KR20180100849A - Animal model for pendred syndrome and method for producing the same - Google Patents

Abstract

Provided are an animal model to which a human p.H723R mutant gene is transplanted, and an animal model of pendred syndrome which is useful for the development of therapeutic agents and further treatment methods for the pendred syndrome. In addition, provided are a method for producing the animal model of the pendred syndrome and a method for screening effective therapeutic agents for the pendred syndrome using the produced animal model.

Description

[0001] The present invention relates to an animal model of Pendrim ' s Syndrome,

The present invention relates to an animal model of Pendream syndrome and a method for producing the same, and more particularly, to an animal model of Pendre syndrome in which a human pendrin mutant gene has been transplanted, a method for producing the animal model and a composition for treating Pendrim syndrome ≪ / RTI >

The SLC26A4 gene is located on chromosome 7 and is known to encode a pendrin protein. In addition, the SLC26A4 gene can be expressed in the inner ear, kidney thyroid. Specifically, the SLC26A4 gene releases iodine from the follicular lumen in the thyroid cells and cleaves Cl ^- / HCO ₃ ^- exchange in the acid - salt control of the intercalated cells of the kidney Mediated, and mediates the exchange of Cl ^- / HCO ₃ ^- exchange with the regulation of the lymphatic fluid in the inner ear. Such changes or mutations in the SLC26A4 gene may cause Pendred syndrome.

Symptoms of Pendridium syndrome include sensory nerve deafness / vestibular dysfunction, observer bone abnormalities, and the appearance of goiter. Pendridium syndrome may be associated with congenital hearing loss, especially in newborns. For example, a newborn born with a mutated SLC26A4 gene from each parent may have congenital hearing loss. Therapeutic agents specific to Pendrim syndrome, including congenital hearing loss, have not been specifically addressed except for thyroid hormones. In congenital hearing loss, it may be difficult to achieve language rehabilitation if it is not treated before the age of 2 years, which is the most important stage of language development, for the treatment of congenital hearing loss. have.

Thus, there is a continuing need to develop an effective early diagnosis method for Pendrim syndrome as well as an effective treatment or treatment method.

BACKGROUND OF THE INVENTION [0002] Techniques as a background of the invention have been made in order to facilitate understanding of the present invention. And should not be construed as an admission that the matters described in the technical background of the invention are present in the prior art.

In the development of therapeutic agents for Pendrim syndrome, Pendrim syndrome animal models can be very important in the pre-clinical phase, demonstrating efficacy against candidate therapeutic agents.

Accordingly, the inventors of the present invention have recognized the importance of developing an animal model of Pendream syndrome and attempted to develop an animal model exhibiting the same symptoms as Pendrim syndrome human patients.

In particular, the inventors of the present invention were able to recognize that the p.H723R mutation among the mutations of the SLC26A4 gene causing Pendrim syndrome is the most frequent in the patients with Pendrim syndrome in Korea.

Accordingly, the inventors of the present invention have recognized that an animal model of Pendrill's syndrome can be produced by knocking in a human SLC26A4 mutation gene, in particular, a mutant gene of p.H723R, and various production methods thereof have been studied.

As a result, the inventors of the present invention have developed an animal model showing degeneration of the deaf and womb duct, which is one of the symptoms of Pendream syndrome.

Accordingly, an object to be solved by the present invention is to provide an animal model of PredrD syndrome transplanted with a human SLC26A4 mutant gene and a method for producing the same.

Another object to be solved by the present invention is to provide an animal model of Pendrill's syndrome in which a human SLC26A4 mutation gene is specifically expressed in the inner ear and a method for producing the same.

Still another object of the present invention is to provide a method for screening a therapeutic composition effective for Pendrim syndrome using an animal model of Pendrim syndrome.

The problems of the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, an animal model with a human SLC26A4 mutant gene is provided, wherein an animal model of Peedle's syndrome induced by cochlear or deafness is provided.

As used herein, the term "SLC26A4 mutant gene" may refer to any SLC26A4 gene having a mutant domain. Here, the mutation refers to all the actions that change the genetic function of the SLC26A4 gene, and specifically, the mutation of the SLC26A4 gene may mean a mutation caused by a quantitative or qualitative change of the SLC26A4 gene among various mutations of the organism . For example, a SLC26A4 mutant gene is a mutant gene in which one or more of its DNA bases is replaced by another base, ie, a change in the level of one or more bases, such as substitution, deletion, addition, inversion, The change in molecular level, such as the frame shift due to insertion or deletion of DNA, results in changes in enzymes or peptides that are not produced or disappear according to the genetic information of DNA, May be an increased or altered gene. The SLC26A4 mutation gene may be, but is not limited to, a human mutant gene of at least one of the p.H723R, p.T410M, p.L676Q, or pM147V mutant genes. At this time, the pendrin protein produced from the SLC26A4 mutation gene may show a decrease in biological activity as compared with the normal pendrin protein, and the abnormal pendrin protein may cause the Pendrite syndrome. In particular, among these mutations, the p.H723R mutation may be more frequent in Pendrim syndrome patients than in other mutations.

Here, the "p.H723R mutation gene" can correspond to the SLC26A4 gene showing a mutation of His723Arg among the mutations that can appear in the SLC26A4 gene. Here, the p.H723R mutation may mean a mutation in the 723th amino acid of the pendrin protein. For example, the p.H723R mutation may be a point mutation in which the CAT is changed to the CGT in the STAS domain of the pendrin protein C-terminus. As a result, the 723th amino acid of the pendrin protein is changed from histidine (histidine) to arginine (Arg, arginine). At this time, the site where the p.H723R mutation has occurred may be an important site for folding of the pendrin protein. Thus, the p.H723R mutation can have a devastating effect on pendrin protein function. Also, the p.H723R mutation in the mutation of SLC26A4 can cause Pendrim syndrome.

As used herein, the term "Pendrim ' s syndrome" may refer to any disease that may occur due to a mutation in SLC26A4. Preferably, Pendream syndrome can be an endocrine, nutritional and metabolic disease. Symptoms of Pendrimed syndrome include sensory neural deafness including congenital hearing loss, vestibular dysfunction, observer bone abnormalities, and the appearance of goiter. Symptoms of Pendrim syndrome include malformations of the cochlea and deafness.

As used herein, the term "anomalies of the cochlea" refers to the abnormalities of the cochlear ducts in the inner ear due to the mutation of the SLC26A4 gene, in particular the p.H723R mutation, It can mean. Here, the cochlear duct is also called the cochlea as an auditory organ. In the cochlea, lymphatic fluids are filled and there may be hair cells that play a central role in accepting sounds. The anomalies of the cochlear ducts in patients with Pendream syndrome may include enlargement of the endolymphatic drift of the cochlea, and enlargement of the endolymphatic sac of the cochlea.

As used herein, the term "hearing loss" may refer to sensory neural deafness, which is caused by an abnormality in the function of detecting the sound of the cochlea or an abnormality of the auditory nerve transmitting the stimulation by sound to the brain. Further, the hearing loss of the present invention may include congenital hearing loss that may occur by receiving the SLC26A4 mutation gene from the parent.

In the development of therapies and therapies for Pendream syndrome, animal models of Pendrim syndrome can be very important in the pre-clinical phase of demonstrating efficacy against candidate therapeutic agents.

As used herein, the term "animal model" refers to a disease animal model. Specifically, an animal model may be an animal model that has acquired a condition that is similar to that of a human disease or that has inherently caused the disease. The animal model herein may be an animal model of Pendream syndrome. In addition, the animal that can be used as an animal model of Pendream syndrome of the present invention may be a non-human animal such as a monkey, dog, cat, rabbit, guinea pig, rat, mouse, cattle, sheep, pig or goat. Preferably, the Pendream syndrome animal model may be, but is not limited to, a mouse.

An animal model of Pendream syndrome according to the present invention may be preferably an animal model in which a human SLC26A4 mutant gene has been transplanted in the development of therapies and therapies for deafness and warts anomalies according to Pendream syndrome. Herein, we describe an animal model of Peddle syndrome using an animal model transplanted with the gene of human p.H723R mutation, which is an SLC26A4 mutation gene, which is exemplified by the greatest incidence in patients with Pendream syndrome. However, an animal model in which a variety of SLC26A4 mutation genes associated with Pendream syndrome are implicated, including but not limited to, can also be provided as an animal model of Pendrim syndrome using the method of producing Pendrim syndrome animal models provided by the present invention . For example, the SLC26A4 mutation gene associated with Pendrim syndrome may be the p.H723R, p.T410M, p.L676Q, and pM147V genes.

Animal models transplanted with the above human SLC26A4 mutant gene can be used as an animal model of Pendrill's syndrome, which may result in deafness and womb duct anomalies in human patients with Pendrim syndrome. Furthermore, the animal model transplanted with the gene of human SLC26A4 mutation is not limited to the above-mentioned symptoms, but may exhibit various symptoms that may occur in human patients with Pendrim syndrome.

As the SLC26A4 gene can be expressed in the inner ear, kidney or thyroid, the human SLC26A4 mutant gene in the Pendream syndrome animal model can also be expressed in the inner ear, kidney or thyroid. However, for an animal model of Pendrill's syndrome in which hearing loss has been induced by SLC26A4 mutation, an animal model may be preferred in which the human SLC26A4 mutant gene is expressed only in the inner ear except for the other regions.

As used herein, the term "expression" may refer to the expression of a gene, and moreover, the production of a protein. For example, the expression of a human SLC26A4 mutation gene in an animal model can be used to generate a transcript that includes mRNA by transcription of the human SLC26A4 gene of the mutation, or to a translation of the pendrin protein by translation of the human SLC26A4 gene with the p.H723R mutation Lt; / RTI >

However, the expression of the human SLC26A4 mutant gene may not occur when loxP is present in the transgene used for the transplantation of the human SLC26A4 mutant gene for the Pendream syndrome animal model. At this time, loxP may be a nucleotide sequence or locus that stops transcription. For example, if loxP is present in front of the locus in which the human SLC26A4 mutation gene is inserted in the transcription step of the gene, expression of the human SLC26A4 mutation gene may not occur. That is, even though the human SLC26A4 mutant gene has been transplanted, these animal models may not exhibit the symptoms that appear in human Pendrite syndrome patients, such as deafness or wow tuberculosis. To solve this problem, a cre-recombinase can be used. The cre-recombinant enzyme is an enzyme capable of recognizing loxP and cleaving it. For example, when a transgene containing loxP and a human SLC26A4 mutation gene is injected into an animal model having a region where cre-recombinase is expressed, loxP is removed and the human SLC26A4 mutant gene is expressed in a cre-recombinant enzyme Lt; / RTI > site. Accordingly, an animal model according to an embodiment of the present invention is an animal model expressing a cre-recombinant enzyme specifically in the cerebrospinal fluid such that a human SLC26A4 mutant gene, particularly a p.H723R mutant gene thereof, can be expressed in the inner ear, Specifically, it may be a mouse expressing a cre-recombinant enzyme.

Furthermore, according to one embodiment of the present invention, the mouse may be a mouse in which the mouse-inherent SLC26A4 gene is knocked out and the human SLC26A4 mutant gene is transplanted.

Thus, the term "SLC26A4 gene deficient mouse" as used herein may mean a mouse lacking the original SLC26A4 gene of the mouse. As a method of disrupting the SLC26A4 gene in a mouse, there may be a method using the above-mentioned loxP and cre-recombinant enzyme. For example, a mouse lacking the SLC26A4 gene of the mouse can be obtained by crossing a mouse in which loxP is inserted on both sides of the SLC26A4 gene of a mouse as a target gene and a mouse expressing a cre recombinase.

According to one embodiment of the present invention, a step of obtaining a first-generation mouse by crossing a mouse expressing a cre-recombinant enzyme specifically in the inner ear with a mouse into which a human SLC26A4 mutant gene has been transplanted to obtain a first generation mouse, To obtain a Pendlet syndrome mouse model. ≪ Desc / Clms Page number 3 >

As used herein, the term "trait injection" may mean that a genetic property of an organism is changed by receiving DNA from the outside. Transformation methods may include, but are not limited to, use of ribosomes, electroporation, chemicals that increase the uptake of the DNA, direct DNA injection into the host cell, particle gun impact, and micro- Can be used as a transformation method.

As used herein, the term "first generation mouse" may refer to a mouse p.H723R mutant gene and a mouse SLC26A4 gene expressed in the inner ear. First-generation mice can be obtained by crossing a mouse expressing the cre-recombinant enzyme and a mouse transfected with the human p.H723R mutant gene as described above, wherein the mouse expressing the cre-recombinant enzyme is Foxg1-cre mouse May be preferred. Specifically, the promoter of Foxg1 may be innate-specific, and the cre-recombinant enzyme of Foxg1-cre mouse can specifically express inner-specificity. In addition, when pax2-cre mice, known as mice expressing cre-recombinase, were used, mice obtained by crossing of pax2-cre mice with human SLC26A4 transgenic mice were used as a control model for Pendrij syndrome animal models Can be used. These mice can express the human SLC26A4 gene in the inner ear of the mouse as well as in the kidney and thyroid. As a result, a mouse obtained by crossing between a pax2-cre mouse and a mouse into which a human SLC26A4 gene has been transfected has a defective folding pendrin protein expressed in the kidney or thyroid, for example, and may have a shorter lifespan than a normal mouse. Thus, pax2-cre mice may be unsuitable for use as Pender syndrome animal models.

As used herein, the term "Pendream syndrome mouse model" may refer to a mouse expressing a human SLC26A4 mutant gene in which the mouse SLC26A4 gene is deficient in the inner ear. That is, among the second-generation mice obtained by crossing the above-described first-generation mouse and the SLC26A4 gene-deficient mouse, the mice that received the SLC26A4 mutation gene from the first-generation mouse can be used as a mouse model of Predride syndrome. Such Pendrimed syndrome mouse models may be heterozygous. That is, the PendriD syndrome mouse model can have chromosome 7, in which the human SLC26A4 mutation gene has been transplanted and the mouse SLC26A4 has been deleted. Thus, the Pendream syndrome animal model produced by the method of the present invention expresses the human SLC26A4 mutation gene in the inner ear and can express the phenotype associated with Pendrim syndrome such as deafness and womb tube malformation have.

Furthermore, of the third generation mice obtained by crossing the animal models of Pendre syndrome produced by the above method, the mice of the homozygous can be more severe than the Pendre syndrome animal model in the degree of deformity of the hearing loss or cochlear duct. That is, the third generation mouse has the homologous human SLC26A4 mutation gene on chromosome 7, so that the phenotype associated with Pendrim syndrome can be more pronounced.

According to one embodiment of the present invention, a transgenic mouse can be matched with a mouse transfected with a cre-recombinase inducible transgene into which a human SLC26A4 mutation gene is inserted.

As used herein, the term "metastatic gene" refers to a gene that has been artificially introduced externally by DNA technology. Such a transgene can transfer an exogenous nucleic acid to a transgenic or transgenic animal. The cre-recombinant enzyme inducible transgene of the present invention can be recognized by a cre-recombinant enzyme including loxp in the structure of the transgene.

Microinjection may be desirable as a method for producing a recombinase-inducible transgene in which a human SLC26A4 mutant gene or a human SLC26A4 gene is inserted for generation of a first generation mouse. For example, a human SLC26A4 mutant gene or a cre-recombinant enzyme-induced transgene in which the human SLC26A4 gene is inserted can be directly microinjected into a mouse embryo. The resulting embryos are transplanted into surrogate mothers, and the resulting mice can be used as mice transduced with the inducible metastasis gene. At this time, a mouse in which the human SLC26A4 mutation gene or the human SLC26A4 gene is transfected can be selected through genotyping.

In addition, the human SLC26A4 mutation gene inserted into the cre-recombinant enzyme inducible transgene may have a different expression level in the transduced mouse depending on the inserted gene locus.

Accordingly, the human SLC26A4 mutant gene according to an embodiment of the present invention may exhibit a high expression level when inserted between the XhoI and NotI nucleic acid internal hydrolase recognition sites in the cre-recombinant enzyme inducible transgene. However, without being limited thereto, the human SLC26A4 mutation gene can be inserted into all gene loci within the cre-recombinant enzyme inducible transgene, which can express it in the mouse.

Furthermore, the cre-recombinant enzyme inducible transgene of the present invention may include a promoter, a human SLC26A4 gene as a foreign gene, and loxp. For example, the cre-recombinant enzyme-inducible transgene contains the chick ß-actin promoter, the CMV-IE-enhancer that enhances expression, the two loxp and loxp NeoCyme Phosphoglucokinase-polyA (PGK-Neo PGK-pA) present in the nucleotide sequence of SEQ ID NO: 1, and may include a nucleotide sequence recognizable by XhoI and NotI restriction enzymes. At this time, it may be preferable that the human SLC26A4 gene or the human SLC26A4 mutation gene is inserted between the XhoI and NotI restriction enzyme sites. In addition, the cre-recombinant enzyme-inducible transgene may further contain a rabbit-ß-Globlin pA that can increase the expression thereof after insertion of the human SLC26A4 gene or the human SLC26A4 mutation gene have. The CRE-recombinant enzyme-derived transgene having the above structure is cleaved by the cre-recombinant enzyme to cleave the loxp-PGK-Neo PGK-pA-loxp complex structure so that the human SLC26A4 gene or the human SLC26A4 mutant gene can be linked to the chick ß- have. As a result, the human SLC26A4 gene or the human SLC26A4 mutation gene in the transfected mouse can be expressed.

According to one embodiment of the present invention, there is provided a method of treating a Pendlet syndrome animal model, comprising administering a candidate substance for treating Pendrim syndrome, measuring the hearing of the animal model administered with the candidate substance, There is provided a method for screening a composition for treating Pendlet syndrome, comprising the step of measuring the size of the lymphocyte.

As used herein, the term "screening" may refer to drug screening and may search for a candidate for a composition for the treatment of Pendream syndrome which has an improvement effect on symptoms of Pendrim syndrome, It does not.

Furthermore, the term "candidate substance" as used herein may mean any substance that can be expected to have an improvement effect on the symptoms according to Pendrim syndrome, and its form and kind are not limited. For example, a candidate substance may be a chemical compound, a recombinant protein, or a natural substance that can inhibit the expression of the SLC26A4 mutant gene, particularly the p.H723R mutant gene, or lower its expression level. Also, in the study of gene therapy, the candidate material may be the normal SLC26A4 gene itself.

As used herein, the term "treatment" is intended to mean that a candidate for Pendream syndrome treatment is administered to an animal model of Pendream syndrome according to one embodiment of the present invention to improve symptoms associated with Pendrim syndrome It means all the activities that enable us to be benefited. For example, when a therapeutic candidate having a therapeutic effect on Pendrim syndrome is administered to an animal model of Pendream syndrome according to an embodiment of the present invention, the animal model may be used to improve hearing loss, May indicate an improvement in malformations.

In order to confirm the hearing loss and the improvement of the anomaly of the cochlear duct, hearing measurement or a method of measuring the size of the lymph node in the inner lumen of the cochlea and the lymphocyte in the cochlear duct may be used. However, And various methods can be used as a method for confirming the effect on the therapeutic candidate substance.

Auditory brainstem response test (ABR) can be used to measure the hearing level. CT images can be obtained by measuring the size of the lymphatic vessels of the cochlear ducts and the lymphocytes in the cochlear ducts.

According to one embodiment of the invention, the administration of the candidate substance may be performed by intraperitoneal administration, intranasal administration, intravenous administration, intramuscular administration, transdermal administration, subcutaneous administration, inhalation administration, oral administration, It can be my shot. The preferred method of administering the candidate agent may be intramuscular injection or intrauterine injection in the oviduct, but not limited thereto, if the candidate for treatment for Pendrim syndrome can reach the desired site, . Further, depending on the age of the Pendream syndrome animal model according to one embodiment of the present invention in which the candidate therapeutic substance is to be administered, or whether it is pregnant, the administration method can be easily selected by those skilled in the art. For example, when the Pendream syndrome model is a mouse at 1 to 2 months of age, the preferred method of administration of the candidate agent may be administration of intramuscular injection or oophorectomy. At this time, the administration of oophorectomy can be performed by exposing the middle ear canal by surgical treatment, exposing the oophorus entering the oophoria, injecting the candidate material directly to the oophorus, or injecting with a micropipette.

In the case of congenital hearing loss due to Pendream syndrome, treatment within two years of birth may be important as described above. Therefore, in order to screen candidates for treatment of congenital hearing loss according to Pendrim syndrome, it is preferable that an animal model of Pendrim syndrome according to an embodiment of the present invention is a mouse of 1 week old to 2 months old have. However, it is not limited thereto.

In addition, the treatment of congenital hearing loss according to Pendrite syndrome can be performed on a woman who is pregnant with a Pendrite syndrome fetus. Thus, the administration of the candidate substance may be a step of crossing the Pender syndrome mouse models to obtain a pregnant mouse, and a step of administering to the pregnant mouse a candidate substance for treating the Pender syndrome. At this time, the method of administration of the candidate substance may be preferably, but not limited to, intrauterine injection. More specifically, intrauterine injection may be a method of microinjecting a candidate substance directly into the uterine vesicle of a mouse.

Here, the Predry syndrome mouse models are as described above, in which only one of the alleles of chromosome 7 is a heterozygote having a human SLC26A4 mutation, or alleles of chromosome 7 are mouse of a homozygous mutant having a human SLC26A4 mutation have. Thus, mice born from these can also be homozygous or heterozygous mouse, expressing phenotypes associated with Pendrim syndrome like parents, and can be used as a congenital hearing impairment model according to Pendrim syndrome.

By measuring the level of the Pendrin protein in the uterus of the pregnant mouse or the expression level of the SLC26A4 gene, the effect on candidate therapeutic agents of Pendrim syndrome can be confirmed. Further, it can be confirmed by measuring the level of the Pendrin protein in the uterus or the expression level of the SLC26A4 gene in a Pendream syndrome animal model according to an embodiment of the present invention. , The efficacy of the therapeutic candidate can be ascertained by measuring a decrease in the expression level of the human SLC26A4 mutation gene in a Pendream syndrome animal model according to one embodiment of the present invention. However, for the evaluation of candidate substances for treatment of Pendrim syndrome, the method of measuring the level of pendrin protein or the expression level of the SLC26A4 gene is not limited to the Pendrim syndrome model of the pregnant mouse described above. For example, by measuring the level of the pendrin protein in the inner ear or the expression level of the SLC26A4 gene when the Pendream syndrome animal model for which the effect on the candidate substance is to be determined is 1 to 2 months, The effect can be evaluated.

The measurement of the expression level of the SLC26A4 gene or the human SLC26A4 mutant gene can be carried out by using reverse transcription-PCR, competitive reverse transcription-PCR, real-time reverse transcription-PCR, RNase protection assay (RPA), Northern blotting, and DNA chip (DNA chip) methods can be used, but the present invention is not limited thereto.

In addition, the level of pendrin protein can be determined by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip (FACS) methods have been used for the treatment of cancer, But is not limited thereto.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention.

The present invention provides an animal model in which a human SLC26A4 mutant gene has been transplanted and provides an animal model of Pendlet syndrome which is useful for the development of a therapeutic agent for Pendrim syndrome and a therapeutic method using the same.

In addition, the present invention has the effect of providing an animal model in which the human SLC26A4 mutant gene is expressed only in the ear, which may be essential for proving treatment for sensory neural deafness, which may be caused by Pendream syndrome .

Furthermore, the present invention can provide a method for producing an animal model of Pendream syndrome, and the animal model produced thereby can be used for screening a composition for treating Pendream syndrome.

The effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the specification.

1 is a diagram showing a method of manufacturing an animal model of Pendream syndrome according to an embodiment of the present invention.
Figure 2 is an experimental result showing the expression level of pendrin protein in five animal models including Pendream syndrome animal models according to one embodiment of the present invention.
FIG. 3A shows the results of histological analysis of the cochlea of a mouse model of mouse-derived SLC26A4 gene.
FIG. 3B is an experimental result showing histological analysis of a cochlear duct of a mouse having the mouse p.H723R mutant gene.
FIG. 3C shows the results of histological analysis of mouse cochlear ducts in which a human SLC26A4 gene has been transplanted and a mouse-derived SLC26A4 gene has been deleted.
FIG. 3D is an experimental result showing histological analysis of the cochlear duct of an animal model of Pendrim syndrome according to an embodiment of the present invention in which the human p.H723R mutant gene is transplanted and the mouse native SLC26A4 gene is deleted mouse .
FIG. 3E shows the results of an experiment in which a mouse SLC26A4 gene was not transplanted and the mouse original SLC26A4 gene was deleted and the mouse cochlear duct was histologically analyzed.
FIG. 3f is an experimental result showing histological analysis of cochlear ducts for four animal models including an animal model of Pendream syndrome according to an embodiment of the present invention.
FIG. 3g shows the results of measuring the size of the endolymphatic and endolymphatic sacs of the cochlea in four animal models including an animal model of Pendream syndrome according to an embodiment of the present invention.
4A is an experimental result showing the results of the ABR test for four animal models including the Pendream syndrome animal model according to one embodiment of the present invention.
Figure 4b is an experimental result showing the results of ABR assays for four animal models including Pendream syndrome animal models according to one embodiment of the present invention.
5A is an experimental result showing the results of histological analysis and ABR test of Southern blot, cochlear duct, and animal model of Peddor syndrome using a mouse having a mouse p.H723R mutant gene.
Figures 5b and 5c are experimental results showing the results of histological and ABR assays of cochlear ducts for pendridial syndrome animal models using pax2 cre-mice.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and the manner of achieving them, will be apparent from and elucidated with reference to the embodiments described hereinafter in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

Hereinafter, an animal model of Pendream syndrome according to an embodiment of the present invention and a mouse model prepared by the method for producing Pendre syndrome animal model, The degree of induced hearing loss is evaluated and explained. However, the effect of the present invention is not limited to the following effects. An animal model of Pendream syndrome according to an embodiment of the present invention and a mouse model prepared by the method of manufacturing Pendre syndrome animal model may be various symptoms . Further, in the following, a mouse transplanted with a human p.H723R mutation gene among SLC26A4 mutation mutants is exemplarily described but not limited thereto. Accordingly, the mouse model prepared by the animal model of Pendream syndrome and the method of producing Pendlet syndrome animal model provided by the present invention can be used as an animal model for various diseases that may be caused by SLC26A4 gene mutation .

Example 1: Evaluation of an animal model of Pendrim syndrome

Manufacture of Pendrite syndrome animal models

Hereinafter, an animal model of Pendream syndrome according to one embodiment of the present invention evaluated and used in Example 1, and a method of producing a pendrided mouse model prepared by the method of producing Pendlet syndrome animal models will be described.

1 is a diagram showing a method of manufacturing an animal model of Pendream syndrome according to an embodiment of the present invention.

Referring to Figure 1 (a), the structure of the transgene for transplantation of human p.H723R mutant gene or human SLC26A4 gene into mouse is shown. Specifically, the transgene for producing an animal model of Pendream syndrome according to an embodiment of the present invention is a cre-induced transgene containing loxp. The cre-recombinant enzyme inducible transgene contains the chick ß-actin promoter, the CMV-IE-enhancer, and the PGK-Neo PGK-pA between the two loxp and loxp. At this time, the human SLC26A4 gene or the human p.H723R mutation gene is inserted between the XhoI and NotI restriction enzyme sites. In addition, the cre-recombinant enzyme inducible transgene contains a rabbit-β-globulin pA capable of increasing its expression after insertion of the human SLC26A4 gene or the human p.H723R mutant gene. The cre-recombinant enzyme-derived transgene having the above structure is cleaved by the cre-recombinant enzyme to cleave the complex structure of loxp-PGK-Neo PGK-pA-loxp, so that the human SLC26A4 gene or the human p.H723R mutant gene Can be connected. As a result, the human SLC26A4 gene or the human p.H723R mutation gene in the transfected mouse can be expressed.

Referring to FIG. 1 (b), a method of manufacturing an animal model of Pendream syndrome in accordance with an embodiment of the present invention is illustrated. Specifically, a cre-induced transgene-transgenic mouse and a cre-foxg1 mouse transplanted with a cre-inducible transgene containing the human SLC26A4 mutant gene are crossed to obtain a first-generation mouse. First generation mice can express the human p.H723R mutant gene and the mouse native SLC26A4 gene in inner ear tissues. At this time, in mice transplanted with a cre-induced transgene containing the human SLC26A4 mutation gene, the human SLC26A4 gene and the mouse-derived SLC26A4 gene can be expressed in the inner ear tissue. Next, a second-generation mouse obtained by crossing the mouse of the first generation with the mouse lacking the SLC26A4 gene is obtained. Mice receiving the p.H723R mutant gene from the first generation of these second generation mice can be used as a model of Peddle syndrome mice. That is, the Pendream syndrome mouse model can be a mouse of heterozygous having the chromosome 7, in which the human p.H723R mutation gene is transplanted and the mouse SLC26A4 is deficient. Accordingly, the animal model of Pendrim syndrome produced by the method according to an embodiment of the present invention expresses a human p.H723R mutation gene in inner ear, and a phenotype associated with Pendrim syndrome such as deafness and womb duct malformation .

Pendrin Protein Levels for Pendrimed Syndrome Animal Models

To determine the level of pendrin protein for the Pendream syndrome animal model, first prepare five animal models. Specifically, the five animal models are mice (hereinafter, referred to as a control group) having a mouse-inherent SLC26A4 gene, mice lacking the human SLC26A4 gene (SLC26A4 (-)) (Hereinafter referred to as human SLC26A4 (+)), the mouse p.H723R mutant gene was not transplanted and the mouse original SLC26A4 gene was deleted (hereinafter, human p.H723R (-)) and a human p.H723R mutant gene of the Pendream syndrome animal model of the present invention is transplanted and the mouse original SLC26A4 gene is deleted (hereinafter, human p.H723R (+)). Levels of pendrin protein were measured in five models of inner ear using western blot.

Figure 2 is an experimental result showing the expression level of pendrin protein in five animal models including Pendream syndrome animal models according to one embodiment of the present invention.

2, the levels of pendrin protein in human SLC26A4 (-), human SLC26A4 (+), human p.H723R (-) and human p.H723R have. Furthermore, in human p.H723R (+), an animal model of Pendrin syndrome according to an embodiment of the present invention, a single band appears unlike normal human SLC26A4 (+). That is, considering that pendrin protein is a glycoprotein, normal pendrin protein can be generated in normal human SLC26A4 (+), and several bands can appear. In human p.H723R (+), mutant pendrin protein So that a saccharide is not formed and a single band may appear. As a result, the p.H723R mutation can be predicted to be a mutation at the glycation site of the SLC26A4 gene.

Histological analysis of the cochlear duct in an animal model of Pendrim syndrome

For the histological analysis of the cochlear duct, we used micro-computed tomography (M-CT) to measure the size of the left endolymphocyte and cochlea of mouse models. At this time, mouse models were used in addition to the mouse models of the control group, human SLC26A4 (-), human SLC26A4 (+), human p.H723R (-) and human p.H723R . A mouse having the H723R mutation gene (hereinafter referred to as mouse p.H723R (+)) is used.

FIG. 3A shows the results of an experiment in which a mouse model (control group) having a mouse-derived SLC26A4 gene was histologically analyzed. FIG. 3B shows the results of an experiment in which the mouse p.H723R mutant gene-bearing mouse (mouse p.H723R (+)) was histologically analyzed. FIG. 3C shows the results of experiments showing histological analysis of the cochlea of a mouse (human SLC26A4 (+)) in which the human SLC26A4 gene has been transplanted and the mouse original SLC26A4 gene has been deleted. Figure 3d illustrates the expression of the Pheid's syndrome animal model (human p.H723R (+)) in the cochlea according to one embodiment of the present invention in which the human p.H723R mutant gene is transplanted and the mouse native SLC26A4 gene is deleted. Histologically, the results of the experiment are shown. FIG. 3E is an experimental result showing histological analysis of the cochlea of a mouse (human SLC26A4 (-)) in which the human SLC26A4 gene was not transplanted and the mouse original SLC26A4 gene was deleted.

3A and 3B, in the control group, the inner diameter of the cochlea was 0.3 mm, and the size of the endolymphatic sac and the size of the cochlea were not enlarged. 3B and 3B, the inner diameter of the cochlear duct was 0.25 mm in the mouse p.H723R (+), and the enlargement of the endolymphatic sac and cochlea was not observed similarly to the normal mouse . In particular, referring to FIGS. 3 (a) and 3 (b), in the human SLC26A4 (+), the inner diameter of the cochlear duct was 0.11 mm and expansion of the endolymphatic sac and cochlear ducts was not observed as in normal mice. As a result, the insertion of normal SLC26A4 in gene therapy for deafness due to Pendrim syndrome may be effective in the treatment of Pendrim syndrome.

Referring to Figs. 3d (a) and (b), an animal model of Pindhid syndrome according to an embodiment of the present invention of human p.H723R (+) showed an inner diameter of the cochlea of 0.53 mm, Extension of the sac and cochlea were observed. Furthermore, in the human SLC26A4 (-) of 3e (a) and (b), the inner diameter of the cochlea was 0.72 mm, and expansion of the endolymphatic sac and cochlea was observed in the same manner as the Pendre syndrome mouse model of the present invention .

Figure 3f is a graph depicting four animal models (human SLC26A4 (-), human SLC26A4 (+), human p.H723R (-) and human p.H723R (-)) containing an animal model of Pendream syndrome according to one embodiment of the present invention +)) Were obtained by histological analysis. Figure 3g shows the results of four animal models (human SLC26A4 (-), human SLC26A4 (+), human p.H723R (-) and human p.H723R (-)) containing an animal model of Pendream syndrome according to one embodiment of the present invention +)) And the size of the endolymphatic and endolymphatic sacs in the cochlear duct.

Referring to FIG. 3F and FIG. 3G, in the human p.H723R (-) in which the mouse-derived SLC26A4 gene was deleted, the size of the lymph node and the endolymphatic sac, that is, the expansion of the womb tube, were shown. In addition, in the human p.H723R (+) and human SLC26A4 (-) animal models of the Pendream syndrome of the present invention, enlargement of the size of the endolymphatic and endolymphatic sacs was shown, similarly to the results of Figs. 3d and 3e. In contrast, human SLC26A4 (+), a human SLC26A4 transgenic mouse, did not exhibit an enlargement of the size of the lymph nodes and endolymphatic sac. This result is the same as the result in Fig. 3C. That is, human p.H723R (+) can be used as a model of human Pendrite syndrome.

Hearing aids for Pendream syndrome animal models

Hearing measurements for Pendream syndrome animal models are performed with ABR assays.

4A is a graph showing the results of four animal models (human SLC26A4 (-), human SLC26A4 (+), human p.H723R (-) and human p.H723R +)), Which is the result of the ABR test.

Referring to Figure 4a, the human p.H723R (+), an animal model of the Pendream syndrome of the present invention, does not respond at 100 dB. Similar to the structural abnormalities shown in FIGS. 3A to 3G, it was confirmed that human p.H723R (+) had hearing abnormality, which is another symptom of Pendrim syndrome. Furthermore, human SLC26A4 (+) transplanted with the normal human SLC26A4 gene responds at about 25dB. Similar to the results in which the cochlear abnormalities were not shown in FIGS. 3A to 3G, the human SLC26A4 (+) confirmed that the hearing was normal.

Figure 4b is a graph showing the effect of ABR on human animal models (human p.H723R (+), human SLC26A4 (+) and mouse p.H723R (+)) including Peddler syndrome animal models according to one embodiment of the present invention. The results of the test are shown.

Referring to Figure 4b (a), the ABR assay results for mouse p.H723R (+) with the mouse p.H723R mutant gene are shown. The SLC26A4 mutation in the mouse occurred, but hearing is normal. Accordingly, a model in which the mouse itself mutates to the SCL26A4 gene has difficulties in being used as an animal model of Pendrim syndrome. Referring to Figure 4b (b), the results of the ABR assay of human p.H723R (+), an animal model of the Peddle syndrome of the present invention in which the human p.H723R mutant gene has been transplanted and the mouse native SLC26A4 gene has been deleted Respectively. An animal model of Pendream syndrome according to an embodiment of the present invention shows that the above-described histopathological analysis of the cochlear duct also revealed hearing loss as well as the result of the abnormality of the cochlear duct. Accordingly, a mouse transplanted with the human p.H723R mutant gene may be preferable as an animal model of Peddle syndrome. Referring to (c) of Figure 4b, the results of ABR assays of mice in which the human SLC26A4 gene has been transplanted and the mouse native SLC26A4 gene has been deleted are shown. It can be seen that the hearing ability of the mouse transplanted with the human SLC26A4 gene appears normal.

As a result of the above Example 1, the mouse model produced by the animal model of Pendream syndrome and the animal model of Pendlet syndrome according to an embodiment of the present invention not only expresses the human p.H723R mutant gene, And anomalies of the cochlear duct are observed and can be provided as animal models for diseases according to Pendream syndrome.

In addition, the present invention can provide a method for screening a composition for treating Pendream syndrome using the Pendream syndrome animal model provided by the present invention. For example, a substance expected to have a therapeutic effect on Pendrim syndrome may be administered intraperitoneally, intranasally, intramuscularly, or transdermally to an animal model of Pendrim syndrome according to an embodiment of the present invention , Subcutaneous administration, inhalation administration, oral administration, intravaginal administration, intramembrane injection or intrauterine injection. At this time, an animal model of Pendream syndrome according to an embodiment may be preferably a mouse at 1 to 2 months old or a mouse pregnant with the Pendlet syndrome mouse model provided in the present invention. In addition, for the evaluation of candidates for treatment of Pendream syndrome, measurement of hearing for the Pendream syndrome animal model according to one embodiment of the present invention, measurement of the size of the endolymphatic or endolymphatic sac of the cochlear duct . Alternatively, if the candidate substance for the treatment of Pendrim syndrome is the normal SLC26A4 gene itself, the evaluation can be performed by measuring the expression level of the SL723R gene or the level of the pendrin protein in the Pendrill's syndrome mouse model have. Further, an evaluation of candidate substances for treating Pendrim syndrome can be performed by measuring the expression level of the SL723R gene in the uterus of the pregnant mouse or the level of the pendrin protein in a mouse model of Pendrim syndrome.

Comparative Example 1: Evaluation of an animal model of Pendream syndrome using conventional techniques

Hereinafter, a mouse model of Pendrim syndrome using mouse p.H723R mutant gene and pax2 cre-mouse will be described. At this time, the mouse models are made in the same manner as the mouse model of Example 1. Specifically, the mouse model includes a pax2 mouse (hereinafter referred to as a control) having a mouse-specific SLC26A4 gene, a pax2 mouse (hereinafter referred to as p human SLC26A4 (+)) in which a human SLC26A4 gene has been transplanted and a mouse- (hereinafter referred to as p human p.H723R (+)), a mouse p.H723R mutant gene (p mouse p H723R (+)) having a mouse p.H723R mutant gene and a mouse native SLC26A4 gene ) And pax2 mouse in which the mouse SLC26A4 gene has been deleted (hereinafter, p mouse SLC26A4 (-)).

5A is an experimental result showing the results of southern blot, histological analysis of the cochlear duct, and ABR assay for an animal model of Pendrill's syndrome using a mouse having the mouse p.H723R mutant gene.

Referring to FIG. 5A, in the mouse mouse p.H723R (+) having the mouse p.H723R mutant gene, which has the SLC26A4 gene of the mouse, as compared with the control group, DNA fragments represented by the A band and the B band are shown . Here, the B band may mean a DNA fragment in which the p.H723R mutation has occurred in the mouse SLC26A4 gene. Referring to FIG. 5 (a), p.H723R (+) of the mouse p.H723R mutant does not cause expansion of the cochlear and lymphatic vessels even when the mutation of p.H723R occurs. However, in the mouse SLC26A4 (-) in which the mouse SLC26A4 gene has been deleted, malformation of the cochlear duct, that is, extension of the lymph node, appears. Referring to (c) of FIG. 5A, it can be seen that the mouse p.H723R (+) of p mouse has normal hearing power as in the control group.

Figures 5 (b) and 5 (b) show the results of histological analysis and ABR assays of the cohort of p human p.H723R (+) transplanted with the human p.H723R mutant gene using pax2 cre-mouse . p human p.H723R (+) can be confirmed to have induced malformation and deafness in the cochlear duct, as in the animal model of Peddler syndrome of the present invention (human p.H723R (+)).

Figures 5 (a) and 5 (b) show histological and histological analysis of the human papillomavirus SLC26A4 (+), in which the mouse SLC26A4 gene was deleted and the human SLC26A4 gene was transplanted with pax2 cre- Lt; / RTI > Referring to this, the p-human SLC26A4 (+) represents the normal of the cochlear structure and the normal hearing. However, referring to (c) of FIG. 5c, it can be seen that the pendrin protein in p human SLC26A4 (+) is expressed not only in the inner ear but also in the kidney as compared to the p mouse SLC26A4 (-). Furthermore, the lifespan of the p human SLC26A4 (+) control mice is observed at 4 weeks of age and is shorter than that of normal mice, which may be unsuitable as an animal model of control for Pendrim syndrome animal models.

As a result of the above Comparative Example 1, it was confirmed that the Pendream syndrome animal model using the conventional technique is unsuitable for use as an animal model of Pendrim syndrome. Thus, the mouse model produced by the animal model of Pendream syndrome and the method of producing Pendlet syndrome animal model, according to an embodiment of the present invention, may be a suitable model for the development of a therapeutic agent or treatment for Pendrim syndrome . Further, the present invention provides a method for screening a composition for treating Pendream syndrome using an animal model of Pendrim syndrome according to an embodiment of the present invention, thereby contributing to the study of various diseases according to Pendrim syndrome .

Although the embodiments of the present invention have been described in detail with reference to the accompanying drawings, it is to be understood that the present invention is not limited to those embodiments and various changes and modifications may be made without departing from the scope of the present invention. . Therefore, the embodiments disclosed in the present invention are intended to illustrate rather than limit the scope of the present invention, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.

Claims (21)

An animal model in which a human SLC26A4 mutant gene has been knocked in, wherein said animal model is a model of Pendred syndrome, which is a model in which cochlear or deafferentation is induced, as opposed to a normal model. The method according to claim 1,
Wherein the human SLC26A4 mutant gene is a human mutant gene of at least one of the p.H723R, p.T410M, p.L676Q, or p.M147V mutant genes. The method according to claim 1,
The human SLC26A4 mutant gene is the human p.H723R gene, an animal model of Pendre syndrome. The method according to claim 1,
The anomaly of the cochlea corresponded to the expansion of the lymphatic vessels in the cochlear duct or the lymphocyte in the cochlear duct. The method according to claim 1,
An animal model of Pendrill's syndrome, wherein the hearing loss is sensory nerve impairment. The method according to claim 1,
Wherein said human SLC26A4 mutant gene is expressed only in the inner ear of said animal model. The method according to claim 1,
Wherein said animal model is a mouse that expresses cre-recombinase specifically in the inner ear, Pendre syndrome animal model. 8. The method of claim 7,
The mouse is an animal model of Pendrill's syndrome in which the SLC26A4 gene is knocked out. Obtaining a first-generation mouse by crossing a mouse expressing a cre-recombinant enzyme specifically in the inner ear with a mouse into which a human SLC26A4 mutation gene has been transduced; And
Wherein the first generation mouse and the SLC26A4 gene-deficient mouse are crossed to obtain a Pendlet syndrome mouse model. 10. The method of claim 9,
Wherein the human SLC26A4 mutant gene is a human mutant gene of at least one of the p.H723R, p.T410M, p.L676Q, or p.M147V mutant genes. 10. The method of claim 9,
Wherein said human SLC26A4 mutation gene is a human p.H723R gene. 10. The method of claim 9,
Wherein the Pendlet syndrome mouse model is a mouse in which the human SLC26A4 mutant gene is expressed only in inner ear. 10. The method of claim 9,
Wherein the mouse expressing the cre-recombinant enzyme is a Foxg1-Cre mouse. 10. The method of claim 9,
Wherein the transfected mouse corresponds to a mouse transfected with a cre-recombinase inducible transgene into which the human SLC26A4 mutation gene is inserted. 15. The method of claim 14,
Wherein said human SLC26A4 mutation gene is inserted between the XhoI and NotI nucleic acid internal hydrolase recognition sites in said cre-recombinant enzyme inducible transgene gene. 9. A method for treating Pendryl syndrome, comprising administering to a Pendlet syndrome animal model according to any one of claims 1 to 8 a candidate substance for treating Pendrim syndrome; And
Measuring the hearing of the animal model to which the candidate substance has been administered or measuring the size of the endolymphatic or endolymphatic sac of the cochlear duct. 17. The method of claim 16,
Wherein said administration is by intraperitoneal, intranasal, intravenous, intramuscular, transdermal, subcutaneous, inhalation, oral or intrauterine injection, for the treatment of Pendrim syndrome. 17. The method of claim 16,
Wherein said Pendream syndrome animal model is a mouse at 1 to 2 months of age at birth. 17. The method of claim 16,
Wherein the administering step comprises crossing the Peddle syndrome mouse models of a homozygote to obtain a pregnant mouse; And
And administering to said pregnant mouse a candidate substance for treating said Pendream syndrome. 20. The method of claim 19,
Wherein said measuring step comprises measuring the level of the Pendrin protein or the level of the SLC26A4 gene in the uterus of said pregnant mouse. 17. The method of claim 16,
Wherein the Pendream syndrome animal model is expressed only in the inner ear of the animal model, wherein the human SLC26A4 mutant gene is expressed in the animal model.

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