JP5240756B2 - Cartilage disease model non-human animal - Google Patents

Description

  The present invention relates to a model non-human animal of cartilage disease characterized by dysfunction of the BBF2H7 gene.

The present invention also relates to a method for screening a therapeutic agent for cartilage disease using the non-human animal or a method for evaluating the efficacy of a therapeutic agent for cartilage disease.
The present invention further relates to a therapeutic agent for cartilage diseases.

  It has become clear that endoplasmic reticulum stress is deeply involved in the development of many intractable diseases such as neurodegenerative diseases and diabetes. The present inventors have previously found OASIS (Old Astrocyte Specific-Induced Substrate), a novel endoplasmic reticulum-localized transcription factor that plays an important role in signal transduction for avoiding endoplasmic reticulum stress. It has been found that by promoting activation or expression, neuronal cell death resulting from endoplasmic reticulum stress can be suppressed, and there is a possibility of treating neurodegenerative diseases (Patent Document 1). Thereafter, in order to elucidate the function of the OASIS gene, the present inventors made an OASIS gene-deficient mouse, and found that the mouse unexpectedly showed a lesion that closely resembled osteoporosis (Patent Document 2).

  In the process of exploring another endoplasmic reticulum localization transcription factor involved in the endoplasmic reticulum stress, the present inventors further newly identified the endoplasmic reticulum localization transcription factor BBF2H7. It was found that it is a stress transducer and can play an important role in preventing the accumulation of non-holding proteins in damaged neurons (Non-patent Document 1).

  According to Non-Patent Document 1, the BBF2H7 gene is highly expressed in the lung, bladder, uterus, spleen, testis, etc., and is also expressed in the heart, liver, kidney, adrenal gland, submandibular gland, cerebrum, pancreas, prostate, etc. Yes. Further, this protein has a structure similar to that of the OASIS protein in terms of a functional domain, and has been shown to be one member of the OASIS family (Non-patent Document 1).

JP-A-2005-52016 WO2007 / 102240 S. Kondo et al., Mol. Cell. Biol. 2007, 27 (5): 1716-1729

  However, the role or function of BBF2H7 in vivo is unknown except for the above findings. For this purpose, the present inventors made a BBF2H7 gene-deficient non-human animal and tried to clarify its characteristics.

  As a result, the present inventors have unexpectedly found that BBF2H7 has an important function closely related to cartilage formation.

Accordingly, the present invention includes the following features.
(1) A model non-human animal of cartilage disease characterized by dysfunction of the BBF2H7 gene.
(2) The model non-human animal according to (1) above, wherein the cartilage disease is accompanied by abnormal cartilage formation.
(3) The model non-human animal according to (2) above, wherein the cartilage disease is further accompanied by skeletal dysplasia.

(4) The model non-human animal according to any one of (1) to (3), wherein the non-human animal is a mammal.
(5) The model non-human animal according to (4) above, wherein the mammal is a rodent.
(6) The model non-human animal according to (5) above, wherein the rodent is a mouse.

(7) A method for screening a therapeutic agent for cartilage disease, comprising administering a candidate drug to the model non-human animal according to any one of (1) to (6) above.
(8) The method according to (7) above, wherein the cartilage disease is accompanied by abnormal cartilage formation.
(9) The method according to (7) above, wherein the candidate drug is a small molecule, peptide, protein or nucleic acid.

(10) The method according to (7) or (9) above, wherein the candidate drug is a BBF2H7 protein, a mutant or a chemically modified derivative thereof.
(11) The method according to (7) or (9) above, wherein the candidate drug is a vector comprising DNA encoding the BBF2H7 protein or a variant thereof.
(12) A method for evaluating the efficacy of a therapeutic agent for cartilage disease, comprising administering the therapeutic agent for cartilage disease to the model non-human animal according to any one of (1) to (6) above.

(13) The method according to (12) above, wherein the cartilage disease is accompanied by abnormal cartilage formation.
(14) A therapeutic agent for cartilage disease comprising, as an active ingredient, a vector comprising DNA encoding BBF2H7 protein or a variant thereof.
(15) A therapeutic agent for cartilage disease comprising BBF2H7 protein, a mutant or a chemically modified derivative thereof as an active ingredient.
(16) The therapeutic agent for cartilage disease according to (14) or (15) above, wherein the cartilage disease is accompanied by abnormal cartilage formation.

Terms used herein include the following definitions.
The term “BBF2H7” in the present specification is one of transmembrane bZIP transcription factors, a protein presumed to be an endoplasmic reticulum stress transducer, and is also referred to as CREB3L2 (human) or Creb3l2 (mouse). (Non-Patent Document 1). The amino acid sequence or nucleotide sequence of this BBF2H7 protein or the DNA encoding it is registered as, for example, GneBank Accession Number NM — 178661 (mouse Creb312), NM — 194071 (human CREB3L2). As used herein, the term “BBF2H7” is intended to include those derived from other animals (ie, homologs) in addition to those derived from humans and mice.

  As used herein, the term “dysfunction” refers to expression of a gene by modifying (eg, substituting, deleting, adding or inserting) part or all of the BBF2H7 gene on the genome of a non-human animal. This refers to a state in which the product BBF2H7 protein does not exert its biological function or hardly exhibits its biological function. Here, the biological function of BBF2H7 refers to a function related to cartilage formation in a living body. In the present specification, a non-human animal having such a malfunction is referred to as a model non-human animal of cartilage disease. Alternatively, such a non-human animal may be referred to as a BBF2H7-deficient non-human animal or a BBF2H7 knockout non-human animal.

  As used herein, the term “cartilage disease” refers to a disease associated with abnormal cartilage formation, and further includes abnormal skeletal dysplasia. Specifically, the cartilage diseases related to the present invention include achondroplasia, hypocartilaginous dysplasia, epiphyseal dysplasia, osteoarthritis and the like. These diseases often have symptoms such as shortened limbs and trunk, bone dysplasia of the forehead, nose, face, limbs, lumbar spine, etc., and short stature.

  As used herein, the term “wild type” refers to a non-human animal having a normal BBF2H7 gene.

  According to the present invention, it was found that the BBF2H7 gene is closely involved in cartilage formation. It was also found that when this gene is deficient, cartilage hypoplastic lesions appear. Based on this finding, when the cause of cartilage disease is due to dysfunction of BBF2H7, improvement or treatment of cartilage disease can be achieved by gene therapy for converting the BBF2H7 gene into a normal form or treatment with BBF2H7 protein in the patient. Is possible.

  Furthermore, based on the above findings, the cartilage disease model non-human animal of the present invention can be provided by deleting all or part of the BBF2H7 gene on the genome in the non-human animal. When used, it provides screening for therapeutic agents for cartilage diseases, or provides special effects such as a tool for pathological analysis or cause elucidation of cartilage diseases.

1. Non-human animal model of cartilage disease The non-human animal model of cartilage disease characterized by dysfunction of the BBF2H7 gene of the present invention is a so-called knockout non-human animal whose BBF2H7 gene on its genome has been deleted or dysfunctional. It is a human animal. That is, by causing deletion, substitution, addition, or insertion in a part of the genomic DNA of the BBF2H7 gene, the function of the gene is completely or substantially impaired or deleted. Since the BBF2H7 gene is not expressed due to the gene deficiency, and as a result, the BBF2H7 protein is not synthesized, the function cannot be performed in vivo. At this time, if it is a protein that plays an important role in the living body, it is considered that the process of development and development is affected and pathological symptoms appear.

The BBF2H7 gene-deficient non-human animal of the present invention has the following characteristics.
That is, in the non-human animal of the present invention, skeletal dysplasia, particularly hypoplasia, is observed in which the limbs and trunk are markedly shortened and the thorax is strongly formed. Due to the low formation of the rib cage, animals may die from respiratory failure immediately after birth. In the histopathological analysis, the growth layer and the hypertrophied layer of the growing cartilage zone are very poorly developed, and the chondrocytes present in this region are observed to have an enlarged cytoplasm and a large number of large vacuoles. Furthermore, as a result of toluidine blue staining, a marked decrease in cartilage mass is observed. From these findings, BBF2H7 is closely related to the endoplasmic reticulum protein quality control of the cartilage matrix, and when this gene is deficient, secretion of the cartilage matrix is impaired, and it is considered that the formation of the skeleton is reduced. Therefore, the model animal of the present invention is a disease model animal that can be an effective tool for analyzing the pathological condition of cartilage disease or elucidating the cause of the onset of the disease.

  In the present invention, the cartilage disease is a disease in which the growth layer and the hypertrophy layer of the growing cartilage zone are poor, the cytoplasm of the chondrocytes is enlarged, and a large number of large vacuoles are observed in the cytoplasm as described above. It is a disease that is characterized physically, and specifically, a disease that involves abnormal cartilage formation. The disease may also be accompanied by skeletal abnormalities. Specific examples of the cartilage disease in the present invention include achondroplasia, hypochondral dysplasia, epiphyseal dysplasia, osteoarthritis and the like.

  Thus, since the non-human animal of the present invention has cartilage malformation such as a hypochondrogenic lesion due to dysfunction of BBF2H7, it is useful as a model non-human animal for cartilage disease. Conventionally, BBF2H7 has been known to play an important role in preventing accumulation of non-holding proteins in damaged neurons caused by endoplasmic reticulum stress (Non-Patent Document 1). There was nothing known about close involvement. Such a characteristic became apparent for the first time when a non-human animal deficient in the BBF2H7 gene could be produced, and was completely unexpected.

  In the present invention, the non-human animals are vertebrates and include fish, reptiles, amphibians, birds and mammals, but preferred animals are mammals. Preferred examples of mammals are rodents, such as mice, rats, guinea pigs and hamsters.

  The non-human animal of the present invention can be generally produced by using a known target gene recombination technology, knockout technology, homologous recombination technology, gene targeting technology, etc. (for example, Methods in Enzymology 225: 803-890). S. L. Mansour et al., Nature 1988, 336: 348-352; M. Zijlstra et al., 1989, Nature 342: 435-438; M. Zijlstra et al., Nature 1990, 344: 742-746; 1991, Nature 1991, 350: 242-246). A specific knockout method will be described below.

  The nucleotide sequence of the non-human animal BBF2H7 gene (or DNA or cDNA) is registered in GenBank as NM_178661, for example, for mice, and the amino acid sequence of the protein and the nucleotide sequence of the DNA are shown in the sequence listing below, respectively. 1, shown as SEQ ID NO: 2. The sequences of animals other than mice can be searched and obtained by accessing gene banks such as GenBank (US NCBI) and EMBL (European EBI) (for example, Toshihisa Takagi and Satoshi Kanahisa, GenomeNet database) Usage, 2nd edition, Kyoritsu Publishing (1998)). Alternatively, a primer is prepared based on the DNA sequence of mouse BBF2H7, and a cDNA prepared from mRNA of tissue of a non-human animal, for example, another rodent, by RT-PCR, is used as a template to prepare a target BBF2H7. DNA can be amplified and sequenced (see, eg, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, 1989; FM Ausubel et al. Methods from Current Protocols in Molecular Biology, 200 ).

Based on this nucleotide sequence, a probe (for example, about 30 to 150 bases) is prepared and labeled with a radioactive (such as ³² P) or fluorescent label (such as fluoresamine, rhodamine, cyanine, such as FITC, Cy), and the BBF2H7 gene genome. It can be used to detect or isolate DNA. After genomic DNA is extracted from cells derived from tissues such as the brain and tail according to a conventional method, the genomic DNA is cleaved with a restriction enzyme, and the target probe is then used by a hybridization method such as Southern hybridization or in situ hybridization. Search for the open reading frame (ORF) of the BBF2H7 gene. If necessary, a restriction enzyme map is prepared, and an arbitrary target site for homologous recombination is determined. Alternatively, a BBF2H7 gene-containing genomic DNA fragment is amplified and sequenced by polymerase chain reaction (PCR) using a primer (eg, about 15-25 bases) prepared based on the base sequence of the BBF2H7 gene (for example, Ausubel et al., Supra). ). The homologous region for homologous recombination is preferably about 1 to 7 kb or more.

  In order to knock out the BBF2H7 gene, foreign DNA such as a drug resistance gene (eg, neomycin resistance gene, hygromycin B phosphotransferase gene, etc.) may be inserted or substituted into the homologous region in the targeting vector, or All or part of the BBF2H7 gene may be deleted. The deletion position or the insertion or replacement position of the foreign DNA is, for example, any exon of the BBF2H7 gene, for example, exon 2 (E2 in FIG. 1). Here, the drug resistance gene as foreign DNA is useful for positive selection of embryonic stem cells having a targeting vector. Moreover, a gene for negative selection may be introduced into the vector, and such genes include, for example, HSV thymidine kinase (HSV-tk) gene, diphtheria toxin A gene and the like. Negative selection is useful for selection of random vector-embedded embryonic stem cells that are sensitive to drugs such as anti-herpes drugs, FIAU, cyclovir, ganciclovir.

The targeting vector may be any vector and includes, for example, pGT-N28 (New England Bio Labs), pBluescript II SK ⁺ (Stratagene), pSP72, pPNT and the like.

  In order to increase the homologous recombination efficiency, for example, the Cre-loxP system (R. Kuhn et al., Science 1995, 269: 1427-1429) can also be used.

  In order to produce the non-human animal of the present invention, there is a method in which vector DNA is directly injected into a fertilized egg and transplanted to a foster parent, but a method using embryonic stem (ES) cells is preferred. Preferred ES cells are rodent ES cells, such as mouse ES cells.

  ES cells are established by culturing blastocysts after fertilization or undifferentiated cells existing in 8-cell stage embryos, and by repeating dissociation and transfer of cell masses. A cell line that continues to grow while maintaining its state. Examples of mouse ES cell lines include, but are not limited to, D3 cell line, E14 cell line, TT2 cell line, AB-1 cell line, J1 cell line, R1 cell line and the like.

  A targeting vector in which the function of BBF2H7 gene is modified as described above to be deleted is introduced into ES cells by a known method. Examples of the introduction method include, but are not limited to, a microcell method, an electroporation method, a liposome method, a calcium phosphate method, and a DEAE-dextran method.

  The obtained recombinant ES cells can be screened for homologous recombination by, for example, a known Southern blot method using a probe or primer, a genomic PCR method, an in situ PCR method, or the like. As a result, cells in which homologous recombination has occurred correctly are selected.

  ES cells in which the BBF2H7 gene is knocked out are introduced into blastocysts or 8-cell stage embryos of wild-type non-human animals. Then, a chimeric animal can be produced by transplanting the embryo containing the ES cells into the uterus of a pseudopregnant foster parent non-human animal and giving birth. Preferred animals are rodents such as mice.

  As a method for introducing an ES cell into an embryo such as a blastocyst, a microinjection method and an aggregation method are known, but any method can be used. In the case of mice, female mice that have undergone superovulation treatment with a hormonal agent are mated with male mice to obtain early embryos. However, when blastocysts are used as embryos for introduction of recombinant ES cells, fertilization is performed. From day 3.5 to day 3.5, if an 8-cell embryo is used, day 2.5 is collected from the uterus on day 2.5. ES cells that have undergone homologous recombination using the targeting vector are injected in vitro to the embryos thus collected to produce a chimeric embryo.

  On the other hand, a pseudopregnant female non-human animal for use as a temporary parent can be obtained by mating a female animal having a normal cycle with a male animal castrated by vagina ligation or the like. A chimeric non-human animal can be produced by transplanting the chimeric embryo produced by the above-mentioned method into the uterus to the produced pseudo-pregnant non-human animal and allowing it to become pregnant and give birth. In order to ensure the implantation and pregnancy of a chimeric embryo, it is desirable to produce a female animal from which a fertilized egg is collected and a pseudopregnant animal as a foster parent from a group of female animals in the same sexual cycle.

  When a non-human animal individual derived from an ES cell-transplanted embryo is obtained from such a chimeric animal, this chimeric animal is crossed with a pure-type animal, and the ES cell-derived hair color appears in the next-generation individual This confirms that ES cells have been introduced into the chimeric animal germline. In order to confirm that the ES cell has been introduced into the germ line, various traits can be used as an index. However, in consideration of ease of confirmation, it is desirable to use a coat color. It is also possible to perform selection by extracting DNA from a part of the body (for example, the tail) and performing Southern blot analysis or PCR assay.

  Thus, by selecting an animal in which a recombinant ES cell transplanted into an embryo is introduced into the germ line and breeding the chimeric animal, an individual deficient in the target gene can be obtained. By crossing the obtained BBF2H7 gene-deficient heterozygous non-human animals (+/−) with each other, a target gene-deficient homozygous non-human animal (− / −) can be obtained at a ratio according to Mendel's law. . The produced heterozygote or homozygote carrying the BBF2H7 mutant gene has a stable BBF2H7 gene deficiency in all germ cells and somatic cells and is transmitted to progeny animals.

  When producing the knockout non-human animal of the present invention, it is also possible to use the Cre / loxP system (R. Kuhn et al., Science 1995, 269: 1427-1429) used in the production of knockout mice. In the Cre / loxP system, a BBF2H7 gene-deficient mouse having a gene in which a foreign DNA sequence sandwiched between loxP sequences is inserted into a target homologous sequence, a Cre enzyme that is a recombinant enzyme derived from E. coli P1 phage is used. When mated with an expressing animal, the Cre enzyme recognizes and deletes the sequence flanked by loxP sequences, making it possible to create an animal lacking that region. By using such a Cre / loxP system and mating with a non-human animal that expresses the Cre enzyme in a tissue-specific manner, a knockout animal having a tissue-specific gene-deficient property can be produced. . A specific example of such a method is called a conditional knockout method.

  For specific methods relating to gene targeting, reference can be made to, for example, Shinichi Aizawa, Gene Targeting—Mutation of Mutant Mice Using ES Cells, Biomanual Series 8, Yodosha (1995).

2. Screening and evaluation of efficacy of therapeutic agents for cartilage diseases The BBF2H7 gene-deficient non-human animal of the present invention is a model animal for cartilage diseases (for example, achondroplasia, hypocartilaginous dysplasia, osteoarthritis, etc.) Therefore, it can be used for pathological analysis of the disease, elucidation of the cause of the onset of disease, screening for therapeutic drugs, evaluation of drug efficacy, and the like.

  Therefore, the present invention provides a method for screening a therapeutic agent for cartilage disease, comprising administering a candidate drug to the model non-human animal.

  The present invention also provides a method for evaluating the efficacy of a therapeutic agent for cartilage disease, comprising administering the therapeutic agent for cartilage disease to the model non-human animal.

  Candidate agents include, but are not limited to, for example, small molecules, (poly) peptides, (sugar) proteins, (poly) nucleotides, nucleic acids, nucleosides, carbohydrates, lipids, and the like. Examples of candidate drugs are BBF2H7 protein, chemically modified derivatives thereof (eg, PEGylated, acylated, alkylated, glycosylated, phosphorylated derivatives, etc.). Candidate drugs include, for example, human BBF2H7 protein (GenBank: NM — 194071; SEQ ID NO: 3), mutants, homologs, chemically modified derivatives, and the like.

  A variant of the human BBF2H7 protein has a sequence having 90% or more, preferably 95% or more, more preferably 98% or more, for example 99% or more identity with the amino acid sequence of the protein, or It has an amino acid sequence and a sequence containing a deletion, substitution or addition of one or several amino acids. Here,% identity means the percentage (%) of the number of identical amino acids relative to the total number of amino acids when the amino acid sequence of human BBF2H7 protein and another amino acid sequence are aligned with or without introducing a gap. . The identity can be determined using a known algorithm such as BLAST or FASTA.

  Another example of a candidate drug is a vector containing DNA encoding a BBF2H7 protein or a variant thereof. The production of this vector and the like are described in “3. Cartilage disease therapeutic agent” below.

  The therapeutic agents for cartilage diseases include growth hormone and the like, but only limited drugs are known. The therapeutic agents in the present invention also include potential agents that may allow improvement of cartilage disease.

Methods for administering the candidate drug or therapeutic agent include, but are not limited to, oral administration or parenteral administration (eg, intravenous administration, intraperitoneal administration, intranasal administration, etc.) and the like. In the case of oral administration, the candidate drug can be administered in a diet.
The dose is not limited, but ranges from about 1 μg to 100 mg.

  Candidate drugs or therapeutic agents can also be administered in combination with conventional pharmaceutically acceptable carriers, excipients such as diluents, additives and the like. Alternatively, the candidate drug or therapeutic agent may be encapsulated in liposomes (for example, positively charged liposomes) or nanoparticles.

  Evaluation is based on macroscopic observation, body weight measurement, using as an index the reduction or improvement of symptoms of cartilage disease, the development of the growth layer and hypertrophy layer of the growing cartilage band, the decrease of vacuoles in the cytoplasm of chondrocytes, and the increase of cartilage base mass , Histopathological observation (for example, microscopic observation after tissue staining) and the like.

3. Cartilage Disease Treatment Agent The present invention further provides a cartilage disease treatment agent comprising, as an active ingredient, a vector comprising DNA encoding BBF2H7 protein or a variant thereof.

  The present invention also provides a therapeutic agent for cartilage disease comprising BBF2H7 protein, a mutant or a chemically modified derivative thereof as an active ingredient.

  Here, the cartilage disease is accompanied by abnormal cartilage formation, and may also be accompanied by abnormal skeleton formation. Cartilage diseases include, for example, achondroplasia, hypocartilaginous dysplasia, epiphyseal dysplasia, osteoarthritis and the like.

  The BBF2H7 protein and the DNA encoding it are derived from animal patients. In the case of a human patient, the human BBF2H7 protein (SEQ ID NO: 3) and the DNA encoding it (SEQ ID NO: 4) are used.

  As a vector as an active ingredient, for example, a commercially available or documented adenovirus vector, adeno-associated virus vector, retrovirus vector, virus vector such as lentivirus vector, plasmid vector, and the like can be used as a basic vector. These vectors should be vectors that are not harmful to patients (including humans), such as not causing canceration or viral replication. In addition to the above DNA, the vector can contain a promoter (eg, a constitutive promoter such as a viral promoter, a tissue-specific promoter, an inducible promoter, etc.), a selectable marker (eg, drug resistance gene, thymidine kinase gene, etc.), etc. .

The vector may be in a form suspended in a suitable excipient such as saline or buffer.
The administration of the vector may be directly injected into the bone tissue of the affected area, or may be injected in a form encapsulated in liposomes (preferably cationic liposomes).

  The BBF2H7 protein as an active ingredient or a mutant thereof can be prepared by gene recombination technology, PCR technology for mutagenesis, etc. (for example, Sambrook et al., Supra; Ausubel et al., Supra).

  DNA encoding the BBF2H7 protein can be amplified and synthesized from a cDNA library prepared from a specific animal tissue (eg, lung, uterus, etc.) by a PCR method using specific primers. After purifying the DNA by agarose gel electrophoresis or the like, an appropriate expression vector (usually a promoter, replication origin, terminator, Shine-Dalgarno sequence or ribosome binding site, multicloning site, selection marker ( For example, drug resistance genes), etc.) and inserted into prokaryotic cells (eg E. coli, Bacillus subtilis, Pseudomonas bacteria) or eukaryotic cells (eg yeast, filamentous fungi, basidiomycetes, insects) Cells, plant cells, mammalian cells, etc.), transforming the cells, cultivating and growing them in an appropriate culture medium, and recovering the target protein from the medium or cells. Can be obtained.

  The mutant has a sequence containing substitution, deletion or addition of one or several amino acids in the amino acid sequence of the natural BBF2H7 protein, or 90% or more, preferably 95% or more of the amino acid sequence, More preferably, it contains a sequence having 98% or more, for example, 99% or more identity. The mutant should retain the biological activity involved in chondrogenesis of the BBF2H7 protein. The mutant can be prepared by a conventional method by site-directed mutagenesis, mutagenesis PCR, or the like.

  An example of a mutation is a conservative amino acid substitution. A conservative amino acid refers to a group of amino acids having similar properties such as structure, chargeability, hydrophobicity or polarity. For example, structurally similar amino acids are aromatic amino acids consisting of phenylalanine, tryptophan and tyrosine, charged similar amino acids are basic amino acids consisting of arginine, lysine and histidine, acidic amino acids consisting of aspartic acid and glutamic acid, hydrophobicity Similar amino acids include hydrophobic amino acids such as alanine, methionine, leucine, isoleucine, valine and proline, polar amino acids such as serine, threonine, cysteine, asparagine, glutamine and glycine.

  Such chemically modified derivatives include, but are not limited to, pegylation, acylation, alkylation, glycosylation, phosphorylated derivatives and the like. In particular, pegylation is the binding of polyethylene glycol (for example, molecular weight 4000-10000) molecules to proteins, thereby providing advantages such as being less susceptible to enzymatic degradation in vivo and reducing the antigenicity of proteins. It has been known. Specifically, such a chemical modification method includes introducing a functional group into the amino group, carboxyl group, threonine residue, serine residue or the like of the protein or mutant by a chemical method.

  The therapeutic agent of the present invention includes excipients, diluents, carriers and additives commonly used in the pharmaceutical industry in addition to the above active ingredients (for example, stabilizers, binders, disintegrants, lubricants, surfactants). Agents, buffers, suspending agents, sweeteners, flavors, etc.). The dosage form of the therapeutic agent is not particularly limited, and includes solutions, suspensions, tablets, pills, capsules, powders, granules, powders, delayed release agents, suppositories, liposome encapsulants and the like. For the preparation of such dosage forms, reference can be made to, for example, Remington, The Science and Practice of Pharmacy, Mack Publishing Company, 1995.

  The administration method of the therapeutic agent of the present invention includes oral administration or parenteral administration. Parenteral administration includes, but is not limited to, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, rectal administration and the like. The administration method also includes a method of injecting directly into the affected area.

  The dosage of the active ingredient of the therapeutic agent of the present invention is a dose that reduces or ameliorates cartilage disease, and the optimal dose depends on the judgment of the doctor and may vary depending on age, sex, weight, medical condition, etc. .

  The therapeutic agent of the present invention can lead to reduction or improvement of symptoms of cartilage disease, improvement of cartilage formation, and the like. Specifically, the therapeutic agent of the present invention makes it possible to develop the growth layer and hypertrophy layer of the growing cartilage zone, decrease the vacuoles in the cytoplasm of chondrocytes, increase the cartilage base mass, and the like.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.

Preparation of targeting vector Using primers prepared based on the mouse BBF2H7 genomic sequence (ENSMUSG00000038648), PCR using 129 mouse-derived genomic DNA as a template (denaturation 94 ° C 1 minute, annealing 55 ° C 1 minute, extension 72 ° C 3 minutes 35 cycles) And DNA fragments 1.5 kb (short arm) and 6 kb (long arm) around exon 2 of the BBF2H7 gene were isolated. The primer used at this time is on the short arm side,
5'-GCGGCCGCTTCGACACTTTGTCTGCCACTC-3 '(SEQ ID NO: 5) and
5′-CTCGAGTCACTCCGAGAAGTGCTGCAAGAAGC-3 ′ (SEQ ID NO: 6),
The long arm side is
5'-CAGAGATGCCCTGAGATCAGCTG-3 '(SEQ ID NO: 7) and
5′-GGTACCCTACACCATGCGCCACCAGCCATG-3 ′ (SEQ ID NO: 8).

  After confirming that no base substitution occurred by sequencing, each DNA fragment was subjected to targeting vector pPNT1.1 (Cell 1991 Jun 28, 65 (7): 1153-1163; Dr. Masaru Okabe (Osaka University, Osaka, Incorporated into the NotI-XhoI site (short arm side) and the KpnI-KpnI site (long arm side) of (given from Japan)) (see FIG. 1).

Production of BBF2H7 knockout mice Undifferentiated cultured mouse ES cells (about 0.8 × 10 ⁷ ) were obtained by electroporation.
The D3 cell line (Doetschman et al., J Embryol Exp Morphor. 1985, 87: 27-45) was introduced with the targeting vector of Example 1 at a rate of 25 μg / ml to obtain transgenic ES cells. Cells were seeded on a plate (medium ESM), G418 after 24 hours, G418 and ganciclovir were added to the medium after 48 hours, and further cultured for 7 to 10 days to obtain colonies resistant to G418 and ganciclovir. Were individually separated and further cultured, and then DNA was extracted and homologous recombinant ES cells were selected by Southern blotting.

Subsequently, this homologous recombinant ES cell was injected into a blastocyst of a C57BL / 6CR SLC strain mouse by a conventional method, transplanted into a temporary parent mouse, and allowed to develop into an individual. As a result, 6 chimeric mice were obtained. Chimera mice were selected based on the chimera ratio (the ratio of brown 129 to black C57BL6), which had a high ratio on the germline. Among the obtained chimeric mice, male individuals and female wild-type C57B / 6 mice were mated to obtain primary (F ₁ ) mice. And subjected to Southern blot of these F ₁ mice. As shown in FIG. 2A, as a result of Southern blotting when the BBF2H7 gene was digested with BamHI and SphI, in the heteromouse genome, 6.4 kbp and 2.7 kbp at the time of BamHI digestion, 11 at the time of SphI digestion. Bands of .6 kbp and 8.1 kbp were detected, respectively. This result indicates that the homologous recombination of the BBF2H7 gene has been successfully performed, and the BBF2H7 gene hetero mouse was successfully produced. Individuals (male and female) whose mutant sequence was confirmed on one of the diploid chromosomes were selected and crossed to obtain second generation (F ₂ ) mice.

As a primer
5′-CTGCAGTGGTCAGATGGACAG-3 ′ (SEQ ID NO: 9),
5′-TGGCTGCGCTGCTGCCCAAGACCCAG-3 ′ (SEQ ID NO: 10),
5'-CTTGACGAGTTCTTCTGAGG-3 '(SEQ ID NO: 11)
As a result of genomic PCR, it was confirmed that the BBF2H7 gene was defective in knockout mice (− / −) (see FIG. 2B). The BBF2H7 gene knockout mouse of the present invention was successfully produced.

Characterization of BBF2H7 knockout mice BBF2H7 knockout mice may die due to hypothorax formation immediately after birth. Embryonic knockout mice showed marked shortening of limbs and trunk, and low skeletal formation with strong thoracic dysplasia.

  When Alizarin Alcian Blue staining of embryonic knockout mice is performed, cartilage formation is hardly observed as shown in FIG. On the other hand, no histological abnormalities were observed in organs / tissues other than the skeletal system. The histopathological examination of the long bones of embryonic day 18.5-day mice showed that the development of the growth layer and hypertrophy layer of the growing cartilage zone was extremely poor (Fig. 4). And many large vacuoles were observed (FIG. 5). Furthermore, as a result of toluidine blue staining, a marked decrease in the cartilage base mass was observed (FIG. 6). From the above findings, BBF2H7 is closely related to the endoplasmic reticulum protein quality control of the cartilage matrix, and when this gene is deficient, secretion of the cartilage matrix is impaired, and it is considered that the formation of the skeleton is reduced.

  According to the present invention, a model non-human animal of cartilage disease is provided. By using the model animal of the present invention, it becomes possible to develop / evaluate a therapeutic agent for cartilage disease, analyze the onset mechanism, and the like. Therefore, this model animal is extremely useful industrially.

The construction of a targeting vector for producing a BBF2H7-deficient mouse is shown. E1 and E2 represent exon 1 and exon 2 of the BBF2H7 gene, respectively. Neo ^r represents a neomycin resistance gene, also HSV-tk is HSV thymidine kinase gene, respectively. The defect confirmation of the BBF2H7 gene by Southern blotting and genomic PCR is shown. In the BBF2H7 gene-deficient genome, a band of 2.7 kbp is detected upon digestion with BamHI and an 8.1 kbp band is detected upon digestion with SphI (FIG. 2A). FIG. 2B shows genomic PCR. + / + Is a wild type, +/− is a hetero mouse, − / − is a knockout mouse. The phenotype of BBF2H7 deficient mice is shown. A: Macroscopic photograph of embryonic day 18.5 days old mouse. B: Alizarin red / Alcian blue staining. Comparison of skeletons after staining with Alcian Blue and Alizarin Red. In gene-deficient mice (KO), almost no cartilage tissue stained with alcian blue (light blue) is observed. Skeletal hypoplasia due to cartilage malformation. Enlarged photographs of alizarin red and alcian blue stained for the forelimbs (C), hindlimbs (D), and spine (E). Hematoxylin and eosin (HE) staining of the forelimbs of BBF2H7-deficient mice (KO). Upper panel: HE staining of embryonic 18.5 day old mice (WT or KO) forelimbs. The humerus is poorly formed and shortened significantly. Lower panel: Magnified image of the part surrounded by a square on the upper panel. BBF2H7-deficient mice have almost no cartilage growth layer and hypertrophy layer. Magnified image of BBF2H7-deficient chondrocytes from embryonic 18.5 day old mice (WT or KO) (right panel) and magnified image of proliferative layer chondrocytes (left panel) (HE staining). Wild type (WT) chondrocytes have a flat cytoplasm and are arranged in a columnar shape. In the chondrocytes of gene-deficient mice (KO), many large vacuoles are observed in the cytoplasm, and the cell arrangement is also disturbed. Toluidine blue staining of BBF2H7-deficient cartilage from embryonic 18.5 day old mice (KO). Wild-type (WT) cartilage has abundant cartilage matrix that stains well with toluidine blue staining, but in the cartilage of gene-deficient mice, the staining is clearly reduced and the cartilage mass is reduced.

Sequence number 5-11: Primer

Claims (13)

  A model non-human animal of cartilage disease characterized by dysfunction of the BBF2H7 gene.   The model non-human animal according to claim 1, wherein the cartilage disease is accompanied by abnormal cartilage formation.   The model non-human animal according to claim 2, wherein the cartilage disease is further accompanied by skeletal dysplasia.   The model non-human animal according to any one of claims 1 to 3, wherein the non-human animal is a mammal.   The model non-human animal according to claim 4, wherein the mammal is a rodent.   The model non-human animal according to claim 5, wherein the rodent is a mouse.   A method for screening a therapeutic agent for cartilage disease, comprising administering a candidate drug to the model non-human animal according to any one of claims 1 to 6.   The method according to claim 7, wherein the cartilage disease is accompanied by abnormal cartilage formation.   The method according to claim 7, wherein the candidate drug is a small molecule, a peptide, a protein or a nucleic acid.   The method according to claim 7 or 9, wherein the candidate drug is a BBF2H7 protein, a mutant or a chemically modified derivative thereof.   The method according to claim 7 or 9, wherein the candidate drug is a vector comprising DNA encoding the BBF2H7 protein or a variant thereof.   A method for evaluating the efficacy of a therapeutic agent for cartilage disease, comprising administering the therapeutic agent for cartilage disease to the model non-human animal according to any one of claims 1 to 6.   The method according to claim 12, wherein the cartilage disease is accompanied by abnormal cartilage formation.

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