JP4898126B2 - Transgenic non-human animal in which TROY signal is inhibited - Google Patents

Description

  The present invention relates to a transgenic non-human animal in which TROY signal is inhibited and uses thereof.

Cytokines and their receptors that act in the brain are important targets for the development of new drugs related to the brain. For example, the following molecules have been identified as cytokines that act in the brain.
Nutritional factor: nerve growth factorβ (P01138)
brain-derived neurotrophic factor (P23560)
neurotrophin-3 (P20783)
neurotrophin-4 (P34130)
neurotrophin-6α (P34132)
neurotrophin-6β (P34133)
neurotrophin-6γ (P34134) etc. Hormone-like cytokine: leptin (P41160)

  The present applicant has also isolated TROY as a TNF receptor-like molecule that is expressed in the brain (WO 00/49149). TROY has a signal sequence at the N-terminus and an amino acid region rich in hydrophobicity that is expected to be a transmembrane region in the middle. The structural features and amino acid sequence of TROY had homology with known proteins belonging to the TNF receptor superfamily. Therefore, TROY was considered to be a protein belonging to the TNF receptor superfamily (Beulter. B, and Huffel. C.V (1994) SCIENCE 264, 667-668 Unraveling function in the TNF ligand and receptor families).

  Furthermore, as a result of Northern blot analysis, the gene encoding mouse TROY protein was found to be strongly expressed in the brain and skin. Moreover, it showed high homology with Edar (Nature Genetics 1999 vol.22. 370-374, 366-369), which is thought to be involved in the development of hair follicles, sweat glands and teeth. Therefore, TROY protein is expected to be involved in the maintenance of brain function and skin development and homeostasis. Purification and cloning of factors related to brain function and skin development, as well as treatment of neurological diseases It was considered to be a useful tool for screening drug candidate compounds.

  In the brain capillaries, it is known that the substance permeability in the cell gap is strictly limited due to the structure in which the endothelial cells are closely connected. The close connection between the endothelial cells is called a tight junction. Proteins such as occuldin, claudin, or ZO-1 have been identified as molecules that constitute tight junctions. The existence of several subtypes in these proteins has also been revealed. The tissue that is formed by this tight junction and restricts the permeability of the substance from the blood to the brain is the blood brain barrier (BBB).

  It is becoming clear that the blood brain barrier has a system that actively transports various substances between blood vessels and the brain, while severely limiting the permeability of the substances. For example, at the blood-brain barrier, there is a system for transferring glucose necessary for survival of the brain from the blood into the brain and transferring lactic acid, which is a waste product, from the brain to the blood. Alternatively, ATP necessary for energy generation is thought to be stored as creatinine in the brain. The blood-brain barrier has also been shown to have a creatinine transporter (CRT) for transporting creatinine.

  The selective transport mechanism of various substances possessed by the blood-brain barrier and the function of limiting the permeation of substances are one of the important functions necessary for protecting and maintaining the function of the brain. Therefore, many brain diseases are accompanied by abnormal functions of the blood brain barrier. For example, it is known that cerebral infarction (particularly in the acute phase) and migraine are often accompanied by increased permeability of the blood brain barrier. In addition, diseases such as brain injury, traumatic cerebral edema, influenza encephalopathy, subarachnoid hemorrhage, AIDS encephalopathy, and multiple sclerosis are also known to have pathologies with increased blood-brain barrier permeability. . Therefore, it is considered that enhancement of the substance permeability of the blood brain barrier is involved in the pathogenesis of these diseases.

On the other hand, in order to predict the effect of a compound on a living body (human), an experiment is generally conducted in which the compound is administered to an experimental animal and the effect is observed. When observing the effects of the compound on the central nervous system through such experiments, the presence of the blood brain barrier causes the experiment to be difficult. To know the effects of a compound on the central nervous system, ie the brain, the compound needs to be sent into the brain. However, many substances cannot easily reach the brain due to the blood-brain barrier. In particular, most of the compounds administered orally or in the blood cannot act on the central nervous system due to the action of the blood brain barrier. Therefore, it is useful to artificially create a state in which the permeability of the blood brain barrier is enhanced in order to know the influence of the compound on the central nerve.
WO 00/49149 SCIENCE 264,667-668,1994 Nature Genetics 1999 vol.22. 370-374

  An object of the present invention is to provide a transgenic non-human animal in which the TROY signal is inhibited. It is another object of the present invention to provide a method for detecting an effect of regulating increased permeability of the blood brain barrier using a transgenic non-human animal in which TROY signal is inhibited. Alternatively, an object of the present invention is to provide a screening method for a therapeutic agent for a disease associated with increased permeability of the blood brain barrier. Another object of the present invention is to provide a model animal with enhanced blood-brain barrier permeability and its use for evaluating the screening method or the effect of the compound on the central nervous system.

  In order to clarify the function of TROY protein at the living body level, the present inventors have attempted to artificially create a state in which TROY signal is inhibited. TROY is a membrane protein with a transmembrane region. In addition, TLQE, which is a binding sequence of TNFR-associated factor (TRAF) 2, exists in the intracellular region of TROY. TRAF2 is a TNF receptor type II signaling molecule. Furthermore, the present inventors have confirmed that NF-kB activation is actually induced by mouse TROY. Thus, TROY signal includes the process of ligand binding to TROY and the resulting signal transduction to the intracellular domain. The present inventor has analyzed the effect of competitive inhibition on endogenous TROY on cells by soluble TROY. As a result, it was found that the permeability of the blood-brain barrier was enhanced in animals that inhibited the TROY signal.

As described above, the present inventor has clarified that inhibition of TROY signal leads to enhancement of substance permeability of the blood brain barrier. Further, the present invention was completed by finding that the animal having enhanced substance permeability of the blood-brain barrier obtained in this manner was used to evaluate the action of regulating the enhancement of substance permeability and to screen for substances having the action. Furthermore, the present invention has been completed by clarifying that the action of a compound on the central nerve can be evaluated using an animal having enhanced substance permeability of the blood brain barrier. That is, the present invention relates to the following transgenic non-human animals and uses thereof.
[1] A transgenic non-human animal in which the TROY signal is inhibited.
[2] The transgenic non-human animal according to [1], wherein the TROY signal is inhibited by either or both of the expression of soluble TROY protein and the suppression of endogenous TROY protein expression.
[3] The transgenic non-human animal according to [1], wherein the non-human animal is a rodent.
[4] The transgenic non-human animal according to [3], wherein the rodent is a mouse.
[5] The transgenic non-human animal according to [2], wherein the soluble TROY protein is any protein selected from the group consisting of the following (a) to (c):
(A) a protein comprising an amino acid sequence that lacks the membrane-binding domain of the TROY protein;
(B) a protein comprising an amino acid sequence lacking a region extending from the membrane binding domain to the C terminus of the TROY protein; and (c) an amino acid sequence lacking a region extending from the membrane binding domain to the C terminus of the TROY protein and TROY. A fusion protein having the amino acid sequence of the protein.
[6] A model animal having enhanced blood-brain barrier permeability, comprising a step of inhibiting the TROY signal by either or both of the expression of soluble TROY protein and the suppression of endogenous TROY protein expression Production method.
[7] The production of a model animal with enhanced blood-brain barrier permeability according to [6], wherein the step of inhibiting the TROY signal includes a step of producing a transgenic non-human animal that expresses soluble TROY protein. Method.
[8] A method for detecting an action of a test compound that regulates the enhancement of permeability of the blood brain barrier, comprising the following steps (1) to (3):
(1) A step of administering a test compound to a transgenic non-human animal whose blood-brain barrier permeability is enhanced by inhibition of the TROY signal;
(2) a step of administering to the transgenic non-human animal a tracer compound to be observed for transition to brain tissue before, after or simultaneously with the administration of the test compound; and (3) a blood brain barrier. The amount of tracer compound that permeated and transferred to the brain was measured. When the amount of tracer compound that transferred to the brain decreased compared to the control, the test compound inhibited the increase in blood-brain barrier permeability. Process to be detected.
[9] A screening method for a compound having the action of suppressing the enhancement of blood-brain barrier permeability, comprising the following steps (a) and (b);
(A) a step of detecting an effect of regulating an increase in the permeability of the blood brain barrier of the test compound by the method according to [8], and (b) an increase in the permeability of the blood brain barrier in the step (a). A step of selecting a test compound in which an effect of regulating the activity is detected.
[10] A screening method for a drug for improving a disease state in which blood-brain barrier permeability is enhanced, comprising the following steps (a) and (b):
(A) a step of detecting an effect of regulating an increase in the permeability of the blood brain barrier of the test compound by the method according to [8], and (b) an increase in the permeability of the blood brain barrier in the step (a). A step of selecting a test compound in which an effect of regulating the activity is detected.
[11] From the group consisting of acute phase of cerebral infarction, migraine, brain injury, traumatic brain edema, influenza encephalopathy, subarachnoid hemorrhage, AIDS encephalopathy, and multiple sclerosis [10] The method according to [10], which is any selected disease.
[12] A method for evaluating the influence of the test substance on the central nervous system, comprising the following steps (i) to (iii):
(I) a step of administering a test compound to a transgenic non-human animal in which the permeability of the blood brain barrier is enhanced by the inhibition of the TROY signal;
(Ii) observing changes in the central nervous system of the transgenic non-human animal, and (iii) providing a test compound to the central nervous system when a change in the central nervous system is observed compared to the control The process in which the impact is detected.
[13] A kit for detecting an effect of a test compound that regulates the enhancement of permeability of the blood brain barrier, comprising a transgenic non-human animal whose permeability of the blood brain barrier is enhanced by the inhibition of the TROY signal and a tracer compound.
[14] A test compound comprising a transgenic non-human animal whose blood brain barrier permeability is enhanced by inhibition of a TROY signal, and a device for monitoring changes in the central nervous system of the transgenic non-human animal Kit for detecting the effect on blood.

  According to the present invention, a transgenic non-human animal in which TROY signal is inhibited is provided. The transgenic non-human animal of the present invention has enhanced substance permeability at the blood brain barrier. Therefore, the transgenic non-human animal of the present invention is useful as a model animal of a disease state in which the substance permeability of the blood brain barrier is enhanced. The model animal based on the present invention can be used to screen for therapeutic agents for diseases associated with increased substance permeability of the blood brain barrier.

  The transgenic non-human animal with enhanced blood-brain barrier substance permeability provided by the present invention is also useful for evaluating the effects of substances on the central nervous system. In general, a substance administered to a living body cannot easily reach the brain due to a substance permeability control function of the blood brain barrier. Therefore, with the exception of intracerebral administration, in experimental animals that maintain normal blood-brain barrier function, the range of compounds that can evaluate the effects on peripheral nervous system by peripheral administration is limited. The transgenic animal of the present invention has enhanced blood-brain barrier permeability. Therefore, the possibility that the compound can reach the brain even when administered orally or in the blood is increased. This means that the influence of the compound on the central nerve can be evaluated with higher sensitivity.

The present invention provides a transgenic non-human animal in which the TROY signal is inhibited. In the present invention, the TROY signal refers to signal transmission via TROY. More specifically, for example, the following mechanism is included in the TROY signal in the present invention.
i) binding of signaling molecules to the extracellular region of TROY,
ii) activation of the intracellular region of TROY associated with the binding of signaling molecules, and
iii) Signal transduction from the intracellular region of TROY to a further downstream mediator In the present invention, the mechanism for inhibiting the TROY signal is not limited. For example, the TROY signals shown in i) and iii) can be inhibited using the following mechanisms, respectively. These mechanisms can be used alone or in combination.

i) Binding of signaling molecules to the extracellular region of TROY
TROY is a TNF receptor-like molecule. Therefore, a signal is transmitted into the cell by binding of a signal transmitter to the extracellular region. The series of processes can be inhibited by TROY-like molecules and signal transduction molecule antagonists. In the present invention, a TROY-like molecule refers to a molecule that binds to a signal transduction substance that binds to the extracellular region of TROY, but has no (or low) signal transduction action into the cell. Specifically, a soluble TROY that retains the ability to bind to a signaling substance can be shown as a TROY-like molecule.

In addition, substances that bind to signal transduction substances and inhibit the binding to TROY are also included in TROY-like molecules. Alternatively, there is a possibility that at least a part of signal transmission to the extracellular region of TROY can be inhibited by using an antagonist having a competitive inhibitory action on a signal transduction substance. An antagonist is a substance that has a binding activity to TROY but has a low or no signal transduction effect on TROY.
Troy has been reported to form a complex with Nogo-66 receptor and LINGO-1 and function as a receptor for myelin inhibitor (myelin-associated glycoprotein, oligodendrocyte myelinglycoprotein) (Neuron, 2005, vol. 45, 345 -351, Neuron, 2005, vol.45, 353-359). Therefore, signal transduction to TROY can be inhibited by a substance that can antagonize the binding between the two.

  By producing a transgenic animal that expresses substances necessary for these inhibition mechanisms, a transgenic non-human animal in which the TROY signal of the present invention is inhibited can be obtained. In the transgenic animal of the present invention, a gene that expresses a substance necessary for inhibiting the TROY signal may be homozygous or heterozygous.

ii) Activation of the intracellular region of TROY upon binding of signaling molecules
TROY has a function of signal transduction into cells. Therefore, TROY signal can also be inhibited by inhibiting this process. For example, signal transduction into cells can be inhibited by modification or deletion of the intracellular region of TROY. Specifically, signal transduction into cells can be effectively inhibited by modification or deletion of TROY2 binding sequence of TROY. Alternatively, the same inhibitory effect can be expected by substituting the intracellular region of TROY with the amino acid sequence of another protein. Furthermore, the TNF receptor superfamily, including TROY, forms a trimer and transmits signals. Therefore, TROY in which a region necessary for formation of a trimer on the cell membrane is modified is also effective in inhibiting signal transduction. Furthermore, the signal can also be inhibited by expressing a variant completely lacking the function of TROY or suppressing the expression of TROY.

  The transgenic animal of the present invention based on these inhibition mechanisms can be obtained by producing a transgenic animal that expresses modified TROY instead of endogenous TROY. In other words, the transgenic animal thus obtained is a transgenic animal in which the modified TROY that suppresses the signal transduction action is knocked in. Alternatively, a knockout animal in which the function of endogenous TROY or expression itself is suppressed can be prepared and used as the transgenic animal of the present invention. In the transgenic animal of the present invention, the gene expressing the modified TROY necessary for the inhibition of the TROY signal may be homozygous or heterozygous. Similarly, in a knockout animal of the endogenous TROY gene, the gene modification may be homozygous or heterozygous.

iii) Inhibition of signal transduction from the intracellular region of TROY to further downstream mediators Activated TROY further transmits signals to intracellular mediators. Therefore, TROY signal can also be inhibited by inhibiting this process. For example, TROY is thought to bind to TRAF2. Therefore, the TROY signal can also be inhibited by modifying the TROY binding domain of TRAF2. The transgenic animal of the present invention based on these inhibition mechanisms can be obtained by producing a transgenic animal that expresses modified TRAF2 instead of endogenous TRAF2. In other words, the transgenic animal thus obtained is a transgenic animal in which a mutant TRAF2 having a suppressed signal transduction activity is knocked in. In the transgenic animal of the present invention, the gene expressing the modified TROY necessary for the inhibition of the TROY signal may be homozygous or heterozygous.

Among the inhibition mechanisms of TROY signal exemplified in i) -iii) above, i) inhibition by expression of soluble TROY is preferable. In the present invention, soluble TROY refers to a mutant of TROY that maintains binding activity with a signal transduction substance in the extracellular region of TROY and is released to the outside of the cell. More specifically, TROY containing a secretion signal for secretion outside the cell and having a mutation or deletion in the cell membrane binding region is included in the soluble TROY in the present invention. The soluble TROY in the present invention includes, for example, the following modified TROY.
(A) a protein comprising an amino acid sequence that lacks the membrane-binding domain of the TROY protein;
(B) a protein comprising an amino acid sequence from which a region extending from the membrane-binding domain to the C-terminus of the TROY protein is deleted,
(C) a fusion protein having an amino acid sequence from which the region extending from the membrane-binding domain to the C-terminal of the TROY protein is deleted and an amino acid sequence of a protein other than TROY

The origin of the TROY protein in the present invention is not limited as long as the endogenous TROY signal can be inhibited in the animal expressing the soluble TROY. For example, it was confirmed that soluble TROY having the structure of the extracellular region of human TROY effectively suppresses TROY signal in mice. Therefore, when a mouse is used as a transgenic animal, a preferable soluble TROY is a TROY mutant containing an extracellular structure derived from mouse or human. The amino acid sequences of mouse and human TROY (and the base sequences encoding them) can be referred to by the following Accession Nos. In human TROY, positions 169-193 correspond to the membrane-binding domain, and the extracellular region corresponds to 30-168.
Mouse GenBank Acession No. BAB03267 (AB040432)
Human GenBank Acession No. AAQ89247 (AB040434)

Furthermore, the soluble TROY in the present invention contains a mutation in the extracellular region as long as the inhibitory action of the TROY signal, that is, the ability to bind to a substance that transmits the signal by binding to the extracellular region of TROY is maintained. be able to. Examples of mutants in the present invention include the following.
I. A protein that is functionally equivalent to a natural protein, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence of a natural TROY
II. A protein that is functionally equivalent to a protein consisting of a natural amino acid sequence, encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence encoding the natural amino acid sequence

  In the present invention, a functionally equivalent protein refers to a protein that maintains the ability to bind to a substance that transmits a signal to TROY. Substances that bind TROY and transmit signals are also called ligands. It can be known that a certain protein maintains the ability to bind to a ligand by actually confirming the binding to the ligand. TROY transmits signals into cells via NF-kB. Therefore, signal transmission can be detected using a reporter plasmid containing an NF-kB binding site. Examples of such reporter plasmids include kB-luc. The reporter plasmid kB-luc has five NF-kB binding sites upstream of the basic promoter dN55 (Cancer, No. 79, 800-804 (1988)) of the HTLV-1 promoter, and a luciferase gene downstream. In cells expressing a TROY mutant, if a reporter plasmid signal is observed in the presence of the ligand, it can be confirmed that the mutant maintains the ability to bind to the ligand. The extracellular region derived from the mutant maintaining the binding ability with the ligand can be used for the soluble TROY in the present invention.

As a method well known to those skilled in the art for isolating such a protein, a method for introducing a mutation into the protein is known. For example, those skilled in the art can prepare proteins functionally equivalent to these proteins by appropriately introducing mutations into the amino acid sequence of the natural Troy protein. The following papers can be referred to as site-directed mutagenesis methods for introducing mutations into amino acid sequences.
Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275,
-Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500,
-Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction.Nucleic Acids Res. 12, 9441-9456,
-Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods.Enzymol.154, 350-367,
-Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492)

  Amino acid mutations can also occur in nature. Thus, proteins having an amino acid sequence in which one or a plurality of amino acids are mutated in the amino acid sequence of the natural Troy protein and functionally equivalent to these proteins are also included in the protein of the present invention. In such mutants, the number of amino acids to be mutated is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids, and even more preferably within 3 amino acids.

In the present invention, the amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid residues that share structural features as shown below can generally be predicted to retain similar properties. Therefore, there is a high possibility that the function of the protein is maintained even if amino acid residues are substituted with each other. Such amino acid substitutions are called conservative substitutions.
Hydrophobic amino acids (A, I, L, M, F, P, W, Y, V)
Hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T)
Amino acids with aliphatic side chains (G, A, V, L, I, P)
Amino acids with hydroxyl-containing side chains (S, T, Y)
Amino acids with side chains containing sulfur atoms (C, M)
Amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q)
Amino acids with base-containing side chains (R, K, H)
Amino acids with aromatic-containing side chains (H, F, Y, W)

  As a method for isolating functionally equivalent proteins, a method using a hybridization technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) is available. Can be mentioned. That is, a person skilled in the art can isolate DNA having high homology with a DNA encoding a natural protein or a part thereof, and singly isolate a protein functionally equivalent to these proteins from the DNA. Separation is also usually possible. Proteins encoded by DNA isolated using such a hybridization technique are included in the functionally equivalent proteins of the present invention. Examples of such proteins include mammalian homologs.

  Hybridization for isolating DNA encoding a functionally equivalent protein can be performed, for example, under conditions of stringency of 10% formamide, 5xSSPE, 1x Denhardt's solution, and 1x salmon sperm DNA. More preferable conditions (more stringent conditions) are 25% formamide, 5xSSPE, 1x Denhardt solution, 1x salmon sperm DNA, and more preferable conditions (more stringent conditions) are 50% formamide, 5xSSPE. , 1x Denhardt's solution, 1x salmon sperm DNA conditions. However, a plurality of factors other than the above formamide concentration are conceivable as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. Moreover, it can also isolate using the gene amplification method which uses a part of DNA which codes natural type protein as a primer instead of hybridization. PCR can be used as a gene amplification method.

  Proteins encoded by DNA isolated by these hybridization techniques or gene amplification techniques usually have high homology in amino acid sequence with natural proteins. High homology is usually 70% or more homology, preferably 85% or more homology, and more preferably 95% or more homology. To determine protein homology, the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730) may be used.

  A preferred transgenic non-human animal in the present invention is, for example, a transgenic non-human animal in which the permeability of the blood ability barrier is enhanced by introducing DNA encoding a soluble TROY protein. TROY has been shown to be a molecule involved in signal transduction in neurons such as the brain. However, it is a novel finding revealed by the present invention that the permeability of the blood ability barrier is enhanced by inhibiting the signal transduction. In particular, it is not known that an animal with enhanced permeability of the blood brain barrier can be produced by the action of soluble TROY.

  That is, the present invention is a model in which the permeability of the blood-brain barrier is enhanced, including a step of inhibiting the TROY signal by either or both of the expression of a soluble TROY protein and the suppression of the expression of an endogenous TROY protein. The present invention relates to an animal production method. Alternatively, the present invention enhances the permeability of the blood-brain barrier of a non-human animal in which the TROY signal is inhibited by either or both of soluble TROY protein expression and endogenous TROY protein expression suppression. To use as a model animal. In the present invention, specifically, the step of inhibiting the TROY signal can be a step of producing a transgenic non-human animal that expresses a soluble TROY protein. A transgenic non-human animal that expresses a soluble TROY protein is produced by (1) introducing a vector that retains soluble TROY into an embryo, and (2) selecting an individual generated from the embryo. can do. A specific method for producing a transgenic non-human animal is as described below.

  In the present invention, the enhanced permeability of the blood brain barrier refers to a state in which the permeability of a substance is enhanced as compared with a normal state. The normal blood-brain barrier, in principle, allows only limited penetration of substances. Many of the artificially administered compounds usually do not penetrate the blood brain barrier. Therefore, in a normal animal, when a substance whose permeability cannot be confirmed permeates through the blood brain barrier of a transgenic non-human animal, it can be confirmed that the permeability is enhanced. Alternatively, it means a transgenic non-human animal in which T cell lineage differentiation or development is suppressed as compared to permeability in normal animals. It can also be confirmed that the permeability is enhanced when the permeability at the blood brain barrier of the transgenic non-human animal is increased. Methods for evaluating the permeability of the blood brain barrier are known.

  For example, the permeability of the blood brain barrier can be assessed by administering a traceable compound into the blood and following the transition to brain tissue. Traceable compounds that are administered into the blood are called “probes”. A dye or protein can be used as a probe for evaluating the permeability of the blood-brain barrier. More specifically, horseradish peroxidase (HRP), or blood proteins such as endogenous albumin and immunoglobulin can be used as probes. As the dye, Evans blue (Azovan blue) and the like are known as probes. Evans blue is sometimes used as a complex with albumin. Probes that have migrated to brain tissue can be detected by microscopic observation of the tissue. Alternatively, the level of translocation can be determined by detecting probes contained in brain homogenates.

  The “non-human animal” in the present invention means a vertebrate or invertebrate that does not include humans. Non-human animals suitable for artificially modifying gene expression using genetic modification techniques include non-human mammals and insects, but more preferably non-human mammals (eg, mice) And rodents such as rats, most preferably mice.

  Methods for producing transgenic animals are known. For example, transgenic animals can be obtained by the method described in Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980). Specifically, for example, DNA encoding soluble TROY is introduced into a totipotent cell of an animal, and this cell is generated into an individual. By selecting individuals from which the transgene has been incorporated into somatic cells and germ cells, the desired transgenic mice can be produced. Examples of totipotent cells into which genes are introduced include fertilized eggs and early embryos, and cultured cells such as ES cells having multipotency. More specifically, a transgenic animal can be produced by the method described in the Examples described later. A person skilled in the art can produce a transgenic animal in which expression of a desired gene is modified by appropriately modifying the above method.

  The above-mentioned “DNA encoding soluble TROY” is generally a recombinant gene construct (expression vector) linked to a promoter that can be expressed in the cells of an animal into which the DNA is to be introduced. The recombinant gene construct of the present invention can be constructed by inserting a DNA encoding the soluble TROY and a promoter upstream thereof into a vector that can be cloned using an appropriate host and cloning. .

  The promoter that can be used in the present invention is not particularly limited as long as it can be expressed in animal cells. In the present invention, soluble TROY may be secreted into the blood. Accordingly, as the promoter, not only a promoter that induces systemic expression but also a site-specific promoter can be used. Furthermore, an inducible promoter can be used. Specifically, viral promoters such as cytomegalovirus, retrovirus, polyoma virus, adenovirus, and simian virus 40 (SV40) can be used. For example, when the SV40 promoter is used, the above construct can be prepared according to the method of Mulligan et al. (Nature (1990) 277, 108). Additional expression control regions such as enhancers and terminators can also be added to the construct.

  As described above, the soluble TROY can also be expressed as a fusion protein with other proteins. For this purpose, a gene encoding another protein is arranged via an appropriate splicing donor sequence and an intron sequence. Alternatively, a construct can be constructed by fusing a gene encoding another protein to the TROY gene. Any protein other than TROY to be fused with TROY can be used. By fusing with a protein that improves retention in blood, the retention of soluble TROY can be expected. For example, immunoglobulin constant regions and proteins containing albumin are thought to contribute to the improvement of blood retention. Therefore, a fusion protein of the extracellular region of TROY with the Fc domain of immunoglobulin or albumin is preferable as soluble TROY in the present invention.

  The vector for introducing the construct into the animal is not particularly limited as long as the expression of the transgene can be induced in the animal body. Expression vectors well known to those skilled in the art can be used. Specifically, a pCAGGS vector (Gene 108 (1991) p193-200) can be used.

  A vector carrying the recombinant gene construct is sufficiently purified and used to produce a transgenic animal. Usually, a transgenic animal is produced by introducing the construct into an unfertilized egg, a fertilized egg, a sperm and a germ cell containing a progenitor cell thereof. As the cells into which the construct is introduced, those at the stage of embryogenesis in the development of non-human mammals, more specifically, at the stage of single cells or fertilized egg cells, usually those before the 8-cell stage are used. Known methods for introducing the construct include, for example, the calcium phosphate method, the electric pulse method, the lipofection method, the aggregation method, the microinjection method, the particle gun method, and the DEAE-dextran method. Furthermore, it is also possible to produce a transgenic animal by fusing the thus obtained transformed cells with the above-mentioned germ cells.

  The construct can be introduced into cells derived from any non-human animal capable of producing a transgenic animal. Specifically, cells such as mouse, rat, hamster, guinea pig, rabbit, goat, sheep, pig, cow, dog or cat can be used. In the case of a mouse, for example, a normal male mouse can be mated with a female mouse administered with an ovulation inducing agent, and a fertilized egg for introducing the construct can be collected. The collected mouse fertilized eggs are generally introduced with a construct by microinjection into the male pronucleus. The cells into which the construct has been introduced are cultured outside the body, and then the cells that have been successfully introduced are transplanted into the surrogate mother's fallopian tube, and a transgenic animal is born. The surrogate mother is usually a female that has been mated with a male whose vagina has been cut into a pseudopregnant state.

  After confirming that the DNA to be introduced is integrated, the born transgenic animal is mated with a normal animal for the birth of the F1 animal. In general, multiple copies of foreign DNA introduced as a construct are integrated in series in the same part of the genome. This is because the larger the number of incorporated copies, the more gene expression is expected and a clearer phenotype can be expected. In the somatic cell genome, it can be confirmed that the DNA to be introduced is integrated in the correct direction by PCR using a primer specific for the construct or Southern blotting using a specific probe.

  Among F1 animals born as a result of this mating, those that have foreign genes (DNA encoding soluble TROY) in somatic cells (heterozygotes) are those that have foreign genes (DNA encoding soluble TROY) in germ cells. ) Is a transgenic animal that can communicate. A homozygote that is an F2 animal can be obtained by selecting an F1 animal that retains a foreign gene (DNA encoding soluble TROY) in a somatic cell and using these as parents.

  The transgenic animal of the present invention may be an animal of any generation of the above-described transgenic animals as long as it expresses soluble TROY. For example, even a transgenic animal that holds heterologous DNA of soluble TROY heterozygously can be used as the transgenic animal of the present invention as long as soluble TROY is expressed. Or even if it is a transgenic chimera animal which does not have a foreign gene in a germ cell, as long as soluble type TROY is expressed, it is contained in a transgenic animal in which TROY signal in the present invention was inhibited. In addition, when the inhibition of TROY signal is realized by the action of an antagonist or the like, the target transgenic animal can be obtained by using DNA encoding the antagonist instead of soluble TROY.

Alternatively, by inhibiting the function and expression of endogenous TROY, a transgenic animal in which the TROY signal in the present invention is inhibited can be produced. Methods for inhibiting the function of endogenous genes are known.
In the present invention, the animal that is the target of inhibition of the function and expression of endogenous TROY is not particularly limited. Preferred are rodents such as mice, rats and hamsters. In particular, when considering use as an animal model, a mouse is preferable. In addition, the ES cell that is the target of modification of the TROY gene is not particularly limited. Specifically, rodents, particularly those derived from mice are preferred. ES cells can be obtained and maintained by well-known methods (see Doetschman et al. (1985) J. Embryol. Exp. Morphol. 87: 27-45).

  Here, “TROY gene expression was suppressed” means complete suppression (null allele) in which expression of both TROY gene pairs was suppressed, as well as failure in which expression of only one of the gene pairs was suppressed. Includes completely suppressed state. In the present invention, means for artificially suppressing the expression of the TROY gene include a method for deleting the TROY gene and a method for deleting the expression control region of the TROY gene. As a method for deleting the endogenous gene and the expression control region, gene targeting can be shown. Among them, a method of inactivating a gene by inserting a foreign gene into one or both of TROY gene pairs (knockout or knockin) is preferable. In addition, inhibition of gene function by RNA interference (RNAi) effect, dominant negative effect, antisense effect or the like can also be used to suppress the expression of TROY gene. The effect of controlling gene expression by RNAi is known (Nature Biothechnol. 2003, 21, 559-561).

  The expression control region of the TROY gene can be obtained from genomic DNA by a known method using the base sequence of TROY, for example. For example, the transcription start point can be specified by methods such as S1 mapping (cell engineering separate volume 8 new cell engineering protocol, Institute of Medical Science, University of Tokyo (1993) pages 362-374, Shujunsha). it can. In general, the expression control region of a gene is detected by screening a genomic DNA library using the 5 'terminal nucleotide sequence of the transcript as a probe. For the probe, a base sequence having a length of 15 to 100 bp, preferably 30 to 50 bp is used. Next, the transcriptional activity can be confirmed by shortening or fragmenting the polynucleotide obtained from the genomic DNA library and examining its expression ability using a reporter gene.

In addition to these experimental methods, the following programs for predicting expression control regions have also been developed. For the expression control regions predicted by these programs, the transcriptional activity can be actually confirmed for the fragmented base sequence using a reporter gene, as in the experimental method.
Program for predicting gene expression control region:
Neural Network (http://www.fruitfly.org./seq_tools/promoter.html); Reese et al. (1996) Biocomputing: Proceedings of the 1996 Pacific Symposium, Hunter and Klein ed., World Scientific Publishing Co.) etc. Program for predicting the minimum activity unit of expression control region of:
http://biosci.cbs.umn.edu./software/proscan/promoterscan.htm

  The genetically modified mouse in the present invention can be prepared, for example, as follows. First, DNA containing the exon part of the TROY gene is isolated from a mouse, an appropriate gene is inserted into this DNA fragment, and a targeting vector is constructed. As a gene to be inserted into the DNA fragment, for example, a marker gene for an antibiotic resistance gene (neomycin resistance gene or the like) can be used. When an antibiotic resistance gene is used as the marker gene, it can be confirmed that the vector has been introduced into the cell by culturing the cell in a medium containing the antibiotic.

  The targeting vector is integrated into the genomic DNA by insertion into the cell so as to prevent or prevent transcription of the native TROY gene. A vector is introduced into an ES cell line by an electroporation method, a microinjection method, a calcium phosphate method, or the like, and a cell line that has undergone homologous recombination is selected. Furthermore, in order to perform efficient selection of recombinant cells, cell lines that have undergone non-homologous recombination can be excluded by binding a thymidine kinase gene or the like to a targeting vector. In addition, a method is also known in which a diphtheria toxin A fragment (DT-A) gene or the like is bound to a targeting vector to eliminate cell lines that have undergone non-homologous recombination. These technologies. Any of them can be used for mutant cells or animals in the present invention. In addition, a homologous recombination assay can be performed by PCR and Southern blotting to efficiently obtain a cell line in which at least one of the TROY gene pairs is inactivated.

  The obtained ES cell line can be injected into a mouse blastocyst to obtain a chimeric mouse. In selection of cell lines that have undergone homologous recombination, there is a risk of gene disruption due to gene insertion other than at the site of homologous recombination. Therefore, it is preferable to produce a chimera using a plurality of clones. By mating this chimeric mouse, a mouse in which one of the TROY gene pair is inactivated can be obtained. Furthermore, by mating this mouse, a mouse in which both TROY gene pairs are inactivated can be obtained. In animals other than mice in which ES cells are established, TROY gene-modified cells and animals can be obtained by the same technique.

An ES cell line in which both TROY gene pairs are inactivated can also be obtained by the following method. That is, by culturing an ES cell line in which one of the gene pair is inactivated in a medium containing a high concentration of antibiotics, a cell line in which an antibiotic resistance marker is introduced into both gene pairs can be selected. . That is, an ES cell line in which both TROY gene pairs are inactivated can be obtained.
Alternatively, by selecting an ES cell line in which one of the gene pairs has been inactivated, introducing a targeting vector again into this cell line, and selecting a cell line that has undergone homologous recombination, the gene pair of the TROY gene can also be selected. ES cell lines in which both are inactivated can be obtained. When performing homologous recombination again, it is preferable to use different marker genes for insertion into the targeting vector for the first homologous recombination and the second homologous recombination. The transgenic animal in which the expression of the TROY gene thus suppressed is used as a transgenic animal in which the TROY signal is suppressed.

According to the present invention, a model animal with enhanced permeability of the blood brain barrier is provided. Therefore, the transgenic non-human animal of the present invention is useful for detecting an action that regulates the enhanced permeability of the blood brain barrier. That is, the present invention provides a method for detecting the action of a test compound that regulates the enhanced permeability of the blood brain barrier, comprising the following steps (1) to (3).
(1) A step of administering a test compound to a transgenic non-human animal whose blood-brain barrier permeability is enhanced by inhibition of the TROY signal;
(2) administering to the transgenic non-human animal a tracer compound to be observed for transition to brain tissue before, after, or simultaneously with the test compound, and (3) a blood brain barrier. The amount of tracer compound that permeated and transferred to the brain was measured. When the amount of tracer compound that transferred to the brain decreased compared to the control, the test compound inhibited the increase in blood-brain barrier permeability. Process to be detected.

If a test compound in which the action of suppressing the increase in blood-brain barrier permeability is detected by the method of the present invention is selected, the compound having the action can be screened. That is, the present invention provides a method for screening a compound having the action of suppressing the enhancement of blood brain barrier permeability, comprising the following steps (a) and (b).
(A) In the step of detecting the effect of regulating the permeability enhancement of the blood-brain barrier of the test compound by the method of the present invention comprising the steps (1) to (3), and (b) in the step (a) A step of selecting a test compound in which an effect of regulating increased permeability of the blood brain barrier is detected.

Increased permeability of the blood-brain barrier is one of the important pathologies of diseases such as acute cerebral infarction and migraine. Therefore, a compound having an action of regulating increased permeability of the blood brain barrier can be expected to be used as a therapeutic agent for a disease accompanied by increased permeability of the blood brain barrier. That is, the present invention relates to a method for screening a drug for improving a disease state in which blood-brain barrier permeability is enhanced, including the following steps (a) and (b).
(A) In the step of detecting the effect of regulating the permeability enhancement of the blood-brain barrier of the test compound by the method of the present invention comprising the steps (1) to (3), and (b) in the step (a) A step of selecting a test compound in which an effect of regulating increased permeability of the blood brain barrier is detected.

  In the present invention, a tracer compound is administered together with the test compound. In general, test compounds and tracers can be administered into peripheral blood. When the test compound can be administered other than in blood, the administration method can be appropriately selected. Specifically, oral, percutaneous, or airway administration can be selected. Similarly, for the tracer compound, an administration method can be selected depending on the properties of the administered compound.

  Examples of the test compound in the present invention include natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, and cell culture supernatants. , Fermented microorganism products, marine organism extracts, plant extracts and the like. If the test compound is a protein, a viral vector having a gene encoding the protein can be constructed, and the gene can be introduced into the transgenic non-human animal of the present invention using its infectivity.

  On the other hand, in the present invention, the tracer compound refers to any compound that can follow the transition to the brain tissue via the blood-brain barrier after administration to a living body. As the tracer compound for evaluating the permeability of the blood-brain barrier, the dyes and proteins exemplified above as probes can be used. That is, in a normal state, a compound in which transfer to the brain tissue via the blood brain barrier is restricted is used as a tracer compound in the present invention. Of these, compounds that are easy to detect are preferred as tracer compounds. In the present invention, the timing of administration of the tracer compound and the test compound is arbitrary. That is, the transgenic non-human animal can be administered with a tracer compound whose transition to brain tissue should be observed before, after, or simultaneously with the test compound.

  More specifically, horseradish peroxidase (HRP), or blood proteins such as endogenous albumin and immunoglobulin can be used as a tracer compound. As the coloring matter, Evans blue (Azovan blue) or the like can be used. Evans blue is sometimes used as a complex with albumin. Tracer compounds that have migrated to brain tissue can be detected by microscopic observation of the tissue. Alternatively, tracer compounds contained in brain homogenates can be detected to determine migration levels. When an endogenous substance such as albumin is used as the tracer compound, it is not necessary to administer an exogenous tracer compound. When the endogenous substance is used as the tracer compound, the transgenic non-human animal can be considered to have been previously administered with the tracer compound.

  In the present invention, the effect of regulating the permeability of a substance at the blood brain barrier can be evaluated by comparison with a control. For example, the level of transfer of the tracer compound to the brain in a transgenic animal that has not been administered the test compound can be used as a control. When the test compound is administered with a solvent such as physiological saline, the transition level when the solvent alone is administered can also be used as a control. Alternatively, the level of transfer of the tracer compound to the brain when a substance apparently having an action of regulating the permeability of a substance at the blood brain barrier is administered in advance can be used as a control. In this case, it is possible to find a substance having a greater regulating action than a certain regulating action.

  The elements necessary for the method of detecting the action of the present invention for regulating the permeability of a substance of the blood brain barrier or the screening method for a compound having the action based on this method can be combined in advance to form a kit. That is, the present invention relates to a kit for detecting an effect of a test compound that regulates the enhancement of permeability of the blood brain barrier, including a transgenic non-human animal whose permeability of the blood brain barrier is enhanced by inhibition of the TROY signal, and a tracer compound. I will provide a. The kit of the present invention can be used as a kit for carrying out a screening method for a compound having the action. The kit of the present invention can additionally be combined with a negative control composed of a compound that is clearly not having the desired action, or a positive control composed of a compound that has been confirmed in advance to have the desired action. . Furthermore, it is possible to combine a protocol describing the detection method and the protocol for carrying out the screening method.

  The compounds selected by the screening method of the present invention are useful for the regulation of increased substance permeability of the blood brain barrier. It is possible to treat a disease associated with an increased substance permeability of the blood brain barrier through regulation of the increased substance permeability of the blood brain barrier. Such diseases include acute cerebral infarction and migraine. That is, the present invention provides a drug for regulating enhancement of substance permeability of the blood-brain barrier, which contains a compound selected by the screening as an active ingredient.

It has been shown that an increase in the substance permeability of the blood brain barrier or the breakdown of the permeability control function of the blood brain barrier is an important disease state constituting the following diseases. Accordingly, the disease model animal provided with the present invention with enhanced blood-brain barrier substance permeability is useful as a model animal for the following diseases. Furthermore, the drug selected through the screening method using the model animal is useful as a therapeutic drug for the diseases shown below.
Acute phase of cerebral infarction: Neuropatol Pol. 1990; 28 (1-2): 1-17.
Migraine: Pathol Biol (Paris). 1992 Apr; 40 (4): 313-7.
Brain injury (traumatic brain edema): Anesteziol Reanimatol. 2002 Nov-Dec; (6): 17-9
Influenza encephalopathy: J Med Invest. 2003 Feb; 50 (1-2): 1-8.
Subarachnoid hemorrhage: Acta Neurochir (Wien). 1985; 77 (3-4): 110-32.
AIDS encephalopathy: Ann Neurol. 1992 Nov; 32 (5): 658-66.
Multiple sclerosis (MS): Mult Scler. 2003 Dec; 9 (6): 540-9.

  That is, the present invention provides a therapeutic agent for a disease associated with a disease state in which the substance permeability of the blood brain barrier is enhanced, containing the compound selected by the screening as an active ingredient. Alternatively, the present invention relates to a method for treating a disease associated with a disease state in which substance permeability of the blood brain barrier is enhanced, comprising a step of administering a compound selected by the screening. Furthermore, the present invention relates to the use of a compound selected by the screening method in the manufacture of a therapeutic agent for a disease associated with a disease state in which substance permeability of the blood brain barrier is enhanced. The present invention also relates to the use of a compound selected by the screening method in the treatment of a disease associated with a disease state in which the substance permeability of the blood brain barrier is enhanced.

  When the compound selected according to the present invention is used as a drug for humans or other animals, the compound of the present invention itself is formulated and administered by a known pharmaceutical method in addition to directly administering to the patient. You can also. For example, it can be orally administered as a sugar-coated tablet, capsule, elixir, or microcapsule as necessary. Alternatively, it can also be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It can be formulated by mixing in a unit dosage form as required for generally accepted pharmaceutical practice, in appropriate combination with a binder and the like. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

  Examples of additives that can be mixed in tablets and capsules include binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry are used. When the dispensing unit form is a capsule, the above material can further contain a liquid carrier such as fats and oils. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

  Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled in a suitable ampoule.

  For example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, percutaneous, or oral administration to patients are performed by methods known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. In addition, if the compound can be encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

  In the case of oral administration, the dose of the compound is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day for an adult (with a body weight of 60 kg). In general, the dose of the compound can be appropriately adjusted depending on the symptoms. When the compound is administered continuously, the dosage can be adjusted according to the therapeutic effect.

  When administered parenterally, the single dose is usually about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day for an adult (with a body weight of 60 kg), for example, in the form of an injection. About 0.1 to 10 mg can be administered by intravenous injection. In the case of other animals, an amount converted per 60 kg body weight or an amount converted per body surface area can be administered. The dose of the compound can be appropriately adjusted according to the administration subject, target organ, symptom, and administration method.

According to the present invention, it was revealed that inhibition of TROY signal enhances permeability of the blood brain barrier. In a transgenic non-human animal in a state where the permeability of the blood brain barrier is enhanced, a compound administered to the periphery reaches the brain more easily than in a state where the function of the blood brain barrier is normal. Such animals are useful for assessing the effects of compounds on the central nervous system. That is, this invention provides the method of evaluating the influence which it has on the central nervous system of a to-be-tested substance including the following process (i)-(iii).
(I) a step of administering a test compound to a transgenic non-human animal in which the permeability of the blood brain barrier is enhanced by the inhibition of the TROY signal;
(Ii) observing a change in the central nervous system of the transgenic non-human animal, and (iii) providing a test compound to the central nervous system when a change in the central nervous system is observed compared to the control The process in which the impact is detected.

In the present invention, as the test compound, any substance whose influence on the central nervous system should be evaluated can be used. For example, in drug development, evaluation of effects on the central nervous system is essential. According to the present invention, since the test compound easily reaches the brain tissue, the influence of the test compound on the central nervous system can be evaluated sensitively and rapidly. According to the present invention, it is possible to know various effects of a compound on the central nerve. For example, in drug development, an effect called central toxicity or neurotoxicity of a compound is important. These toxicities refer to a nerve center, a function center necessary for survival such as a respiratory center and a digestive center, or a disorder effect on a center for voluntary movement such as a motor center. The present invention is useful for detecting central toxicity or neurotoxicity of a compound.
In laboratory animals with normal blood brain barrier function, there is always the possibility that the test compound will not reach the brain. This means that compounds that do not cross the blood brain barrier cannot be evaluated for effects on the central nervous system. Alternatively, even if the test compound passes through the blood-brain barrier, if it takes time to pass or the level of the passing compound is insufficient, it is difficult to observe the influence on the central nerve sensitively. According to the method of the present invention, even such a compound can be rapidly and sensitively evaluated for its influence on the central nervous system.

  In the transgenic non-human animals to which the test compound is administered, changes in the central nervous system are observed after the administration. Any method capable of knowing changes in the central nervous system can be used in the present invention. In general, evaluation on the central nervous system is evaluated by observing changes in animal behavior. For example, as a method of evaluating central nervous system toxicity, a modified Irwin method (Samuel Irwin, Psychopharmacologia (Berl.), 13, p222-257 (1968)) or a functional observational evaluation method (Functional observational battery) for rats and mice : FOB) is widely used. These methods are methods for comprehensively evaluating changes in animal behavior and biological responses.

According to the present invention, it has been revealed that inhibition of the TROY signal results in increased permeability of the blood brain barrier. Based on this finding, it is possible to realize a method for administering a drug to the brain. That is, the present invention provides a drug delivery method to brain tissue, which includes the following steps.
(1) The step of inhibiting the TROY signal, and (2) The step of administering the drug to be delivered to the brain tissue In the present invention, as the step of inhibiting the TROY signal, for example, a step of administering soluble TROY is shown. Can do. That is, the drug can be delivered to the brain tissue by administering the soluble TROY and the drug to be delivered to the brain tissue. Or this invention provides the pharmaceutical composition for delivering a chemical | medical agent to the brain which mix | blended the following components.
(A) an inhibitor of TROY signal, and (b) a drug to be delivered to brain tissue

In the pharmaceutical composition of the present invention, the TRO signal inhibitor can specifically represent soluble TROY. In the method for delivering a drug to brain tissue of the present invention or the pharmaceutical composition for delivering a drug to the brain, the drug to be delivered to the brain tissue can be used for treatment and / or prevention of brain-related diseases. Any drug can be used. In the present invention, the permeability of the blood-brain barrier is enhanced by the inhibition of the TROY signal. As a result, even when a drug alone cannot be expected to be transferred to brain tissue, it can be rapidly delivered to the brain tissue. The present invention has realized a new approach for drug delivery to the brain (Drug Delivery).
Hereinafter, based on an Example, this invention is demonstrated more concretely.

[Example 1] Production of transgenic mice incorporating soluble TROY
In order to investigate the role of TROY in the central nervous system, transgenic mice in which soluble TROY was incorporated under the CAGG promoter were prepared. It is a transgenic mouse intended to suppress the action of TROY ligand by continuously expressing soluble TROY.

1) Construction of plasmid In order to produce a gene encoding a fusion protein of soluble TROY and Fc of human IgG1, a gene with a splice donor sequence added immediately after Thr at 168 was constructed (Fig. 1). ). The base sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.
Using the BamHI site (the underlined part), a gene fused with soluble TROY and Fc was constructed by inserting into the intron immediately before the exon encoding the hinge of the Fc gene of human IgG1. Hereinafter, the constructed fusion gene is referred to as TROY-Fc. The amino acid sequence of the fusion protein encoded by this gene (SEQ ID NO: 2) is as shown in FIG. 2 (the underlined portion is soluble TROY).
This was inserted into the EcoRI site of an animal cell expression vector pCAGGS (Gene 108 (1991) p193-200) to prepare pCAGGS-TROYFc (FIG. 3). This plasmid was used for the production of transgenic mice.

2) Gene introduction A DNA fragment for injection was prepared as follows. First, a pCAGGS vector (pCAG-TROYFc; FIG. 3) into which TROY-Fc was inserted was treated with SalI and PstI, and then a fragment (about 4 kb) containing the TROY gene was excised. This fragment was recovered by Gel Extraction Kit (QIAGEN) and diluted with PBS- to give 3 ng / μl to obtain a DNA fragment for injection.

  Mouse pronuclear eggs injected with DNA fragments were collected as follows. That is, superovulation treatment was performed by first intraperitoneally administering 5 i.u PMSG to C57BL / 6J female mice (Claire Japan), and further 48 hours later, intraperitoneally administering 5 i.u hCG. This female mouse was bred with a male mouse of the same strain. The next morning after mating, the oviduct of the mouse in which the plug was confirmed was perfused to recover the mouse pronuclear egg.

The DNA fragment for injection was injected into the pronuclear egg using a micromanipulator (the latest technology of gene targeting (Yodosha), 190-207, 2000). DNA fragments were injected into 335 C57BL / 6J embryos, and the next day, 281 embryos that had developed at the 2-cell stage were transferred to the oviducts of recipient females on the first day of pseudopregnancy at around 10 per side (per mouse About 20) were transplanted.
Receiving females who did not deliver pups even on the scheduled delivery date were subjected to caesarean section and fostered by foster parents. As a result, 65 offspring were obtained, 3 of which were transgenic mice introduced with the TROY-Fc gene.

3) Confirmation of transgene
At the age of 3 weeks, DNA was extracted from the tail of the mouse using an automatic nucleic acid separator (KURABO), and the transgene was confirmed by Southern blotting and PCR. The method for confirming the transgene by Southern blotting is as follows. That is, 15 μg of genomic DNA was treated with EcoRI, migrated, and transferred to a nylon membrane. An approximately 2 kb fragment containing TROY-Fc was used as a probe and hybridized with the transferred DNA. The transgene was confirmed based on the presence or absence of bands specific to 1.7 kb and 0.2 kb transgenic mice (FIG. 4).

4) Confirmation of expression of TROY-Fc in TROY-Fc transgenic mice Blood TROY was measured by enzyme immunoassay (ELISA) to confirm the expression of soluble TROY. In the mouse of line CAG2 in which the expression of soluble TROY was confirmed, it was confirmed that the detected soluble TROY was a fusion protein having human Fc by ELISA for human IgG Fc. Goat affinity purified antibody to human IgG Fc (Cappel) was diluted 5000 times with PBS and immobilized on OPAQUE plate, (Costar, 3922). After blocking with PBS containing 2% BSA, mouse serum appropriately diluted with this buffer was added. Further, diluted human IgG1 was similarly added as a standard for calculating the receptor concentration. After washing 3 times, Biotin-SP-conjugated Affinipure F (ab ') 2 Fragment Goat Anti-Human IgG, Fc g Fragment Specific, (Jackson Laboratory) was reacted, and after washing 3 times, Streptavidin-AP conjugate, ( Roche) was reacted, washed three times, and then developed with CDP-Star, (TROPIX) as a substrate, and measured with MicroLumat, LB96P, (EG & G Berthod) (FIG. 5). This line CAG2 was used for phenotypic analysis.

5) Typing of transgenic mice Transgenic mice were typed by Southern blot (Fig. 6a) and PCR analysis (Fig. 6b). Production of the transgenic mice was in accordance with Mendel's law, and no abnormalities were observed macroscopically. Observation with an optical microscope revealed no difference in brain size or structure between wild-type and transgenic mice.

[Example 2] Changes in perivascular glial cells in transgenic mice
TROY is expressed in astrocytes in addition to progenitor cells in the subventricular zone of the adult central nervous system (striatum, cerebral cortex, hippocampus, etc.) after birth. Furthermore, in the adult central nervous system, expression was also observed at the end of GFAP-positive perivascular astrocytes in the hippocampus (FIGS. 7A-C) and cerebral cortex (FIGS. 7E-G). Therefore, the astrocytes of transgenic mice were examined morphologically. When immunostaining was performed using an antibody against GFAP, which is an astrocyte marker, no obvious abnormality was observed in the parenchyma and at the end of the astrocytes in the transgenic mice (FIGS. 7D and H). Furthermore, the expression level of GFAP in the cerebral cortex and hippocampus was examined by Western blot analysis, but no difference was found between wild-type and transgenic mice.

However, as a result of observation with an electron microscope, abnormalities in the morphology of the astrocytes around the blood vessels were observed in the transgenic mice. In wild-type mice, glial endings completely covered the vessel wall, whereas in transgenic mice, glia endings swelled, vacuoles and electron-rich debris (FIGS. 8A and B). As shown in FIG. 8C, as a result of examining the ratio of blood vessels containing abnormal glial endings, it was found that in transgenic mice and wild-type mice, blood vessels with glial endings containing vacuoles were observed. The percentage of blood vessels with glial endings, including electron-rich debris, was as follows: Cell degeneration accompanied by reconstitution of intracellular skeleton such as actin filaments was considered.
Transgenic mouse Wild type vacuole 57.8 ± 8.3% 3.0 ± 1.3%
High electron density 62.9 ± 7.7% 4.5 ± 2.2%

[Example 3] Increased permeability of blood-brain barrier (BBB) in transgenic mice There are many reports that astrocytes around the cerebral blood vessels are involved in the structure and function of vascular endothelial cells. . These reports suggest that astrocytes play an important role in the development and maintenance of BBB. In order to examine the effect on endothelial cells due to the change of glia cells around blood vessels in transgenic mice, we examined the permeability of BBB using horseradish peroxidase (HRP) as a tracer.

  5 minutes after administration of HRP, significant leakage of HRP out of the blood vessels was observed in the hippocampus (FIG. 9B) and cerebral cortex of the transgenic mice with an optical microscope. On the other hand, no leakage of HRP was observed in the wild type (FIG. 9A). Even at the ultrastructural level, significant leakage of HRP out of the blood vessels was observed in the hippocampus of the transgenic mice (FIG. 9D, arrow). In wild-type mice, HRP reaction products were recognized as endothelial-containing vesicles (pinocytotic vesicles) (FIG. 9C, arrow), but no leakage into the subendothelium or parenchyma was observed.

  It is known that tight junctions of endothelial cells play a central role in the control of paracellular permeability. Therefore, the expression of tight junction proteins, ZO-1 and Occludin in transgenic mice was examined. As a result of Western blot analysis and immunostaining, no difference in expression level was observed between wild-type and transgenic mice. These results indicate that changes in perivascular glial cells in transgenic mice were accompanied by increased permeability of BBB, but not by decreases in tight junction proteins ZO-1 and Occludin. It was.

[Example 4] Reduction of SSeCKS expression in perivascular glial endings in transgenic mice Furthermore, in order to investigate the molecular mechanism of changes in perivascular glial endings in transgenic mice We investigated AQP4 and SSeCKS, which are expressed in, and are thought to be involved in BBB differentiation and maintenance.
Consistent with previous reports, SSeCKS and GFAP fluorescent double staining showed that SSeCKS was expressed at the end of GFAP-positive perivascular astrocytes (FIG. 10). Therefore, the expression of SSeCKS in the transgenic mice was examined by Western blot analysis. As shown in FIG. 11, the expression of SSeCKS in the hippocampus was significantly reduced in the transgenic mice. On the other hand, there was no difference in AQP4 expression between wild-type and transgenic mice. These results suggest that changes in the perivascular glial limbs in transgenic mice may be involved in the reduction of SSeCKS expression in the perivascular glial limbs.

  The transgenic non-human animal provided by the present invention is useful for detecting an action that regulates enhancement of substance permeability of the blood-brain barrier and screening for a substance having the action. A compound having such an action is useful for the treatment of a disease associated with increased substance permeability of the blood brain barrier. Such diseases include the acute phase of cerebral infarction, migraine, brain injury, traumatic brain edema, influenza encephalopathy, subarachnoid hemorrhage, AIDS encephalopathy, and multiple sclerosis.

  The present invention also provides a model animal in which the substance permeability of the blood brain barrier is enhanced. The model animal of the present invention is useful as a test animal for evaluating the effect of a compound on the central nervous system. Evaluation of the effects of compounds on the central nervous system using experimental animals is essential in drug development. By using the model animal of the present invention, it is possible to evaluate the influence of a substance that cannot permeate (is difficult to permeate) at the normal blood-brain barrier sensitively and quickly on the central nervous system.

It is a figure which shows the gene which added the splice donor sequence to the soluble TROY gene. A splice donor sequence was added immediately after the 168th Thr (underlined). The underlined portion indicates the BamHI site. It is a figure which shows the amino acid sequence of the protein encoded by the gene which fuse | melted soluble TROY and Fc. The underlined portion indicates soluble TROY. It is a figure which shows plasmid pCAGGS-TROYFc which inserted the gene which fuse | melted soluble TROY and Fc. It is a photograph which shows the result of transgene confirmation by Southern blotting. It is a figure which shows the measurement result of the blood TROYFc density | concentration of the transgenic mouse by ELISA. It is a photograph which shows the result of typing of a transgenic mouse. A shows the Southern blot, and B shows the result of PCR analysis. It is the result of the immuno-staining which shows the expression distribution of TROY in the brain of a normal mouse. 7A-C are micrographs showing the hippocampus, and FIGS. 7E-G are micrographs showing the cerebral cortex staining results. It is the photograph and figure which show the change of the glial cell around the blood vessel in a transgenic mouse. A and B show electron micrographs around the blood vessels of wild type mice and transgenic mice, respectively. C shows the proportion of blood vessels containing abnormal glial limbs in wild type and transgenic mice. The scale bar is 1 μm. It is a photograph which shows the observation result of BBB which used HRP as a tracer in a transgenic mouse. A and B show the observation results of BBB in a wild-type mouse and a transgenic mouse, respectively, using an optical microscope. C and D show the ultrastructure of BBB in wild type mice and transgenic mice, respectively. The scale bars in the figure are FIG. 9-B: 140 μm and FIG. 9-D: 1 μm (corresponding to 300 nm in the enlarged view), respectively. It is a photograph showing the result of fluorescent double staining of SSeCKS and GFAP around blood vessels in a transgenic mouse. It is the photograph and figure which show the analysis of the expression level by Western blotting of SSeCKS around the blood vessel of a transgenic mouse.

Claims (19)

A transgenic non-human animal model for screening a compound for the treatment of a pathological condition accompanied by an increase in permeability of the blood brain barrier, in which a TROY signal is inhibited. Pathology with increased blood-brain barrier permeability selected from the group consisting of acute phase of cerebral infarction, migraine, brain injury, traumatic brain edema, influenza encephalopathy, subarachnoid hemorrhage, AIDS encephalopathy, and multiple sclerosis The transgenic non-human animal model according to claim 1, which is any one of the diseases described above. The transgenic non-human animal model according to claim 1 or 2, wherein the TROY signal is inhibited by either or both of expression of soluble TROY protein and suppression of endogenous TROY protein expression. The transgenic non-human animal model according to any one of claims 1 to 3, wherein the non-human animal is a rodent. The transgenic non-human animal model according to claim 4, wherein the rodent is a mouse. The transgenic non-human animal model according to claim 3, wherein the soluble TROY protein is any protein selected from the group consisting of the following (a) to (c):
(A) a protein comprising an amino acid sequence that lacks the membrane-binding domain of the TROY protein;
(B) a protein consisting of an amino acid sequence lacking the region extending from the membrane-binding domain of the TROY protein to the C-terminus, and (c) an amino acid sequence lacking the region extending from the membrane-binding domain of the TROY protein to the C-terminus and TROY A fusion protein having the amino acid sequence of the protein. Compound for treatment of pathological condition accompanied by increased permeability of blood-brain barrier, comprising the step of inhibiting the expression of soluble TROY protein and / or suppressing the expression of endogenous TROY protein, or both A method for producing a transgenic non-human animal model for screening. Pathology with increased blood-brain barrier permeability selected from the group consisting of acute phase of cerebral infarction, migraine, brain injury, traumatic brain edema, influenza encephalopathy, subarachnoid hemorrhage, AIDS encephalopathy, and multiple sclerosis The method according to claim 7, which is any of the diseases described above. The method according to claim 7 or 8, wherein the step of inhibiting the TOY signal comprises a step of producing a transgenic non-human animal that expresses a soluble TROY protein. A method for detecting an action of a test compound that regulates the enhanced permeability of the blood brain barrier, comprising the following steps (1) to (3):
(1) A step of administering a test compound to a transgenic non-human animal whose blood-brain barrier permeability is enhanced by inhibition of the TROY signal;
(2) administering to the transgenic non-human animal a tracer compound to be observed for transition to brain tissue before, after, or simultaneously with the test compound, and (3) a blood brain barrier. The amount of tracer compound that permeated and transferred to the brain was measured. When the amount of tracer compound that transferred to the brain decreased compared to the control, the test compound inhibited the increase in blood-brain barrier permeability. Process to be detected. A screening method for a compound having an action of suppressing the enhancement of blood-brain barrier permeability, comprising the following steps (a) and (b);
(A) a step of detecting an effect of regulating an increase in permeability of the blood-brain barrier of a test compound by the method according to claim 10; and (b) an increase in permeability of the blood-brain barrier in step (a). A step of selecting a test compound in which an effect of regulating the activity is detected. A method for screening a drug for improving a disease state with enhanced blood-brain barrier permeability, comprising the following steps (a) and (b):
(A) a step of detecting an effect of regulating an increase in permeability of the blood-brain barrier of a test compound by the method according to claim 10; and (b) an increase in permeability of the blood-brain barrier in step (a). A step of selecting a test compound in which an effect of regulating the activity is detected. The condition with enhanced blood-brain barrier permeability was selected from the group consisting of acute phase of cerebral infarction, migraine, brain injury, traumatic brain edema, influenza encephalopathy, subarachnoid hemorrhage, AIDS encephalopathy, and multiple sclerosis The method according to claim 12, which is any disease. A method of using a transgenic non-human animal in which TROY signal is inhibited as a disease state model animal accompanied by enhanced permeability of the blood brain barrier. Pathology with increased blood-brain barrier permeability selected from the group consisting of acute phase of cerebral infarction, migraine, brain injury, traumatic brain edema, influenza encephalopathy, subarachnoid hemorrhage, AIDS encephalopathy, and multiple sclerosis 15. The method of claim 14, wherein the disease is any of the diseases identified. 16. The transgenic non-human animal is an animal in which the TROY signal is inhibited by either or both of expression of soluble TROY protein and suppression of endogenous TROY protein expression. the method of. The method according to any one of claims 14 to 16, wherein the non-human animal is a rodent. The method according to claim 17, wherein the rodent is a mouse. The method according to claim 16, wherein the soluble TROY protein is any protein selected from the group consisting of the following (a) to (c):
(A) a protein comprising an amino acid sequence that lacks the membrane-binding domain of the TROY protein;
(B) a protein consisting of an amino acid sequence lacking the region extending from the membrane-binding domain of the TROY protein to the C-terminus, and (c) an amino acid sequence lacking the region extending from the membrane-binding domain of the TROY protein to the C-terminus and TROY A fusion protein having the amino acid sequence of the protein.