KR20080042058A - Iduronate-2-sulfatase knock-out mouse and its using method - Google Patents

Abstract

An iduronate-2-sulfatase knock-out mouse is provided to diagnose and treat Hunter syndrome caused by defect of iduronate-2-sulfatase and develop a treating agent of Hunter syndrome by performing physiological experiments between the knock-out mouse and a control mouse. An iduronate-2-sulfatase knock-out mouse is produced by (i) preparing a targeting vector by substituting the allele region(12133-21031 bp) of a wild iduronate-2-sulfatase gene(110079 bp) by a gene cassette(9220 bp) containing a left arm(2027 bp), PGK(phosphoglycerine kinase)-neo(1800 bp), and a right arm(5393 bp), (ii) introducing the targeting vector into an embryonic stem cell of mouse, (iii) injecting the transformed embryonic stem cell into a blastocyte of an embryo, (iv) obtaining a chimeric mouse from the transformed embryo, (v) hybridizing the chimeric mouse with a normal mouse to obtain a heterozygote mouse, and (vi) hybridizing a male with a female of the heterozygote mouse, wherein the targeting vector deletes exon 2 and exon 3 of the iduronate-2-sulfatase gene.

Description

Iduronate-2-sulfatase knock-out mouse and its using method}

The present invention relates to a duronate-2-sulfatase knockout mouse, a method for producing the same, and a use thereof as an animal model for developing a hunter syndrome treatment using the duronate-2-sulfatase knockout mouse.

More specifically, the present invention relates to genetically engineered mice useful as models for common lysosomal accumulators, in particular, for Iduronate-2-sulfatase (IDS) defects and tissue specificity of therapeutics and therapeutic agents for such disorders. The use of such genetically designed mice in the evaluation of therapeutic agents for evaluation of adaptive delivery systems.

Mucopolysaccharide is a hereditary metabolic disease caused by a defect in an enzyme present in lysosomes. Lysosomes are organelles involved in various degradation processes, including electron transport, oxidative phosphorylation and pyruvic acid, fatty acids, enzymes involved in several amino acid metabolism, and various hydrolytic enzymes, especially muco Related to degradation of polysaccharides (mucopolysaccharides), mucolipids and sphingolipids. In addition, many enzyme defects and gene abnormalities have been reported in their metabolic processes with respect to mucopolysaccharide.

Specifically, mucopolysaccharides have various symptoms clinically, including skeletal, circulatory, and mental function, and can be divided into seven categories on clinical, genetic, and biochemical basis. This is caused by the accumulation of sulfated polysaccharides such as dermatan sulfate, heparan sulfate, or keratan sulfate, which are sulfated polysaccharides above the enzymes involved in polysaccharide metabolism. Indicates.

Hunter II, the type II of the mucopolysaccharides, is caused by a deficiency of eduuronate-2-sulfatase. Hunter syndrome is a mild case in which juvenile forms and mental retardation, which have severe symptoms since childhood, are not seen. It is divided into late forms of symptoms. Specifically, the infant type shows symptoms of mental retardation and physical abnormalities, and most of them die before the age of 15, and the late type shows a dwarf, an ugly appearance, hepatosplenomegaly, and lives long. Hunter syndrome is a relatively common disease in Korea and is inherited in an X-linked recessive pattern associated with the X chromosome.

Hereditary metabolic diseases are very diverse, and the distribution of diseases varies greatly among ethnic groups. Therefore, various diseases that are of interest in each country are different, and many diseases that are specific and convenient to study are being studied. Hunter syndrome has been reported in Korea in the past due to the characteristic clinical symptoms, and can be found recently confirmed by enzyme diagnosis.

Therefore, the present invention is intended to develop an animal model for the diagnosis and treatment of Hunter syndrome, accordingly to prepare a duronate-2-sulfatase knockout mouse and to perform various physiological experiments between the knockout mouse and the control mouse. Through the establishment of an animal model for the development of a hunter syndrome treatment to complete the present invention.

The problem to be solved by the present invention is to develop an animal model for the diagnosis and treatment of Hunter syndrome and to prepare a duronate-2-sulfatase knockout mouse and to perform various physiological experiments between the knockout mouse and the control mouse. To provide an animal model for the development of therapeutic agents for Hunter syndrome.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for measuring the pharmacological effect of eduronate-2-sulfatase as a therapeutic agent for Hunter syndrome using an animal model using a mouse knocked out of eduronate-2-sulfatase function. The pharmacological effect is measured as an animal model of pharmacological effects selected from direct enzyme replacement of eduronate-2-sulfatase, expression of eduronate-2-sulfatase myoblasts through gene therapy techniques, cell transplantation and vector mediated gene transfer. It is to provide a method for doing so.

On the other hand, the mice knocked out of the eduronate-2-sulfatase function are shown in (i) the allele region (12133-21031 bp) of the wild-type eduronate-2-sulfatase gene (110079 bp) on the left arm (2027 bp). Preparing a targeting vector substituted with a gene cassette (9220 bp) consisting of PGK-neo (1800 bp) and right arm (5393 bp); Ii) introducing the targeting vector into embryonic stem cells of a mouse; Iii) injecting the transformed embryonic stem cells into the blastocyst of the fertilized egg; Iii) obtaining a chimeric mouse via said transformed fertilized egg; Iii) crossing said chimeric mice with normal mice to obtain heterozygous mice; And iii) crossing the male and female of the heterozygous mouse to obtain a mouse knocked out of the duronate-2-sulfatase gene.

In addition, the targeting vector is characterized by the deletion of exon 2 and exon 3 of the duronate-2-sulfatase gene.

An effect of the present invention is to provide an animal model for the diagnosis and treatment of Hunter syndrome, and to prepare a eduron-2-sulfatase knockout mouse, and through various physiological experiments between the knockout mouse and the control mouse, Hunter syndrome treatment agent To provide an animal model for the development of the.

Hereinafter, the present invention will be described in more detail.

Mice knocked out of the duronate-2-sulfatase function of the present invention prepare knockout mice by preparing targeting vectors as shown in FIGS. 1 and 2 and introducing them onto chromosomes.

1 is a schematic of a targeting vector for the preparation of eduronate-2-sulfatase knockout mice. Figure 2 is a detailed view for gene construction of the targeting vector for the preparation of eduronate-2-sulfatase knockout mice.

As shown in FIGS. 1 and 2, the targeting vector used in the present invention includes a gene cassette consisting of a left arm (2027 bp), PGK-neo (1800 bp), and a right arm (5393 bp). Homologous recombination occurs in the complementary fragments, resulting in the deletion of a portion of the duronate-2-sulfatase exon 2 and exon 3 resulting in the loss of the gene, thereby preventing the normal duronate-2-sulfatase gene from being expressed.

In order to manufacture a knockout mouse of the present invention, the above-mentioned targeting vector is first introduced into embryonic stem cells of a mouse, cultured embryonic stem cell clones identified with gene hits, and injected into blastocysts of blastocysts. The development of chimeric mice was induced by grafting into the uterus of 2.5 pcs of fertility surrogate mice. The chimeric mice were crossed with normal mice to obtain heterozygote mice with IDS +/− genotypes, and again, females and males of the heterozygote mice were crossed to prepare transgenic mice with IDS − / − genotypes. . The genetically modified mice have a normal lifespan and have the same lifespan as normal mice, and both males and females have normal fertility when crossed with normal mice.

The present inventors developed Hunter syndrome and its therapeutic agent through various physiological experiments using the knockout mice in which a part of the duronate-2-sulfatase gene was deleted and the duronate-2-sulfatase function was inhibited. The animal model was completed.

Because of the various phenotypes and pathological manifestations of IDS − / − genotypes, knockout mouse models that are the object of the present invention may be applied to the testing and development of enzyme replacement therapy for Hunter syndrome. Mice of the invention can also be used to evaluate therapeutic agents for use in treating Hunter syndrome. This assessment can be achieved by administering a therapeutic agent to homozygous transgenic mice for IDS gene defects and evaluating the mice for histopathology or other indicators associated with iduronate-2-sulfatase defects.

First, the inventors conducted various physiological experiments between the knockout mice and the normal mice through IDS-/-mutant mice. The results are as follows.

Homozygous mutant mice are smaller than 4 week old pups. At this age, the snout is short and has an initial effect on the facial form with hair loss symptoms. These facial features are the same as those initially found in β-glucuronidase deficient (MPS shock, Sly syndrome) mice. No severe features appearing in type 7 mucopolysaccharide knockouts, namely severe growth retardation, arthrosis and obvious facial dysplasia were observed until about 6-8 weeks of age. Radiographs of IDS-/-and-/ + mice at 4 weeks of age show evidence of craniofacial dystrophy and alopecia found early in Hunter syndrome. The spine appeared normal and there was no apparent limb shortening noted in type 7 mucopolysaccharide mice.

Early skeletal changes in patients with ironon-2-sulfatase defects may be difficult to distinguish from more malignant bone pathologies in early childhood. Thus, studies of the skeleton of IDS-/-mice over age are required to determine whether skeletal pathology of this model mimics human defects.

We performed pathological examination of various organs in 4 and 8 week old IDS +/- and-/-mice. All tissues analyzed showed evidence of abnormal lysosome accumulation. By 8 weeks of age, the liver of the IDS-/-mouse was more pale and there was no pale pink glow between the normal livers, but initially there was no evidence of phenotypic grafts. Figure 5 is a photograph showing the results of histological analysis of the liver of the eduronate-2-sulfatase knockout mice and the control group.

6 is a photograph showing the results of histological analysis of the kidney of the eduronate-2-sulfatase knockout mouse and the control group.

The most significant pathology at 4 weeks of age occurs in the reticuloendothelial system, manifested by lysosomal accumulation in Kupffer cells, spleen sinusoid lining cells, lung macrophages and glial cells. Lysosome-loaded Cooper cells in the liver readily observed significant hepatocyte accumulation at 4 weeks of age. By 8 weeks of age there was further progression of accumulation in the reticulum endothelial system and there was evidence of significant hepatocellular vacuogenesis. At 8 weeks of age, 15-20% of the cytoplasm of hepatocytes was accumulated by lysosomes, as opposed to very few lysosomes in normal liver specimens. Splenocytes derived from 8-week-old IDS-/-mice showed markedly expanded lysosomes in most cells of the sinusoid.

At 4 weeks of age abnormal lysosomal accumulation caps are found in glial cells of the cortex that do not exhibit lysosomal accumulation. However, at 8 weeks of age, cytoplasmic vesicle formation can be observed not only in meningoid cells but also in Purkinje cells of the cerebellum, neurons of the cerebral cortex, and glial cells. Evidence of disturbed cell structure on the total cytoplasmic volume was detected through abnormal cytoplasmic vacuoles by 8 weeks of age in all Purkinie cells in the cerebellum of IDS-/-mice. Lysosomes were not detected in 8 week old normal mice. Massive lysosomal accumulation also occurred in chondrocytes found on both the bone and articular surfaces of the bone as well as in the organs of the initial four weeks.

Levels of GAG in urine of IDS +/- mice and IDS-/-mice showed a substantial increase in the amount of GAG relative to creatinine in urine in IDS-/-mice. Thus, the measurement of GAG can serve as a non-invasive means of monitoring the mouse during the treatment regimen before sacrificing the mouse for tissue and organ analysis.

3 is a graph showing the amount of glycosaminoglycan (GAG) excretion of the eduronate-2-sulfatase knockout mice. As shown in FIG. 3, the heterozygous transgenic mice at 16 weeks of age showed 30 to 35 μg / ml of GAG excretion, and the heterozygous transgenic mice at 38 weeks of age showed 15 to 20 μg / ml of GAG excretion. .

On the other hand, homozygous transgenic mice that had all of the duronate-2-sulfatase gene deleted showed 130-135 μg / ml at 16 weeks of age and 160-165 μg / ml at 38 weeks of age. The homozygous transgenic mice are completely deficient in the duronate-2-sulfatase enzyme, which is thought to excrete all GAGs.

On the other hand, Figure 4 is a graph showing the change in organ weight of the iduronate-2-sulfatase knockout mice and the control group. As shown in FIG. 4, in the duronate-2-sulfatase knockout mice, liver growth was particularly retarded compared to normal mice, and growth of organs such as the spleen and lungs was also delayed so that the weight of the organs was not sufficiently increased. .

Hereinafter, the effect of hunter syndrome on eduron-2-sulfatase was measured through knockout mice.

Administration of iduronate-2-sulfatase can be carried out by any route including intravenous, intramuscular, oral or intraperitoneal administration. "Administration" includes the use of autologous or xenograft of tissue or pseudo-organ. Autologous transplantation of genetically modified fibroblasts, or neo-organs, is known to produce stable expression of lysosomal enzymes that are effective in improving symptoms in beta-glucuronidase-deficient MPS Ⅶ mouse models. Moullier et al., Nature Genet. 4: 154-159 (1993). "Administration" also encompasses the use of any other agent that is viral, infectious or chemical that enhances the delivery of a protein or nucleic acid to a cell.

Examples of hunter syndrome therapeutic forms that can be assessed using the mice and methods of the invention include iduronate-2-sulfatase (IDS) or IDS analogues, direct enzyme replacement, and in vivo or ex vivo gene therapy techniques In the use of IDS or IDS-analogues, myofiblot expression of IDS or IDS analogs, transplantation and vector-mediated gene delivery.

FIG. 7 is a graph showing glycosaminoglycan excretion in urine following administration of eduronate-2-sulfatase to micelles and controls of eduronate-2-sulfatase. As shown in FIG. 7, the urine GAG content of the knockout mice after the administration of eduronate-2-sulfatase was significantly decreased in comparison with the control group, and the urine GAG content in the urine was less than half after 4 weeks of administration. Through this, the IDG is administered to the knockout mice, thereby reducing the GAG content in the urine to a normal range.

FIG. 8 is a graph showing body weight change after administration of eduuronate-2-sulfatase 11 weeks after birth in eduuronate-2-sulfatase knockout mice and controls. As shown in FIG. 8, knockout mice and normal mice showed the same body weight gain at 16 weeks of age, 5 weeks after IDS was administered to the knockout mice after 11 weeks of age. However, in the knockout mouse control group not administered IDS, body weight after 16 weeks was only 80% of normal mice. It was found that by administering IDS to the iduronate-2-sulfatase knockout mice, the growth could be maintained the same as normal mice.

Extensive histopathology arising in mice of the present invention in the absence of eduronate-2-sulfatase in addition to the Hunter syndrome therapeutics test would make these mice useful for testing targeted delivery systems used for the treatment of other diseases and conditions. For example, if one wants to test a target system for delivery of a therapeutic agent to the brain, this target system is combined with an iduronate-2-sulfatase and administered to an IDS knockout mouse according to the invention. After a period of time mouse brain tissue was examined for pathology related to eduronate-2-sulfatase defects and eduronate-2-sulfatase activity. For example, brain tissue can be examined for abnormal lysosomal accumulation by microscopic observation or biochemically analyzed for eduronate-2-sulfatase activity. The absence of this pathology (or reduction relative to the control) indicates that the target system is effective in effectively delivering therapeutic agents to the brain, particularly in view of the observation that administration of IDS in IDS-/-mice results in increased enzyme activity in the brain. Investigation of other tissues for pathology related to eduronate-2-sulfatase defects provides an indication of the selectivity of the target system.

Because of the presence of the blood brain barrier and the many types of cells that make up the central nervous system, the investigation of the target system into the brain is a very significant application of the method of the present invention, but the method of the present invention may be applied to other types of cells. Thus, for example, target systems that wish to deliver therapeutic agents to kidney cells, liver cells, or other organs and tissues of the body can be investigated using the model systems and methods of the present invention.

The method of investigating the target system can be applied to any type of target system, including target systems with target-specific affinity labels, such as hormones or antibodies bound to therapeutic agents, and target systems that introduce expressible DNA into cells. Accordingly, the present invention is directed to viral expression vectors such as retroviral vectors, adenoviruses, adeno-associated viruses and herpes virus vectors, as well as herpes applicons custom-designed to deliver the iduronate-2-sulfatase gene or cDNA. Useful for testing.

The invention is also useful for testing lysosomal target systems by incorporating purified iduronate-2-sulfatase enzymes that can be combined with cationic lipids or other liposome components and administering the product to the model. Other target systems that can be investigated include systems for the delivery of raw DNA for use in genetic immunization and infection vectors.

The model mice of the present invention in each of these target systems allow investigation of the tissue specific distribution pattern and duration of efficacy of the target system. For example, the model mouse of the present invention provides a mechanism for studying the stability of therapeutic transduction.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Example 1 Preparation of Knockout Mouse

Alternative forms of targeting vectors were constructed from genomic fragments of mice and genes cloned into the vector Bluescript ^™ . An overview of the targeting vector is shown in FIG. 1 and the restriction enzyme cleavage site of the targeting vector is shown in FIG. 2. The cassette of the targeting vector was introduced into the Bluescript ^™ backbone. Nine passaged gene targets are introduced into R1 embryonic stem cells and grown on fibroblasts. 25 mg of the targeting vector DNA was added to embryonic stem cells diluted to a cell concentration of 7 × 10 ⁷ cells / ml and subjected to electric shock at 400 V / 25 uF. After electroporation, cells were selected for neomycin and Ganciclovir resistance on gelatin coated culture dishes.

Gene-targeted clones were 3.5 days p.c. of C57BL6 mice. 9-10 cells were injected into blastocysts. More specifically, 3.5 days old females after male and female mating were sacrificed by cervical dislocation and the uterus was extracted and injected with 1 ml syringe using 20 mM HEPES, 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol and DMEM. 1 ml of solution was perfused. Embryos were isolated from uterine tissue using microglass tubes under a dissecting microscope, and the isolated embryonic embryos were transferred onto 35 mL Petri dishes and the embryonic stem cell clones selected above were introduced into blastocysts of the blastocysts.

The clone-implanted blastocysts were transplanted into gestational surrogate mouse uterus to induce the development of chimeric mice, a type of heterotypic hybrids formed from embryonic stem cell clones and C57BL6 mice. As described above, the embryonic stem cells-implanted blastocysts were transplanted into the surrogate mouse uterus and cultured for about 19 days, thereby transforming the embryonic stem cell-derived transformed cells and the mouse blastocyst-derived cells to IDD +/- genotype. Genie produced chimeric mice.

Thereafter, the chimeric mice obtained above were mated with C57BL6 mice at least six times to prepare IDS + / + and IDS-/-mice at the F1 stage, and finally to prepare IDS-/-knockout mice in which all IDS genes were knocked out. It was.

Example 2 Physiological Characterization of IDS Knockout Mice

G) GAG content in mouse urine

At 38 weeks of age, the homozygous knockout mice lacking all of the IDS genes had a GAG content of 140-200 ㎍ / ml, and the heterozygous knockout mice lacking only one IDS gene. Was about 5-40 µg / ml. Therefore, the GAG content in the urine of homozygous knockout mice is about 5-20 times higher than that of heterozygous knockout mice. This is due to the absence of IDS enzymes in homozygous knockout mice, resulting in increased excretion in urine because GAG is not degraded.

3 is a graph showing the amount of glycosaminoglycan (GAG) excretion of the eduronate-2-sulfatase knockout mice. As shown in FIG. 3, the heterozygous transgenic mice at 16 weeks of age showed 30 to 35 μg / ml of GAG excretion, and the heterozygous transgenic mice at 38 weeks of age showed 15 to 20 μg / ml of GAG excretion. .

Ii) body weight of mouse organs

FIG. 4 is a graph showing the change in organ weight of eduronate-2-sulfatase knockout mice and controls. As shown in Figure 4, in the duronate-2-sulfatase knockout mice, liver growth was particularly slower than normal mice, and growth of organs such as the spleen and lungs was also delayed so that the weight of the organ was not sufficiently increased. Did.

Iii) liver and kidney tissue cells

Figure 5 is a photograph showing the results of histological analysis of the liver of the eduronate-2-sulfatase knockout mice and the control group. 6 is a photograph showing the results of histological analysis of the kidney of the eduronate-2-sulfatase knockout mouse and the control group. In IDS knockout mice, not only did cells develop sufficiently in liver and kidney tissues, but also significant lysosomal accumulation in tissues was observed.

Example 3 Effect of Incorporation of the Dururonate-2-Sulfatase Enzyme on IDS Knockout Mice

Ironon-2-sulfatase enzyme was added to IDS knockout mice and the change of GAG content in urine was measured. FIG. 7 is a graph showing glycosaminoglycan excretion in urine after administration of eduronate-2-sulfatase to control mice with eduronate-2-sulfatase knockout mice and controls. As shown in FIG. 7, the urine GAG content of the knockout mice after the administration of eduronate-2-sulfatase was significantly decreased compared to the control group, and the urine GAG content of the urine was less than half after 4 weeks after administration.

In more detail, the mice treated with IDS 0.15 mg / kg showed a marked decrease in GAG excretion after 4 weeks compared to the control group, and the mice treated with IDS 0.5 mg / kg showed a sharp decrease in GAG excretion after 3 weeks. Indicated. Through this, the IDG is administered to the knockout mice, thereby reducing the GAG content in the urine to a normal range.

FIG. 8 is a graph showing body weight change after administration of eduuronate-2-sulfatase 11 weeks after birth in eduuronate-2-sulfatase knockout mice and controls. As shown in FIG. 8, knockout mice and normal mice showed the same body weight gain at 16 weeks of age, 5 weeks after IDS was administered to the knockout mice after 11 weeks of age. However, in the knockout mouse control group not administered IDS, body weight after 16 weeks was only 80% of normal mice. It was found that by administering IDS to the iduronate-2-sulfatase knockout mice, the growth could be maintained the same as normal mice.

1 is a schematic of a targeting vector for the preparation of eduronate-2-sulfatase knockout mice.

Figure 2 is a detailed view for gene construction of the targeting vector for the preparation of eduronate-2-sulfatase knockout mice.

3 is a graph showing the amount of glycosaminoglycan (GAG) excretion of the eduronate-2-sulfatase knockout mice.

FIG. 4 is a graph showing the change in organ weight of eduronate-2-sulfatase knockout mice and controls.

Figure 5 is a photograph showing the results of histological analysis of the liver of the eduronate-2-sulfatase knockout mice and the control group.

Figure 6 is a photograph showing the results of histological analysis of the kidney of the eduronate-2-sulfatase knockout mice and the control group.

FIG. 7 is a graph showing glycosaminoglycan excretion in urine after administration of eduronate-2-sulfatase to control mice with eduronate-2-sulfatase knockout mice and controls.

FIG. 8 is a graph showing body weight change after administration of eduuronate-2-sulfatase 11 weeks after birth in eduuronate-2-sulfatase knockout mice and controls.

Claims (3)

In a method for measuring the pharmacological effect of eduronate-2-sulfatase as a treatment for Hunter syndrome using an animal model using a mouse knocked out of eduronate-2-sulfatase function, the pharmacological effect is Methods for Measuring Selected Pharmacological Effects as Animal Models in Direct Enzyme Replacement of Nate-2-Sulfatase, Expression of Eduronate-2-Sulfatase Myoblasts via Gene Therapy Technology, Cell Transplantation and Vector Mediated Gene Transfer According to claim 1, wherein the mouse is knocked out of the duronate-2-sulfatase function (i) left the allele region (12133 ~ 21031 bp) of wild type duronate-2-sulfatase gene (110079 bp) Preparing a targeting vector substituted with a gene cassette (9220 bp) consisting of an arm (2027 bp), PGK-neo (1800 bp) and a right arm (5393 bp); Ii) introducing the targeting vector into embryonic stem cells of a mouse; Iii) injecting the transformed embryonic stem cells into the blastocyst of the fertilized egg; Iii) obtaining a chimeric mouse via said transformed fertilized egg; Iii) crossing said chimeric mice with normal mice to obtain heterozygous mice; And iii) crossing the male and female of the heterozygous mouse to obtain a mouse knocked out of the duronate-2-sulfatase gene. The method of claim 2, wherein the targeting vector deletes exons 2 and exons 3 of the duronate-2-sulfatase gene.

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