (BENI) can be used.

  In another aspect, the present invention provides a system for the treatment or prevention of conditions associated with mesoderm differentiation. The system comprises A) means for adjusting the level of BENI. It will be appreciated that as a more preferred embodiment, any embodiment described in (BENI) can be used.

(use)
In another aspect, the present invention also provides the use of a BENI-encoding nucleic acid molecule or BENI polypeptide or a factor specific thereto for the manufacture of a diagnostic agent for a mesodermal differentiation state.

  Alternatively, in another aspect, the present invention relates to a medicament for treating or preventing a condition, disorder or disease associated with mesoderm differentiation of a nucleic acid molecule or BENI polypeptide encoding BENI or a factor specific thereto. Providing use for manufacturing.

  In another aspect, the present invention provides the use of a BENI-encoding nucleic acid molecule or BENI polypeptide or a factor specific thereto for the manufacture of a diagnostic agent for a mesodermal differentiation state.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  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. Animals were handled according to the spirit of animal welfare, in compliance with the standards stipulated at the University of Tokyo Animal Experiment Facility.

(Example 1: Isolation of BENI)
The present inventors searched fertilized eggs of Xenopus laevis by a 22K exhaustive microarray method for genes whose expression is significantly increased when activin A is added.

(Materials and methods)
(Preparation of fertilized eggs)
Eggs were obtained by injecting Xenopus into human chorionic gonadotropic hormone (Gestron: Denka Pharmaceutical Co., Ltd., Kawasaki City). A fertilized egg was prepared by artificial insemination, and gelatinous material was removed using sodium mercaptoacetate. The outbreak progressed to room 9 at room temperature. The fertilized eggs were then added with various concentrations (0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 10 ng / ml, 100 ng / ml of activin A, incubated for 1 hour, washed, and activin A was removed.

(Preparation of RNA and microarray analysis)
Total RNA was extracted using ISOGEN (catalog number 317-02503; Nippon Gene Co., Ltd., Toyama City). Cy-dye labeling of the extracted RNA was amplified according to the instructions of the Low RNA Input linear amplification kit (catalog number 5184-3523, Agilent Technologies, Tokyo). In summary, dsDNA was synthesized from the extracted RNA using an oligo dT primer containing the T7 promoter. Next, cDNA was synthesized with T7 polymerase using any of Cy3-dCTP and Cy-5dCTP (catalog numbers PA53021, PA55021, GE Healthcare Biosciences, Tokyo). Labeled cRNA was hybridized to a DNA microarray. The microarray was washed in 6 × SSC, 0.005% Triton X-102 at 20 ° C. and then in 0.1 × SSC, 0.005% Triton X-102 at 4 ° C. Data analysis was performed using the Rosetta Luminator system (Rosetta Biosoftware, http: // www
w. rosetabio. com). The Xenopus oligo microarray (60-mer oligo DNA) used was made to order from Agilent Technologies' microarray design service.
(result)
As a result, a gene registered under GenBank as the registration number BC043737 was found as such a gene. Although this BC043737 gene was known to exist, its function was unknown until now. As will be described in detail later, according to the study by the present inventors, BC043737 gene induces differentiation of mesoderm in early development while mainly suppressing expression of Brachyury, which is a mesoderm-specific factor, in the nucleus. I found out that it was adjusting. Accordingly, the present inventors have referred to as a BENI (Brachyury Expression N uclear Inhibitor) and BC043737 gene.

  BENI was isolated from the Xenopus laevis 17-19 embryo IMAGE cDNA clone library (Open Biosystems, USA). The Open Reading Frame (ORF) of the isolated BENI gene is 2,025 base pair, and the predicted protein has a sequence of 674 amino acids. By BLASTP search, BENI homologous genes exist not only from closely related species, Xenopus tropicalis, but also from humans to rats (R. norvegirus), mice (M. musculus), and greenfish (T. nigroviridis). I found out. As shown in FIG. 1, the homology between Xenopus BENI and human homologous genes is as high as 57% -93% in the N-terminal 1-58 amino acid sequence. On the other hand, homology on the C-terminal side is low.

  When a known functional domain was examined in the BENI gene sequence, a nuclear translocation signal enclosed in a box in FIG. 1 was identified (one on the N-terminal side and two on the C-terminal side). For other sequences, no known functional domains could be identified.

(Example 2: Elucidation of the function of BENI)
The present inventors investigated to what extent BENI was expressed in fertilized eggs of Xenopus laevis by the reverse transcription PCR (RT-PCR) method.

(Materials and methods)
Total RNA was extracted using the same method as in Example 1. 1 μg of total RNA was treated with RNase-free DNase I (GIBCO / BRL). The extracted total RNA was then pretreated with DNaseI. Reverse transcription was performed at 25 ° C. for 10 minutes, 42 ° C. for 50 minutes, and 70 ° C. for 15 minutes to synthesize cDNA. Originine decarboxylase (ODC) was used as a house-keeping gene (control). In the PCR reaction, denaturation reaction was performed at 95 ° C. for 1 minute, and annealing was performed under appropriate conditions, respectively, and the extension reaction was performed at 72 ° C. for 30 seconds. This was taken as one cycle, and PCR products were obtained by performing each appropriate number of cycles. The primer pair used for amplification was prepared by a commissioned primer synthesis service of Funakoshi Co., Ltd. (Tokyo). Each PCR product (5 μl) was separated by 3% agarose gel electrophoresis and visualized by cyber-gold (Wako Pure Chemicals, Osaka). ODC without RT-PCR was included to confirm genomic DNA contamination.

(result)
As a result, BENI is not expressed at all in the unfertilized egg derived from the maternal, and its expression in the epithelial portion rapidly increases from the 10th stage after the fertilization to the 12th stage and gradually expresses after the 12th stage. Was found to increase (FIG. 2).

  FIG. 2 shows the amount of BENI expressed in each Xenopus fertilized egg examined by RT-PCR (top). The middle orthine decarboxylase (ODC) was used as a so-called house-keeping gene (control). Moreover, the lower part shows ODC which does not perform RT-PCR.

  Similarly, fertilized eggs from the 10th stage to the 42nd stage were examined by the whole-mount in situ hybridization (WISH) method.

(Materials and methods)
As samples, fertilized eggs of 8th, 9th, 10th, 11th, 12th, 13th, 15th, 20th, 23th, 26th, 30th, 35th, 40th and 45th stages were used. . The WISH method was performed according to the method of Harland (Harland, RM (1991) Methods Cell Biol 36: 685-95) using a digoxigenin labeled antisense probe. For cell lineage tracking, albino embryos were injected with 250 pg β-gal mRNA. The injected region was treated with reaction buffer (1 mM MgSO ₄ , 10 mM K ₃ Fe (CN) ₆ , 10 mM K ₄ Fe (CN) ₆ , 0.1 M phosphorylation buffer and 0.1% Triton X- 100) and stained with red-gal (Research Organics, Inc). The probe used was produced by a probe synthesis contract service of Funakoshi Co., Ltd. (Tokyo). In contrast, a sense probe was used. The signal was detected using BM purple (Roche).

  As a result, it was found that the BENI gene was specifically expressed in the ectoderm epithelial part (FIG. 3), and that it was localized especially at the animal pole and non-depressed site from the analysis of the cross section ( FIG. 4).

  FIG. 3 shows the expression site of the BENI gene in Xenopus embryos by the WISH method (the photo shown at the left end of each panel is the result of a control experiment using a sense probe).

  FIG. 4 shows a cross-sectional section of Xenopus laevis embryos stained by the WISH method (upper stage) and an overall view of the cross-sectional section of the embryo (lower stage).

  Next, BENI-antisense morpholino RNA (BENI-MO) was injected into a frog embryo at the blastomere stage to examine the BENI-MO effect on normal development of the embryo (FIG. 5).

(Materials and methods)
The BENI antisense morpholino RNA (BENI-MO) was synthesized by an antisense synthesis contract service of Funakoshi Co., Ltd. (Tokyo). Fertilized eggs were prepared by the same method as in Example 1, and the development was advanced to the 40th blastomere stage. BENI-MO (34 ng) was injected into the frog embryo at the 40th bulbar stage on the back side. As a control, the same amount of StMO (Gene Tools, LLC, OR, USA) was injected.

  The result is shown in FIG. FIG. 5 shows that BENI-MO (34 ng) was injected into the back of the frog embryo at the blastomere stage and observed at the 35th to 36th stage (right panel). The left panel shows a control experiment in which the same embryo was injected with the same amount of unaffected (StMO).

  The embryos injected with BENI-MO are rounded and atrophied compared to the control embryo (left). From this result, it was found that BENI controls gastrulation movement by some mechanism.

  Next, the effect of BENI mRNA (BENI mRNA) and BENI-MO on extensional movement by activin A inducing action was examined using an animal cap which is an undifferentiated cell population of frogs (FIG. 6).

(Materials and methods)
The BENI antisense morpholino RNA (BENI-MO) was synthesized by an antisense synthesis contract service of Funakoshi Co., Ltd. (Tokyo). A fertilized egg was prepared in the same manner as in Example 1, and the animal cap of the 8th stage embryo was cut out at about 0.4 × 0.4 mm. The animal cap was treated in a frog saline Steinberg solution (SS) containing Activin A 25 ng / ml for 1 hour. Animal caps treated with activin A were treated with BENI mRNA (1 ng) or BENI-MO (34 ng), respectively. Subsequently, pre-culture was performed in SS without activin A for 0 hours, 12 hours, 3 hours, and 4 hours.

  The result is shown in FIG. FIG. 6 shows the effects of BENI mRNA and BENI-MO in the presence of activin A.

  An animal cap (upper right) treated with activin A (25 ng / mL) undergoes an extension movement relative to an animal cap not treated with activin A (upper left). On the other hand, when BENI mRNA (1 ng) is treated to the animal cap treated with activin A (lower left), the animal cap is inhibited from stretching motion, but normal stretching motion is treated with BENI-MO (34 ng) treatment. Indicates. Thus, BENI has an action of inhibiting the extension movement induced by activin A. Careful observation of the BENI-MO results showed that the embryos were more extended than the control embryos treated with activin A. Brachyury mesoderm inducer (Smith, JC, Price, B.M., Green, JB, Weigel, D. & Herrmann, BG (1991) Cell 67, 79-87. ), A transcription factor that regulates transcription in the nucleus (Kispert, A., Koscholz, B. & Herrmann, B. G. (1995) EMBO J. 14, 4763-4772.). Overexpression of Brachyury has already been reported to cause abnormal growth of the embryo, similar to the action of BENI-MO (Kwan, KM & Kirschner, MW (2003) Development 130, 1961 -1972.). This suggests that the effect of BENI on embryo elongation movement is related to the action of Brachyury.

(Example 3: Further elucidation of inhibition of gastrulation movement)
In this example, the inhibition of gastrulation movement was further elucidated.

  Based on the results thus far, BENI was considered to have an inhibitory control effect on gastrulation movement induced by activin A. Possible mechanisms of inhibition of gastrointestinal invagination include mediated mesoderm malformation and others. Furthermore, the relationship between the action of BENI and Brachyury has been suggested. Therefore, in order to investigate whether the inhibition control action of gastrulation movement shown by BENI is related to mesoderm formation, mesoderm factor in the absence and presence of activin A (Mix-1, Google, Chordin, The expression of Brachyury was examined by the reverse transcriptase PCR (RT-PCR) method (FIG. 7).

(Materials and methods)
BENI mRNA (1 ng) or BENI-MO (34 ng) was previously injected into the frog embryo at the 40th blastomerium stage. The embryo was raised until the 8th stage and the animal cap was isolated. Subsequently, animal caps isolated in the absence or presence of activin A (25 ng / mL) were cultured for 3.5 hours. The state of gene expression of mesoderm factor in the so-treated animal cap was identified by RT-PCR. Mix-1 is known as a mesendoderm marker, Goodsecoid and Chordin are dorsal mesoderm markers, and Brachyury is known as a pan-mesoderm marker.

  Total RNA was extracted from each animal cap using the same method as in Example 1. 1 μg of total RNA was treated with RNase-free DNase I (GIBCO / BRL). The extracted total RNA was then pretreated with DNaseI. Reverse transcription was performed at 25 ° C. for 10 minutes, 42 ° C. for 50 minutes, and 70 ° C. for 15 minutes to synthesize cDNA. ODC was used as a house-keeping gene (control). In the PCR reaction, denaturation reaction was performed at 95 ° C. for 1 minute, and annealing was performed under conditions appropriate for each, and the extension reaction was performed at 72 ° C. for 30 seconds. This was regarded as one cycle, and a PCR product was obtained by performing an appropriate number of cycles for each. Each primer pair used for amplification was produced by a primer synthesis contract service of Funakoshi Co., Ltd. (Tokyo). Each PCR product (5 μl) was separated by 3% agarose gel electrophoresis and visualized by cyber-gold (Wako Pure Chemicals, Osaka). ODC without RT-PCR was included to confirm genomic DNA contamination.

(result)
FIG. 7 shows identification of gene expression by RT-PCR (ODC was used as a positive control sample and rightmost rain was used as a control sample for normal embryos).

  As can be seen from the figure, the expression of any mesoderm marker is induced by activin A. Among them, only the expression of Brachyury is inhibited by the injection of BENI mRNA. Similarly, only the expression of Brachyury is enhanced by the injection of BENI-MO.

(Summary)
From all the above results, BENI regulates the expression of Brachyury in a suppressive manner, thereby controlling mesoderm induction and, as a result, governs embryonic shaping including gastrulation. There was found. As described above, BENI has a total of three nuclear translocation signals on both the N-terminal side and the C-terminal side (ie, 7-12 sequence, 653-668 sequence, 668-674 sequence ( See also Figure 1. Brachyury has been shown to dominate the induction of mesoderm differentiation during early vertebrate development as a nuclear factor (Kispert, A., Koschorz, B. & Herrmann, BG). (1995) EMBO J. 14,4763-4772.).

(Example 4: Function in the nucleus)
In this example, whether or not BENI really exists in the nucleus and functions is determined by binding EGFP to the C-terminal of the original BENI mRNA sequence (BENI-EGFP mRNA), nuclear translocation on the N-terminal side. A BENI mRNA sequence lacking only a signal is bound to EGFP (BENI-ΔN-NLS-EGFP mRNA), and a BENI mRNA sequence lacking only the nuclear translocation signal on the C-terminal side is bound to EGFP (BENI). -△ C-NLS-EGFP mRNA), which is obtained by binding EGFP to BENI mRNA sequence lacking both N-terminal and C-terminal nuclear translocation signals (BENI-ΔNLS-EGFP mRNA), respectively. Was investigated whether or not the mRNA of was localized in the nucleus.

  As BENI-ΔN-NLS-EGFP mRNA, BENI-ΔC-NLS-EGFP mRNA, and BENI-ΔNLS-EGFP mRNA, those produced by a synthetic contract service of Funakoshi Co., Ltd. (Tokyo) were used. Development of fertilized eggs obtained by the same method as in Example 1 was advanced to the 8th stage embryo. According to a conventional method, subcloning was performed on the multicloning site of the EGFP-C1 vector (manufactured by Clontech). These recombinant vectors were introduced into the eighth stage embryo using the calcium phosphate method. Then, according to a conventional method, immunization staining was performed and observed with a fluorescence microscope.

(result)
FIG. 8 shows a study of nuclear localization of BENI mRNA lacking the nuclear translocation signal of the nucleic acid sequence. EGFP mRNA having no BENI sequence was used as a control sample (EGFP mRNA).

  When EGFP mRNA alone does not have a BENI sequence, mRNA is taken up throughout the cell. On the other hand, mRNA having the entire BENI sequence is localized in the nucleus. The BENI-ΔN-NLS-EGFP mRNA lacking only the N-terminal nuclear translocation signal is localized in the nucleus, but the BENI mRNA lacking the C-terminal nuclear translocation signal has the presence or absence of the N-terminal nuclear translocation signal. Existed throughout the cell regardless of (BENI-ΔC-NLS-EGFP mRNA, BENI-ΔNLS-EGFP mRNA). From this, it was found that BENI is a nuclear factor, and its nuclear translocation depends on the C-terminal nuclear translocation signal.

(Summary)
BENI (ie, BC043737 gene) is a nuclear factor that regulates the induction of mesoderm differentiation while mainly suppressing the expression of Brachyury, a mesoderm-specific gene, in the nucleus during early vertebrate development. I understood that.

  According to the present invention, the function of inducing differentiation of mesoderm which has been unknown until now has been revealed. Conventionally, Mix-1, Google, Chordin, and Brachyury are known as factors that specifically induce mesoderm differentiation, but BENI specifically inhibits Brachyury expression and induces mesoderm differentiation. Is controlling. Moreover, the homology of 57% or more in the 1-57 amino acid sequence is recognized among the three human homologous genes found by the inventor, and in particular, the homology of 93% is recognized with the human BAB14718 gene. Thus, it is suggested that BENI is involved in human genetic diseases that occur due to inhibition of Brachyury's normal function.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range from the description of specific preferred embodiments of the present invention based on the description of the present invention and common general technical knowledge. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

(Industrial applicability)
By the present invention, BENI (ie, BC043737 gene) mesoderm differentiation-inducing function, which has been unknown until now, has been clarified. Conventionally, Mix-1, Goosecoid, Chordin, and Brachyury are known as factors that specifically induce mesoderm differentiation, but BENI specifically inhibits Brachyury expression and induces mesoderm differentiation. Is controlling. In addition, 57% or more homology with the 1-57 amino acid sequence was found among the three human homologous genes found by the inventor, and in particular, 93% homology was found with the human BAB14718 gene. Therefore, it is suggested that BENI is involved in the human genetic disease that occurs because it inhibits the normal function of Brachyury.

FIG. 1 shows the amino acid sequence of the isolated Xenopus BENI and the amino acid sequence of the BENI homologous gene found in other organisms (the upper row represents the N-terminal region and the lower row represents the C-terminal region). FIG. 2 shows the expression level of BENI at each developmental stage of Xenopus fertilized eggs examined by RT-PCR (top). The middle ornithine decarboxylase (ODC) was used as a so-called house-keeping gene (control). The lower row shows ODC without RT-PCR. FIG. 3 shows the expression site of the BENI gene in Xenopus embryos by the WISH method (photos shown at the left end of each panel are the results of a control experiment using senseprobe). FIG. 4 shows a cross-sectional section of Xenopus laevis embryos stained by the WISH method (upper stage) and an overall view of the cross-sectional section of the embryo (lower stage). FIG. 5 shows BENI-MO (34 ng) injected into the back of the frog embryo at the blastomere stage and observed from the 35th to 36th stage (right panel). The left panel shows a control experiment in which the same embryo was injected with the same amount of unaffected (StMO). FIG. 6 shows the effects of BENI mRNA and BENI-MO in the presence of activin A. An animal cap (upper right) treated with activin A (25 ng / mL) causes an extension movement relative to an animal cap not treated with activin A (upper left). On the other hand, when BENI mRNA (1 ng) is treated to the animal cap treated with activin A (bottom left), the animal cap is inhibited by stretching motion, but the BENI-MO (34 ng) treatment shows normal stretching motion. (Bottom right). FIG. 7 shows the identification of gene expression by RT-PCR (ODC was used as a positive control sample, and the rightmost rain was used as a control sample for normal embryos). Next to that, from the right side, the state of gene expression of BENI-MO, the state of gene expression in the case of BENI mRNA, and those not injected were shown in order. FIG. 8 shows a study of the nuclear localization of BENI mRNA lacking the nuclear translocation signal of the nucleic acid sequence. EGFP mRNA without BENI sequence was used as a control sample (EGFP mRNA). Upper row: dark field, lower row: bright field

(Explanation of sequence listing)
SEQ ID NO: 1: Xenopus laevis BENI nucleic acid sequence SEQ ID NO: 2: Xenopus laevis BENI amino acid sequence SEQ ID NO: 3: Xenopus tropicalis BENI nucleic acid sequence SEQ ID NO: 4: Tropicalis (Xenopus tropicalis) BENI amino acid sequence SEQ ID NO: 5: Human (Homo sapiens) BENI nucleic acid sequence SEQ ID NO: 6: Human (Homo sapiens) BENI amino acid sequence SEQ ID NO : 7: Human (Homo sapiens) BENI nucleic acid sequence <br / > SEQ ID NO: 8: human (Homo sapiens) of BENI amino acid sequence <br/> SEQ ID NO 9: BENI nucleic acid sequence <br/> SEQ ID NO: 10 of human (Homo sapiens) Human (Homo sapiens) of BENI amino acid sequence <br/> SEQ ID NO: 11: Mouse (Mus musculus) BENI nucleic acid sequence SEQ ID NO 12: Mouse (Mus musculus) of BENI amino acid sequence SEQ ID NO 13: BENI mouse (Mus musculus) Nucleic acid sequence SEQ ID NO : 14: Mus musculus BENI amino acid sequence SEQ ID NO: 15 : Ratnus norvegirus BENI nucleic acid sequence SEQ ID NO: 16 : Rat norbegirus BENI amino acid sequence No. 17: Nucleic acid sequence of BEN I of Tetradon nigroviridis SEQ ID NO: 18 BENI (Acter of Nitroviridis) Session number: CAG01099) amino acid sequence SEQ ID NO: 19: Tetraodon nigroviridis BEN I nucleic acid sequence SEQ ID NO: 20: Tetradon nigroviridis BENI (accession number: CAG04344) amino acid sequence number 21: Activin A (accession number: NM _ 002192 (human inhibin βA)) of the nucleic acid sequence SEQ ID NO: 22: activin a (accession number: NM _ 002192 (human inhibin βA)) of the amino acid sequence SEQ ID NO: 23: activin a (accession number : X68250 (Xenopus activin A)) SEQ ID NO: 24: Activin A (accession number: X68250 (Xenopus laevis) The amino acid sequence of Kuchibin A))

Claims (15)

For inhibiting the differentiation into mesoderm, a composition comprising a vector comprising a nucleic acid or a nucleic acid encoding the Brachyury Expression N uclear Inhibitor (BENI) , said BENI is
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity Or (c) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
A composition. The composition of claim 1, wherein the nucleic acid encodes the sequence shown in SEQ ID NO: 2. The composition of claim 1, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 1. For inhibiting the differentiation into mesoderm, a composition comprising a Brachyury Expression N uclear Inhibitor (BENI) polypeptide, said BENI is
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence set forth in SEQ ID NO: 2, one or more or one or several amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and suppression of Brachyury expression A polypeptide having activity; or (c) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
A composition. 5. The composition of claim 4, wherein the BENI has the sequence shown in SEQ ID NO: 2. 5. The composition of claim 4, wherein the BENI comprises the sequence shown in SEQ ID NO: 1. A composition for inducing mesoderm differentiation, the composition comprising:
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity Or (c) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
A composition comprising an antisense nucleic acid molecule or RNAi molecule of a nucleic acid molecule encoding or a vector comprising said antisense nucleic acid molecule or RNAi molecule.   8. The composition of claim 7, further comprising means for delivering the antisense nucleic acid molecule or RNAi molecule to a specific site.   The composition according to claim 8, wherein the means expresses the antisense nucleic acid molecule or RNAi molecule specifically at the specific site.   10. A composition according to claim 9, wherein the means is a promoter.   9. The composition of claim 8, wherein the means is a directional liposome. A method for screening a regulator of mesoderm differentiation induction,
A) providing a candidate substance,
B) subjecting it to an assay system to determine whether the candidate substance modulates the activity of BENI;
C) comprising the step of identifying a factor that modulates BENI among the candidate substances, and determining that the factor that modulates BENI is a regulator of BENI,
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence set forth in SEQ ID NO: 2, one or more or one or several amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and suppression of Brachyury expression A polypeptide having activity; or (c) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1. The method according to claim 12, wherein the candidate substance is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof , and combinatorial chemistry libraries . (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity A polypeptide; or (c) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1
Or a mesodermal gastrulation inhibitor comprising a nucleic acid molecule encoding the polypeptide of (a), (b) or (c). (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have Brachyury expression suppression activity Or (c) a polypeptide encoded by an allelic variant of the base sequence set forth in SEQ ID NO: 1;
A mesodermal gastrulation promoter , comprising an antisense nucleic acid molecule or an RNAi molecule of a nucleic acid molecule encoding