Animal model of disease exhibiting excessive intravascular coagulation symptoms, and method for using same

Abstract

[Problem] To prepare an animal model of a disease exhibiting excessive intravascular coagulation symptoms, such as "disseminated intravascular coagulation (DIC)" or "multiple organ failure". [Solution] An animal model of a disease exhibiting excessive intravascular coagulation symptoms, said model animal being characterised in that the expression or function of CUL3 protein is suppressed in a manner specific to vascular endothelial cells; and a method for producing said animal model.

Description

Animal models of diseases exhibiting excessive intravascular coagulation and methods for their use

[0001] This invention relates to animal models and methods for utilizing disease models that are associated with excessive "intravascular coagulation (thrombosis)" and have a high risk of death, such as "DIC (disseminated intravascular coagulation syndrome)," or "multiple organ failure" caused by a lack of oxygen and nutrients to tissues due to thrombosis.

[0002] (What is DIC?) "DIC (Disseminated Intravascular Coagulation)" is also called "disseminated intravascular coagulation," and it is a disease in which the blood coagulation reaction, which should normally occur only at the site of bleeding, occurs in a disorderly manner in the blood vessels throughout the body. Because countless tiny blood clots (microthrombi) form in the blood vessels throughout the body, in the case of small blood vessels, the vessels become blocked and blood flow is obstructed, causing damage to organs such as the kidneys and lungs that cannot receive the necessary oxygen and nutrients, thus posing a serious threat to life. In addition, as the body tries to dissolve the formed blood clots, the activity of plasmin increases, resulting in the following symptoms (Non-Patent Literature 1).

[0003] (1) Platelets and coagulation factors that make up blood clots are consumed in large quantities and become deficient, resulting in a decrease in the ability to stop bleeding in the event of an injury. (2) Antithrombin, a coagulation control factor, is also consumed in large quantities during the blood coagulation process and becomes deficient, which conversely leads to further progression of the coagulation reaction and makes blood clots more likely to form. (3) Plasmin, which has a fibrin-degrading effect, exerts its thrombolytic ability excessively, resulting in the dissolution of blood clots that are actually necessary to stop bleeding, thus increasing the tendency to bleed.

[0004] Thus, because "blood coagulation" and "thrombolytic (fibrinolysis)" occur simultaneously and in a disordered manner, and clinically, seemingly contradictory symptoms such as "thrombosis" and the resulting "multiple organ failure" and "bleeding symptoms" coexist, "DIC" is considered a very difficult disease to treat.

[0005] This is because two types of approaches are needed: "improving the tendency to thrombotic or removing thrombi" and "improving the tendency to bleed or stopping bleeding." Furthermore, given that the true cause is currently unknown, the difficulty lies in the fact that only symptomatic treatment can be given for these symptoms that occur simultaneously in multiple locations.

[0006] Furthermore, because DIC is a disease with a poor prognosis and a high mortality rate, early diagnosis and treatment are considered extremely important, but currently there is no cure.

[0007] One reason for this is the lack of "DIC" animal models that can be used for drug screening and other purposes.

[0008] (Relationship between CUL3 and blood vessels) Meanwhile, in recent years, research has progressed on the ubiquitin system as one of the protein degradation systems in the body, and the enzymes involved have been identified as "ubiquitin-activating enzyme (E1)", "ubiquitin-conjugating enzyme (E2)", and "ubiquitin ligase (E3)".

[0009] Furthermore, E3, which acts in the final stage of the ubiquitin system, is known to be a complex composed of multiple components that have the following various roles.

[0010] 1) The main components of E3 (Cullin (CUL) proteins such as CUL1, CUL2, and CUL3): These components possess ligase activity and also serve as a platform for connecting other components. E3, including these main components, may be expressed as follows: CUL1-RING E3 ubiquitin ligase (CRL1) CUL2-RING E3 ubiquitin ligase (CRL2) CUL3-RING E3 ubiquitin ligase (CRL3)

[0011] Furthermore, RING stands for Really Interesting New Gene, and refers to Rbx1, etc., as described in 3) below. 2) Adapter site: This is the site that recognizes the substrate (the protein to be degraded), and by changing this site, diversity of the target protein is achieved. 3) Other sites (Rbx1, Skp1, etc.)

[0012] As described above, CUL proteins are known to regulate various biological functions in vivo by being responsible for the degradation systems of various proteins in vivo. For example, the present inventors have confirmed that "administration of CUL3 siRNA reagent" (transient knockdown suppression) to mouse eyeballs (vitreous body) inhibits the lumen-forming ability (angiogenic ability) of vascular endothelial cells (Patent Document 1).

[0013] In addition, in vitro experiments conducted by the present inventors have shown that "when the CUL3 gene is knocked down by siRNA in cultured human umbilical vein endothelial cells (HUVEC), the expression level of "VE-cadherin", which is involved in adhesion between vascular endothelial cells, decreases, and vascular structures tend to become unstable" (Non-Patent Document 2).

[0014] However, since various repair systems exist in vivo for both "angiogenesis inhibition" and "inhibition of adhesion between vascular endothelial cells", it has been difficult to predict that these processes would immediately progress to excessive "thrombus formation" as observed in conditions such as "DIC", and this does not suggest the model animals of the present invention for conditions such as DIC achieved through regulation of CUL3.

[0015] In particular, many aspects of the ubiquitin system involving CUL3 remain unclear as described below, and there were concerns that regulation of CUL3 expression would exert excessively large effects on the living body, which forced researchers to be cautious regarding in vivo suppression of CUL3, especially constitutive suppression (knockout).

[0016] - There are multiple E3 adapters, and it is considered that many adapters remain unelucidated, so the scope of the influence of CUL3 has been unknown. - It is not guaranteed that there is only one degradation substrate protein (polyubiquitination target protein) corresponding to one adapter, and even if all adapters were elucidated, the scope of the influence of CUL3 would still not be determined. - For knockout, which is a constitutive inactivation, the greater the influence of the target gene in vivo, the more necessary it is to take into account the risk that the model animal will die before various verifications are carried out.

[0017] In fact, when CUL3 was knocked out at the stage of mouse "fertilized eggs", there was a fact that even an individual did not develop at all.

[0018] Japanese Patent No. 5665213

[0019] DIC (disseminated intravascular coagulation) (Japan Blood Products Organization, http: / / www.ketsukyo.or.jp / disease / dic / dic.html) Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function (Tomohisa Sakaue et al., Cancer Science 108 (2017) 208-215)

[0020] The present inventors have carefully investigated the influence of CUL3 on vascular-related symptoms through various approaches. As a result, they unexpectedly found that by restricting CUL3 "only to vascular endothelial cells" at "any time point after ontogenesis",: X) symptoms such as "DIC" and "multiple organ failure" exhibit excessive "Thrombus formation" simultaneously with "Bleeding"; and Y) the mechanism of action causing X) is based on the temporal and site-specific suppression of the expression and function of "CUL3 protein", thereby arriving at the present invention. The object of the present invention is to produce a model animal of severe diseases (lethal diseases) such as "DIC" or "multiple organ failure", which have been difficult to produce before, exhibiting "excessive intravascular coagulation symptoms" and almost leading to "death" in a short period of time.

[0021] The above object is achieved by the following first to seventeenth inventions.

[0022] <First Invention> A model animal for a disease exhibiting excessive intravascular coagulation symptoms, wherein the expression or function of CUL3 protein is suppressed specifically in vascular endothelial cells.

[0023] <Second Invention> The disease model animal according to the first invention, wherein the suppression means is gene knockout.

[0024] <Third Invention> This is a disease model animal according to the first invention, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is disseminated intravascular coagulation (DIC) or multiple organ failure.

[0025] <Fourth Invention> An animal for creating a disease model exhibiting excessive intravascular coagulation symptoms, characterized by incorporating a system that can induce the suppression of CUL3 protein expression or function in a time-specific and vascular endothelial cell-specific manner.

[0026] <Fifth Invention> The fourth invention is an animal for creating a disease model, characterized in that the inhibitory induction system is a genetically modified system using a recombinase.

[0027] <Sixth Invention> The animal for creating the disease model described in the fifth invention is characterized in that the recombinase is a Cre enzyme.

[0028] <Seventh Invention> This is an animal for creating a disease model as described in the fourth invention, characterized by using a promoter that is specifically expressed in vascular endothelial cells.

[0029] <Eighth Invention> The disease model animal described in the seventh invention is characterized in that the promoter specifically expressed in vascular endothelial cells is the promoter of the VE cadherin gene (Cdh5).

[0030] <Ninth Invention> This is an animal for creating a disease model as described in the fourth invention, characterized in that the time-specific induction is induced by the administration of an estrogen receptor antagonist corresponding to a mutant estrogen receptor.

[0031] <Tenth Invention> This is an animal for creating a disease model according to the ninth invention, characterized in that the estrogen receptor antagonist is tamoxifen.

[0032] <The Eleventh Invention> This is an animal for creating a disease model according to the Fourth Invention, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is DIC or multiple organ failure.

[0033] <Twelfth Invention> A method for producing an animal for creating a disease model exhibiting excessive intravascular coagulation symptoms, characterized by having the step of i) below.

[0034] i) The process of mating the animals A) and B) below.

[0035] A) Animals into which a time-specific inducible recombinase gene and a vascular endothelial cell-specific expression promoter gene have been incorporated. B) Animals in which at least a portion of the CUL3 gene has been genetically modified so that it is sandwiched between recombinase recognition sequences.

[0036] <The thirteenth invention> A method for producing a disease model animal according to the first invention, characterized by having steps i) and ii) below.

[0037] i) A step of mating the animals A) and B) below. ii) A step of inducing time-specific induction in the animals obtained in i).

[0038] A) Animals into which a time-specific inducible recombinase gene and a vascular endothelial cell-specific expression promoter gene have been incorporated. B) Animals in which at least a portion of the CUL3 gene has been genetically modified so that it is sandwiched between recombinase recognition sequences.

[0039] <Invention No. 14> A screening method for agents for the prevention and / or treatment of diseases exhibiting excessive intravascular coagulation symptoms, characterized by having the following steps (1) to (3).

[0040] (1) A step of administering a candidate substance to a disease model animal described in the first invention or to an animal for creating a disease model described in the fourth invention. (2) A step of inducing time-specific induction if the animal administered in (1) is an animal for creating a disease model. (3) A step of comparing the phenotype of each animal before and after administration of the candidate substance, or between the unadministered group and the administered group.

[0041] <Fifteenth Invention> A screening method according to the fourteenth invention, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is DIC or multiple organ failure.

[0042] <Invention XVII> A preventive and / or therapeutic agent for diseases exhibiting excessive intravascular coagulation symptoms, characterized by containing the CUL3 protein and / or its gene.

[0043] <Invention 17> The preventive and / or therapeutic agent described in Invention 16, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is DIC or multiple organ failure.

[0044] [Explanation of Terms and Abbreviations] The terms and abbreviations used in this invention have the following meanings.

[0045] E3: Ubiquitin ligase CUL3: Cullin3 Vascular Endothelial Cells (sometimes abbreviated as VEC or EC) ECKO: Knockout in Vascular Endothelial Cells CUL3 ECKO: Knockout of CUL3 in vascular endothelial cells VE Cadherin: Vascular Endothelial-Cadherin Cdh5: May indicate the Cadherin-5 (VE Cadherin) gene or its promoter. Estrogen Receptor Tamoxifen-inducible mutant (ERT, Tamoxifen-inducible mutant estrogen receptor (gene) ERT2: Second-generation ERT (protein or its gene) Cre enzyme: Cre recombinase (Cyclization recombination enzyme) loxP sequence: locus of X-over P1 sequence (sequence recognized by Cre enzyme) Flip enzyme: Flip recombinase (may be written as Flippase, FLP, etc.) FRT sequence: Flippase Recognition Target (sequence recognized by Flip enzyme) Int enzyme: Int recombinase (may be written as Integrase.) att sequence: attachment site, sequence recognized by Int enzyme CreERT: Cre enzyme (protein or its gene) + ERT (protein or its gene) CreERT2: Cre enzyme (protein or its gene) + ERT2 (protein or its gene) Cre-ERT2 system: system combining Cre-loxP system and ERT2 receptor Cdh5CreERT2: VE cadherin promoter gene + Cre enzyme gene + ERT2 gene Floxed CUL3 (CUL3 flox / flox): sequence in which all or part of the CUL3 gene is flanked by loxP sequences TMX: tamoxifen EGFP: Enhanced Green Fluorescent Protein HE staining: hematoxylin-eosin staining PAS staining: Periodic Acid-Schiff staining PTAH staining: Phosphotungstic Acid Hematoxylin staining (phosphotungstic acid hematoxylin staining) MSB staining: Martius Scarlet Blue staining Fibrin thrombus: fibrin thrombus mAb: monoclonal antibody pAb: polyclonal antibody HRP: horseradish peroxidase HUVEC: human umbilical vein endothelial cells.

[0046] By using the "disease model animals" and "disease model animals" of the present invention for diseases exhibiting excessive intravascular coagulation symptoms such as "DIC" or "multiple organ failure," it is possible to accelerate the development of "preventive and / or therapeutic agents" for these diseases, which have been difficult to develop until now.

[0047] It is a diagram showing an example of a scheme for producing the "animal for preparing a disease model" (Example 1) and the "disease model animal" (Example 2) of the present invention, which were produced using the Cre-ERT2 system. It is a diagram showing an example of a scheme for producing the "animal for preparing a disease model" (Example 3) and the "disease model animal" (Example 4) of the present invention, which were produced by using the Cre-ERT2 system and further introducing a reporter gene. It is a diagram showing a specific example of a scheme for producing the "disease model animal" of the present invention from the "animal for preparing a disease model" of the present invention. CUL3 with vascular endothelial cell-specific knockout of the CUL3 gene ECKO It is an immunofluorescence staining image showing that the expression level of CUL3 protein is indeed reduced at the expression site of CD31 protein (vascular endothelial cell adhesion molecule, indicating the position of blood vessels) in the intestinal tract of mice. It is a diagram showing HE-stained images of tissue sections from each part of the digestive tract (stomach, duodenum, jejunum, terminal ileum, rectum). CUL3 ECKO It is a diagram showing the state of "bleeding" from the terminal ileum in mice. CUL3 ECKO It is a diagram showing the number (proportion) of mice among CUL3 mice that developed "bleeding" from the terminal ileum. CUL3 ECKO It is a volcano plot of the results of gene expression analysis (RNAseq) in the terminal ileum of CUL3 mice, showing that the expression levels of "inflammatory factors" (TNF, TGFβ1, AGT, IL6), which are precursors of "bleeding", were clearly increased. CUL3 ECKO It is a diagram showing HE staining and GFP immunostaining images of tissue sections of brain, lung, heart, liver, intestine, and kidney of (ROSA26EGFP) CUL3 mice. CUL3 ECKO It is a diagram showing high-magnification images of HE staining and GFP immunostaining in the kidney of (ROSA26EGFP) CUL3 mice. CUL3 ECKO It is a diagram showing the thickening of the renal (glomerular) basement membrane and the state of fibrin deposition (thrombus formation) in the glomerulus of CUL3 mice. CUL3 ECKOThis figure shows the number of thrombi formed in the kidneys (glomeruli) of mice (average value for 4 mice in each group). This figure shows the time course of intestinal tissue observed by HE staining, corresponding to the number of days after TMX injection. This figure shows the time course of kidney tissue observed by PTAH staining and MSB staining, corresponding to the number of days after TMX injection. This figure shows the results of counting the number of glomeruli containing fibrin thrombi in kidney tissue, corresponding to the number of days after TMX injection. CUL3 ECKO This figure shows the results of measuring various parameters in the blood of mice. Survival rate graph: CUL3 ECKO This graph shows that all mice died 11 days after TMX injection. Body temperature decrease graph: CUL3 ECKO This figure shows that the body temperature of mice rapidly decreased starting 11 days after TMX injection. CUL3 ECKO This figure shows immunohistochemical images of various tissues (brain, liver, kidney, lung, and heart) of (ROSA26EGFP) mice, along with the results of vascular density measurements.

[0048] The present invention will be described in detail below.

[0049] [Animal model of the present invention] The "animal model of a disease exhibiting excessive intravascular coagulation symptoms" of the present invention is characterized in that the expression or function of "CUL3 protein" is suppressed specifically in vascular endothelial cells.

[0050] (Diseases exhibiting excessive intravascular coagulation) "Excessive" means beyond the amount necessary for survival, etc. This is because a minimum amount of intravascular coagulation is necessary to stop bleeding, etc. Examples of "diseases exhibiting excessive intravascular coagulation" include "DIC" and "multiple organ failure," which are serious diseases (fatal diseases) in which the supply of oxygen and nutrients to various tissues is disrupted as a result of "excessive intravascular coagulation," or "thrombus formation," and the risk of death is high.

[0051] Furthermore, the causes of "thrombus formation" mainly include "damage to the blood vessel wall," "stagnation of blood flow," and "changes in blood coagulation properties." However, considering the reports of in vitro tests by the inventors mentioned above (Non-Patent Literature 2), it is thought that in the "disease model animal" of the present invention, the cause is mainly "damage to the blood vessel wall," that is, an excessive decrease in the connectivity between vascular endothelial cells (increased vascular permeability), which results in thrombi that should normally form outside the blood vessels advancing into the blood vessels.

[0052] As mentioned above, there is also the finding that "knocking down CUL3 in HUVEC cultured cells reduces the expression level of VE cadherin, a vascular endothelial cell adhesion factor" (Non-Patent Literature 2).

[0053] Furthermore, in addition to confirming "thrombus formation" itself, a major characteristic of these diseases is an extreme deficiency of platelets due to "excessive intravascular coagulation." Changes in "hematological parameters" such as "platelet count (PLT)," "platelet criterion (PCT)," and "mean platelet volume (MPV)," which will be discussed later, are also used as one of the diagnostic indicators.

[0054] (Suppression of vascular endothelial cell-specific expression or function) The "suppression of vascular endothelial cell-specific expression or function of CUL3 protein" can be achieved, as detailed in the "Method for producing animals for disease model production" of the present invention described later, for example, when employing a "recombination system using recombinase" as a suppression means, by selecting a vascular endothelial cell-specific expression "promoter" as the expression promoter for the recombinase gene, as described later in the "Method for producing animals for disease model production of the present invention".

[0055] (Inhibition Methods) The means of inhibiting the expression or function of the CUL3 protein are not particularly limited. These include "knockout (KO)" which completely suppresses the expression of the CUL3 protein, "knockdown (KD)" which transiently suppresses expression, and methods that suppress the function of the CUL3 protein (in whole or in part) after protein expression. However, "knockout (KO)" is preferred for reliable suppression of expression.

[0056] Furthermore, since suppressing the expression or function of the CUL3 protein carries a very high risk of inhibiting individual development itself at the fertilized egg stage, it is preferable to use a method that can specifically suppress it in vascular endothelial cells after the development of the animal individual, but the method is not limited to any particular specific method.

[0057] Methods for knockout include removing all or part of the target gene through homologous recombination or genome editing.

[0058] Specifically, this can be carried out by using "recombinase-based genetic engineering systems," such as the "Cre-loxP system" described later, or "RNA-induced site-directed genome editing systems," such as the CRISPR-Cas9 system, either individually or in appropriate combinations.

[0059] A "recombinase-based genetic recombination system" is a system that removes a region sandwiched between two "recombinase-recognition sequences" through homologous recombination by the recombinase.

[0060] Examples of combinations of "recombinase" and "recombinase recognition sequence" include the following, but the Cre-loxP system is preferred because it is widely used and has a proven track record.

[0061] "Cre enzyme" - "loxP sequence" "Flip enzyme" - "FRT sequence" "Int enzyme" - "att sequence"

[0062] KD methods include, for example, using factors that suppress the expression of the target gene, such as antisense RNA, siRNA, and shRNA.

[0063] Methods for suppressing (all or part) the function of the CUL3 protein after protein expression include methods using antibodies, etc.

[0064] Examples of animals that can be used include those listed in (a) to (g) below, but rodents, particularly mice, as in (a) are preferred because they have a long track record of use and are easy to raise and manage. Specifically, in creating disease models exhibiting intravascular coagulation symptoms as in the present invention, the C57BL / 6 strain, DBA / 2, etc., which are widely used in testing and research on vascular diseases, are preferred, among which the C57BL / 6 strain is preferred because it is easy to introduce genes and is genetically stable, but the invention is not limited to these.

[0065] (a) Rodents such as mice, rats, and guinea pigs (b) Chickens (c) Rabbits (d) Cattle (e) Sheep (f) Monkeys (g) Donkeys

[0066] (Preparation Method) The "disease model animal" of the present invention can be produced by introducing the system that can perform the suppression described above, but the method described in the "method for producing a disease model animal" of the present invention, which will be described later, is preferred because it can reliably suppress the expression of the CUL3 gene and can establish a stable strain of disease model mice.

[0067] (Applications) The "disease model animals" of the present invention are useful for studying the pathogenesis of the above-mentioned diseases, which have been difficult to treat until now, and for developing "preventive and / or therapeutic agents" for the above-mentioned diseases.

[0068] [Animals for creating disease models of the present invention] The "animals for creating disease models exhibiting excessive intravascular coagulation symptoms" of the present invention are characterized by incorporating a system that can induce the suppression of CUL3 protein expression or function in a time-specific and vascular endothelial cell-specific manner.

[0069] (Time-Specific Suppression) "Time-specific suppression" means suppressing the expression or function of a specific protein at any given time. Specific methods include administering estrogen receptor antagonists (such as TMX) to mutant estrogen receptors (such as ERT2), as detailed in the "Method for Producing Animals for Disease Model Production" of the present invention, described later.

[0070] (Endothelial cell-specific suppression) "Endothelial cell-specific suppression" means suppressing the expression or function of a specific protein only in endothelial cells. Specific methods include using a promoter that is specifically expressed in endothelial cells as the promoter of the enzyme used in the suppression induction system described below, as detailed in the "Method for creating disease models of animals" of the present invention.

[0071] Examples of "systems capable of inducing the suppression of CUL3 protein expression or function" include "recombinase-based genetic engineering systems" as described in "Method for Producing Animals for Disease Model Production of the Present Invention" below, such as the "Cre-loxP (Cre-ERT2) system," but are not limited to these. For example, "RNA-induced site-directed genome editing systems" such as the "CRISPR-Cas9 system" shown below can also be used.

[0072] (Use of RNA-Induced Site-Specific Genome Editing) By using an "RNA-induced site-specific genome editing system" such as CRISPR-Cas9 at any time after the development of an animal, it is possible to suppress (knock out) the expression of the CUL3 gene by directly editing (destroying) it. The specific method of using an "RNA-induced site-specific genome editing system" will be explained below using "CRISPR-Cas9" as a representative example.

[0073] Examples of methods for incorporating the "CRISPR-Cas9 system" after individual development include the following:

[0074] Introduction to specific tissues or cells using various vectors such as "viral vectors" and "plasmid vectors" Introduction to specific tissues or cells by "injection" Introduction to specific tissues or cells by "electroporation" Introduction to specific tissues or cells by "lipofection"

[0075] The success or failure of the introduction can be confirmed by standard methods such as PCR and sequencing.

[0076] Furthermore, in order to utilize the above-mentioned "CRISPR-Cas9 system" specifically for vascular endothelial cells, as described above, this can be done by selecting a vascular endothelial cell-specific expression "promoter" for the "Cas9 protein (nuclease)" responsible for cleaving the CUL3 gene in this system, similar to the one described in the "Method for Creating Animals for Disease Model Production of the Present Invention" below.

[0077] It should be noted that "Cas9" is merely an example of a nuclease representative of the system, and other "Cas proteins (CRISPR-associated proteins)" such as "dCas9," "nCas9," "Cas12," and "Cas13" can also be used.

[0078] Furthermore, since it is not necessary to introduce a "system" for each individual target animal, and once a strain of the introduced animal is established, the system can be activated at any time simply by injecting TMX, etc., a "genetic recombination system using recombinase" is preferable, and among these, the "Cre-loxP (Cre-ERT2) system" is preferred due to its extensive track record.

[0079] On the other hand, RNA-induced site-directed genome editing systems are preferred because they do not necessarily require the mating and selection processes necessary to establish a lineage into which the target system has been introduced, and they also offer high efficiency in introducing foreign genes. Among these, the CRISPR-Cas9 system is preferred due to its extensive track record.

[0080] Examples of "diseases exhibiting excessive intravascular coagulation symptoms" include serious diseases (fatal diseases) such as "DIC" or "multiple organ failure" accompanied by excessive thrombus formation, as described in the description of the "disease model animal" of the present invention.

[0081] (Manufacturing Method) More specific methods for manufacturing the "animal for disease model production" of the present invention include, but are not limited to, the methods described in the "method for manufacturing the animal for disease model production" of the present invention, which will be described later.

[0082] (Applications) The "animal for disease model production" of the present invention is used to produce the "animal for disease model production" of the present invention described above more quickly and reliably.

[0083] [Method for producing animals for creating disease models of the present invention] The "method for producing animals for creating disease models exhibiting excessive intravascular coagulation symptoms" of the present invention is characterized by having the following step i).

[0084] i) The process of mating the animals A) and B) below.

[0085] A) Animals into which a time-specific inducible recombinase gene and a vascular endothelial cell-specific expression promoter gene have been incorporated. B) Animals in which at least a portion of the CUL3 gene has been genetically modified so that it is sandwiched between recombinase recognition sequences.

[0086] First, let's describe the animals A and B that will be used for mating.

[0087] (Method for creating animal A) A "time-specific induction recombinase gene" is, for example, a combination of a "recombinase gene" and a "receptor gene for a substance that can time-specifically induce the translocation of recombinase into the nucleus."

[0088] Having this gene allows the recombinase to move into the nucleus only in the presence of the substance in question, thus enabling time-specific induction of recombinase activity.

[0089] Estrogen, a type of hormone, is known as a substance that can induce the translocation of recombinase into the nucleus in a time-specific manner. However, since estrogen is naturally present in cells, strict time-specific induction is difficult. Therefore, it is preferable to use TMX or other substances that are not present in the body as the substance that induces the nuclear translocation of recombinase, and as the receptor, a "TMX-inducible mutant estrogen receptor" that does not react to estrogen but reacts to TMX, i.e., ERT or ERT2 (second generation), which has been mutated to react to TMX.

[0090] While recombinases include the Cre enzyme, Flip enzyme, and Int enzyme mentioned above, the Cre enzyme is preferred because it is widely used and has a proven track record.

[0091] Therefore, preferred "time-specific inducible recombinase genes" include "CreERT" and "CreERT2," but "CreERT2" is more preferred among them due to its high sensitivity to TMX.

[0092] Examples of "vascular endothelial cell-specific expression promoter genes" include the promoter gene for Cdh5 (VE cadherin), the promoter gene for PECAM-1 (CD31), and the promoter gene for eNOS (endothelial nitric oxide synthase). However, the promoter gene for Cdh5 (which may be simply referred to as "Cdh5" in the following examples) is preferred because it exhibits the highest stability of expression in vascular endothelial cells.

[0093] Animal A can be produced by introducing the "Cdh5 CreERT2 gene," which contains both the "time-specific inducible recombinase gene" and the "vascular endothelial cell-specific expression promoter gene" mentioned above, into the embryonic stem cells (ES cells) of the target animal according to a standard method such as "homologous recombination," and then repeating selection and mating as appropriate.

[0094] Specifically, when using Cre enzyme as the recombinase, it can be done, for example, as follows.

[0095] Construction of targeting vectors by homologous recombination: A targeting vector is constructed containing homologous sequences (homologic arms) corresponding to the target gene on both sides of the "Cdh5 CreERT2 gene".

[0096] Introduction into ES cells: The "targeting vector" is introduced into the embryonic stem cells (ES cells) of the target animal using methods such as electroporation or lipofection.

[0097] Selection of recombinant cells: ES cells that have successfully undergone homologous recombination are selected using antibiotic resistance markers, PCR, etc.

[0098] Chimeric mouse creation: Selected ES cells are introduced into fertilized eggs of the target animal using conventional methods to create pseudo-embryos (chimeric mice).

[0099] Mating and Selection: Chimeric mice are bred, and individuals possessing the desired "Cdh5 CreERT2 gene" are selected.

[0100] Homozygotes can be produced by mating heterozygotes with each other.

[0101] Furthermore, more genetically stable homozygotes can be produced by mating homozygotes with each other.

[0102] Generally, homozygous individuals are preferred as introduced organisms because they exhibit stable genetic modification. However, in the case of Cre enzymes, heterozygous individuals may be preferred to reduce the risk of nonspecific homologous recombination.

[0103] As mentioned above, animal A can be produced by conventional methods of genetic engineering, but for example, the "Cdh5CreERT2" mouse (C57BL / 6-Tg [Cdh5-cre / ERT2]) can also be obtained from Taconic Biosciences, Ximbio, etc.

[0104] (Method for creating animal B) In order to reliably suppress the expression or function of the CUL3 protein, it is sufficient to surround most of the CUL3 gene with a recombinase recognition sequence, but at least a portion of the region may suffice.

[0105] While theoretically, enclosing the entire CUL3 gene with recombinase recognition sequences would reliably inhibit its expression, in order to increase the efficiency of recombinase inhibition, it may be better to select only the minimum necessary region and shorten the length between the recombinase recognition sequences.

[0106] The phrase "at least a portion of the CUL3 gene" flanked by two recombinase recognition sequences refers to an area where the function of CUL3 can be reliably lost by a genetic recombination system using recombinase. For example, a preferred example would be "exons 4-7" ("Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence", MOLECULAR AND CELLULAR BIOLOGY, May 2007, p. 3651-3666), which are known to be necessary for the expression of the CUL3 protein.

[0107] Furthermore, "at least a portion of the CUL3 gene" may be at least one of the following domains, which are highly useful for the function of CUL3(E3).

[0108] E3 substrate binding domain (e.g., BTB protein) RING finger domain (RING binding domain) NEDD8 binding domain E2 binding domain

[0109] Animal B can be created by introducing "at least a portion of the CUL3 gene" flanked by two recombinase recognition sequences into the target animal using the same method as for animal A.

[0110] While the introduced animals may be heterozygous, homozygous animals are generally preferred because they have undergone stable genetic modification.

[0111] As mentioned above, animal B can be produced by conventional methods of genetic engineering, but for example, a "Floxed CUL3" mouse, in which the "exon 4-7 region" of CUL3 is flanked by a "loxP sequence," can also be obtained from The Jackson Laboratory, etc.

[0112] Next, we will explain how to carry out step i).

[0113] (Mating) Mating of animals A) and B) can be carried out by selecting individuals in which genes from A) and B) have been appropriately introduced into one or both of their two chromosomes, according to conventional methods. However, in order to produce a genetically stable population and ensure that the animals have the necessary properties as "animals for producing disease models" of the present invention, it is preferable to further mat the resulting mice to establish a stable lineage.

[0114] [Method for producing disease model animals of the present invention] The "method for producing disease model animals" of the present invention is characterized by comprising the steps i) and ii) below.

[0115] i) The process of mating the animals A) and B) below.

[0116] ii) A process to induce time-specific induction in the animals obtained in i).

[0117] A) Animals into which a time-specific inducible recombinase gene and a vascular endothelial cell-specific expression promoter gene have been incorporated. B) Animals in which at least a portion of the CUL3 gene has been genetically modified so that it is sandwiched between recombinase recognition sequences.

[0118] Here, the methods for creating each animal in A) and B), and the method for carrying out step i), are as described in the "Method for Creating Animals for Disease Model Creation" above.

[0119] Next, we will explain how to carry out step ii).

[0120] Method for carrying out step ii) "Time-specific induction" can be carried out by administering a "recombinase nuclear transport inducer" that can bind to the "mutant estrogen receptor" derived from the "time-specific induction type recombinase gene" possessed by animal A, that is, an "estrogen receptor antagonist" such as TMX mentioned above.

[0121] While the following methods are available for administering TMX and similar drugs, intraperitoneal administration is preferred because it minimizes the reduction in effectiveness due to metabolism (decomposition) in the bloodstream.

[0122] Intraperitoneal injection: A method of injecting TMX, etc., into the abdominal cavity. Since it is mainly absorbed through the peritoneum and then gradually released into the bloodstream, the breakdown of TMX can be suppressed.

[0123] Oral administration: This method involves mixing TMX, etc., into the animal's drinking water or feed. It has the advantages of being simple and easy to administer over a long period of time.

[0124] Intravenous injection: A method of directly injecting TMX, etc., into a vein. This allows for rapid distribution throughout the body.

[0125] [Screening method for preventive and / or therapeutic agents of the present invention] The "screening method for preventive and / or therapeutic agents for diseases exhibiting excessive intravascular coagulation symptoms" of the present invention is characterized by having the following steps (1) to (3).

[0126] (1) A step of administering a candidate substance to the "disease model animal" of the present invention or the "animal for creating a disease model" of the present invention. (2) A step of inducing time-specific induction if the animal administered in (1) is the "animal for creating a disease model". (3) A step of comparing the phenotype of each animal before and after administration of the candidate substance, or between the unadministered group and the administered group.

[0127] The following describes each of the steps (1) through (3).

[0128] The method of administering the candidate substance in step (1) varies depending on the type and properties of the candidate substance and cannot be broadly limited. Examples include intraperitoneal injection, oral administration, and intravenous injection as described above, but intravenous injection is preferred because it allows for rapid distribution throughout the body.

[0129] Furthermore, while step (1) does not necessarily need to be performed before step (2), in the case of screening for "preventive agents," it may be preferable to use "animals for creating disease models" rather than "animal disease models" and administer the candidate substance before performing time-specific induction in step (2). This is because the "animal disease models" of the present invention exhibit an extremely rapid onset of excessive intravascular coagulation symptoms, and prevention may not be possible after suppression induction with TMX, etc.

[0130] Step (2) The method for "time-specific induction" is as described in the "Method for creating disease model animals" above. Note that this step (2) may be performed before step (1), as described above.

[0131] Step (3) "Phenotype comparison" The "phenotypes" used in this step may include those exemplified below (3-a) to (3-k), either individually or in combination thereof, but it is preferable to have at least two types. Among these, the presence or absence of "thrombus (fibrin)" in (3-a) and / or the number (ratio) thereof, and the mortality rate in (3-k), are considered the most important screening points because they are characteristic phenotypes in the above-mentioned diseases.

[0132] The main problem with "DIC" and "multiple organ failure" is that they have a poor prognosis and a high risk of death, making it urgent to improve mortality rates.

[0133] Furthermore, comparisons of phenotypes before and after administration of the candidate substance, or between the non-administered group and the administered group, can be performed using conventional methods.

[0134] (3-a) Presence or absence of thrombi in the tissues and organs of the administered animals and / or number (ratio) of thrombi (3-b) Presence or degree of bleeding in the tissues and organs of the administered animals (3-c) Presence or degree of inflammation in the tissues and organs of the administered animals (3-d) Concentration of the following blood parameters in the blood of the administered animals (3-e) Expression level of vascular endothelial adhesion molecules in the tissues and organs of the administered animals (3-f) Body temperature of the administered animals (3-g) Body weight of the administered animals (3-h) Expression of the HIF-1α (hypoxia-inducible factor-1α) gene in the tissues and organs of the administered animals (3-i) Vascular density in the tissues and organs of the administered animals (3-j) Pleural effusion volume of the administered animals (3-k) Mortality rate of the administered animals

[0135] The following provides further explanation regarding some of the above phenotypes.

[0136] (3-c) The presence or absence of inflammation can be determined by the expression levels of the following "inflammatory factors" (TNF, TGFβ1, AGT, IL6, Ccl2, Cd14, etc.).

[0137] (3-d) Examples of blood parameters include the following: • White blood cell (WBC) count • Red blood cell (RBC) count • Hemoglobin (HGB) • Hematocrit (HCT) • Mean corpuscular volume (MCV) • Mean corpuscular hemoglobin (MCH) • Mean corpuscular hemoglobin concentration (MCHC) • Platelet count (PLT) • Red blood cell distribution width (RDW) • Platelet criterion (PCT) • Mean platelet volume (MPV) • Platelet distribution width (PDW)

[0138] By using the above blood flow parameters individually or in combination, screenings such as the following become possible.

[0139] Since a decrease in RBC and HCT suggests anemia or a tendency toward major bleeding, if the decrease in these values ​​is clearly suppressed by the administration of a candidate substance, it can be selected as a candidate for prophylactic and / or therapeutic agents.

[0140] A significant decrease in PLT and PCT also suggests a state in which major bleeding or chronic bleeding is occurring or at high risk; therefore, if the decrease in these values ​​is clearly suppressed by the administration of a candidate substance, it can be selected as a candidate for prophylactic and / or therapeutic agents.

[0141] Since an increase in MPV indicates the rapid loss of platelets and the subsequent production of new, larger-than-normal platelets, if the increase in MPV is clearly suppressed by the administration of a candidate substance, it can be selected as a candidate for prophylactic and / or therapeutic agents.

[0142] (3-e) Examples of "vascular endothelial cell adhesion factors" include VE cadherins listed below.

[0143] VE-cadherin (Vascular Endothelial-Cadherin): VE-cadherins expressed on the surface of individual vascular endothelial cells (VECs) effectively bind to each other in response to the support provided by F-actin expressed within the VECs, thereby maintaining the stability of the vascular structure. It has been observed that the expression level of this VE-cadherin protein decreases when the vascular wall is damaged.

[0144] Therefore, if the decrease in VE-cadherin expression is clearly suppressed after administration of the candidate substance compared to before administration, or in the administered group compared to the non-administered group, it can be selected as a candidate for "preventive and / or therapeutic agent."

[0145] (3-h) Since HIF-1α (hypoxia-inducible factor-1α) is known to be expressed to resolve hypoxia, if the increase in these values ​​is clearly suppressed after administration of a candidate substance, it can be selected as a candidate for "preventive and / or therapeutic agent." This is because in "DIC" and "multiple organ failure," excessive thrombi are generated due to a breakdown in the control of vascular permeability, and as a result, oxygen does not reach tissues and organs, often leading to a hypoxic state.

[0146] (3-i) If the decrease in vascular density in the tissues and organs of the administered animals is clearly suppressed after administration of the candidate substance, it can be selected as a candidate for "preventive and / or therapeutic agent." This is because in "DIC" and "multiple organ failure," excessive thrombosis occurs due to a breakdown in the control of vascular permeability, and as a result, the number of blood vessels in the tissues and organs is often extremely reduced.

[0147] (3-j) If the excessive retention of pleural fluid in the administered animals, exceeding normal levels, is clearly reduced after administration of the candidate substance, it can be selected as a candidate for "preventive and / or therapeutic agent." This is because the excessive increase in pleural fluid is thought to be caused by hypercoagulation in "DIC" and "multiple organ failure," which leads to insufficient oxygen and nutrient supply to the lung tissue, damage to the pulmonary blood vessel walls, and leakage of plasma components into the pulmonary interstitium (pleura).

[0148] Examples of "diseases exhibiting excessive intravascular coagulation symptoms" include serious diseases (fatal diseases) such as "DIC" or "multiple organ failure" accompanied by excessive thrombus formation, as described in the description of the "disease model animal" of the present invention.

[0149] [Preventive and / or therapeutic agent of the present invention] The "preventive and / or therapeutic agent for diseases exhibiting excessive intravascular coagulation symptoms" of the present invention is characterized by containing the CUL3 protein and / or its gene.

[0150] The disease symptoms of the "disease model animals" of the present invention are clearly attributable to the suppression of CUL3 protein expression or function, as demonstrated by various test examples described later.

[0151] Examples of "diseases exhibiting excessive intravascular coagulation symptoms" include serious diseases (fatal diseases) such as "DIC" or "multiple organ failure" accompanied by excessive thrombus formation, as described in the description of the "disease model animal" of the present invention.

[0152] [Example 1, Comparative Example 1: Mice for creating disease models of the present invention (before TMX administration)] CUL3 ECKO We created a user mouse and a control mouse.

[0153] As shown in Figure 1-A, from among the mice obtained in "Step 3" by appropriately crossing the two mouse lines A) and B) below, a tamoxifen-inducible EC-specific CUL3 knockout mouse (Cdh5CreERT2: Floxed CUL3) with a C57BL / 6 genetic background was selected and used as the "animal for disease model creation" of the present invention (Example 1).

[0154] Similarly, from the mice in "Step 3," we selected "Floxed CUL3 mice" that did not possess the Cre gene and used them as control mice (Comparative Example 1).

[0155] Figure 1-A is just one example of a crossbreeding method, and is not necessarily the only method.

[0156] A) Cdh5CreERT2 mice: The Cdh5CreERT2 mice (C57BL / 6-Tg [Cdh5-cre / ERT2]) were provided by Keio University.

[0157] B) Floxed CUL3 (CUL3 flox / wt) mouse: A mouse into which the Floxed CUL3 gene has been introduced (Cul3 tm1Jdsr The mouse ( / J) was purchased from The Jackson Laboratory (Bar Harbor, ME, USA; Stock No. 028349). In this mouse, exons 4-7 of CUL3 were sandwiched between two loxP sequences.

[0158] (Results) As described above, by introducing a time-specific and site-specific knockout mechanism, it became possible to create knockouts of CUL3, an important gene involved in various in vivo systems, without incurring risks such as pre-developmental death.

[0159] [Example 2, Comparative Example 2: Disease Model Mice of the Present Invention (After TMX Administration)] Tamoxifen (TMX) was administered to the mice of Example 1 and Comparative Example 1 according to the scheme shown in Figure 2 (see Figures 1-A and 2).

[0160] i) Preparation of TMX solution 0.2 g of TMX powder (Sigma-Aldrich, catalog number T5648) was dissolved in a mixture of 9 mL of corn oil (Sigma-Aldrich, catalog number C8267) and 1 mL of ethanol to prepare a solution with a final concentration of 20 mg / mL.

[0161] ii) Administration of TMX The TMX solution described in i) above was administered intraperitoneally once a day for three consecutive days to each mouse aged 8 to 12 weeks, at a dose of 0.1 mg of TMX per gram of body weight (equivalent to 100 μL of solution per 20 g of mouse), thereby inducing CUL3 deletion in vascular endothelial cells and producing the mice of Example 2 and Comparative Example 2 described below.

[0162] Example 2: Example 1 + TMX...CUL3 ECKO Mouse Comparative Example 2: Comparative Example 1 + TMX...Control (genetically identical to Comparative Example 1) mouse

[0163] (Results) As described above, we were able to create sets of mice for Example 2 and Comparative Example 2 that differed in the presence or absence of CUL3 expression.

[0164] [Example 3, Comparative Example 3: Reporter-introduced mice (before TMX administration)] CUL3 ECKO To more rigorously prove that the mouse-specific phenotype is solely due to CUL3 knockout (suppression of expression), we created Example 3 and Comparative Example 3 (Figure 1-B) by further introducing a reporter gene (EGFP) after ensuring that the genetic background (other than the presence or absence of the Cre gene) was as similar as possible.

[0165] The mouse in Comparative Example 2 described above is the same as the Floxed CUL3 mouse in Comparative Example 1 before TMX administration used in its preparation, in that the Cdh5CreERT2 gene is not incorporated, and is therefore the same as the "CUL3" mouse in Example 2. ECKO This is because, strictly speaking, they have a different genetic background than "mice."

[0166] Specifically, as "animals for creating disease models" before TMX administration, appropriate combinations were selected from the offspring of a cross between the mouse from Example 1 and the reporter mouse described in C) below, and these combinations were further crossed to select individuals with the following genetic backgrounds.

[0167] Example 3: Cdh5CreERT2;CUL3 (flox / flox) + EGFP / wt Comparative Example 3: Cdh5CreERT2;CUL3 (wt / wt) + EGFP / wt

[0168] WT stands for wild type.

[0169] Furthermore, in reporter mouse C), the reporter gene (EGFP) expression system is incorporated into the ROSA26 region, as described below.

[0170] C) Reporter mouse (ROSA26EGFP): This is a mouse model in which an EGFP gene (reporter) capable of time-specific expression by administration of Cre enzyme is incorporated into the "ROSA26Sor region," a gene locus suitable for introducing foreign genes, in a wild-type mouse used for genetic experiments.

[0171] These mice were created by purchasing the following commercially available mice, which incorporate two types of reporters expressed upon administration of either Cre enzyme or Flip enzyme, and then pre-administering Flip enzyme to remove the sequence c3) below.

[0172] Cg-Gt(ROSA)26Sortm1(CAG-lacZ,-EGFP)Glh / J mouse (Stock number 012429) (Manufactured by The Jackson Laboratory)

[0173] This mouse has the following sequence incorporated into it.

[0174] c1) loxP-flanked PGK-Neo-4x PolyA STOP cassette c2) CAG promoter c3) FRT-flanked nuclear localized β-galactosidase (nlslacZ)-3x PolyA c4) EGFP-1x PolyA

[0175] This is a mouse in which two reporter genes, c3) the lacZ gene and c4) the EGFP gene, have been introduced into "ROSA26Sor" along with c2) the CAG promoter.

[0176] Note that c1) is a sequence incorporating a drug resistance marker gene.

[0177] Furthermore, "Glh / J" indicates that it is a mouse from The Jackson Laboratory, belonging to the C57BL / 6 strain.

[0178] In this mouse, the expression of "β-galactosidase (lacZ)" in c3) and "EGFP" in c4) by the CAG promoter in c2) is normally inhibited by the action of "PolyA," a termination signal present in c1), thereby suppressing their function as reporters. However, a double mechanism is in place to bypass this control, as described below.

[0179] Addition of "Cre enzyme": The sequence flanked by loxP in c1) is removed, which causes transcription of c3) and c4) by the CAG promoter in c2). This makes it possible to observe the expression of two types of reporter genes in a single mouse model: "lacZ, which is used for blue-white selection (X-gal staining)" and "EGFP, which is used for identification using green fluorescence, etc."

[0180] Addition of "Flip enzyme (Flippase)": By removing the sequence flanked by the FRT (Flippase Recognition Target) sequence, it becomes possible to control the expression of the lacZ sequence of c3).

[0181] In other words, as in this case, by adding the "Flip enzyme" beforehand and removing the c3) sequence in advance before mating with other mice, it is possible to obtain mice that can express the EGFP reporter in a Cre enzyme-dependent manner.

[0182] (Results) Before TMX administration, these mice had suppressed EGFP gene expression due to the presence of poly A (termination signal) in c1).

[0183] [Example 4, Comparative Example 4: Reporter-introduced (ROSA26EGFP) mice (after TMX administration)] Following the scheme shown in Figure 2, tamoxifen (TMX) was administered to the mice of Example 3 and Comparative Example 3 as follows to produce the mice of Example 4 and Comparative Example 4 (see Figures 1-B and 2).

[0184] Example 4: Example 3 + TMX...CUL3 ECKO(ROSA26EGFP) Mouse Comparative Example 4: Comparative Example 3 + TMX...CUL3 (wt / wt) (ROSA26EGFP) Mouse (Control) (Genetically identical to Comparative Example 3)

[0185] (Results) As described above, we were able to create mouse sets for Example 4 and Comparative Example 4, in which the genetic background other than the presence or absence of CUL3 expression was even more uniform than that of Example 2 and Comparative Example 2.

[0186] [[Test Examples (Effects of CUL3 Knockout or Knockdown)]] The following test examples confirmed the effects of suppressing the expression or function of the CUL3 protein.

[0187] Furthermore, in the various test examples described below, as a general rule, the various analyses were performed 10 to 12 days after TMX-induced CUL3 KO, as shown in the scheme in Figure 2.

[0188] [Test Example 1: Confirmation of suppression of CUL3 protein expression by CUL3 knockout in mouse vascular endothelial cells (in vivo)] (Protocol) CUL3 was knocked out in a time-specific and location-specific manner (vascular endothelial cells) by TMX administration. ECKO In the mice of Example 2 and Control (Comparative Example 2), the expression level of CUL3 was confirmed at the locations where vascular endothelial cell adhesion factor (CD31) is expressed (indicating the location of blood vessels). Specifically, the expression levels of CD31 and CUL3 in small intestinal sections of each mouse were confirmed by immunofluorescence staining images prepared according to a standard method.

[0189] Furthermore, the following reagents were used to perform fluorescence observation of each protein in vascular endothelial cells.

[0190] CD31 detection: (Primary antibody) Anti-CD31 rat mAb (DIANOVA, DIA 310) (Secondary antibody) Alexa Fluor 488 (green fluorescence) conjugated anti-rat IgG goat pAb (Thermo Fisher, A-11006)

[0191] Detection of CUL3: (Primary antibody) Anti-CUL3 mouse mAb (Sigma, SAB4200180) (Secondary antibody) Alexa Fluor 568 (red fluorescence) conjugated anti-mouse IgG goat pAb (Thermo Fisher, A-11031)

[0192] Furthermore, nuclear staining was performed simultaneously using DAPI (4',6-diamidino-2-phenylindole) staining (blue fluorescence).

[0193] Control mouse (Comparative Example 2) and CUL3 ECKO Figure 3 shows the results of the vascular structure analysis in the intestinal tract (small intestine) of a mouse (Example 2) (scale bar = 50 μm).

[0194] The panel on the far right is a magnified view of the area enclosed by the frame in the third row of images, with the CUL3 expression reduction area highlighted by an arrow.

[0195] (Results) From Figure 3, CUL3 ECKO In mouse vascular endothelial cells, the expression level of the CUL3 protein was found to be significantly reduced at the CD31 expression site. Furthermore, the CD31 expression site was partially fragmented.

[0196] (Discussion) Not only is the expression of CUL3 indeed reduced at the location of CD31 expression (location of blood vessels), but the results also show that the vascular structure itself is partially disrupted due to a decrease in the adhesive strength between vascular endothelial cell adhesion factors (CD31). These results strongly suggest that the mice of the present invention, in which the expression of CUL3 in vascular endothelial cells is suppressed, can serve as a model mouse for diseases involving bleeding, such as DIC.

[0197] [Test Example 2: CUL3] ECKO [Confirmation of the effects on the gastrointestinal tract] CUL3 knocked out in a time-specific and location-specific manner (vascular endothelial cells) ECKO Figures 4-A to 4-D show the results of examining the effects on the "tissue structure," "appearance," and "inflammation-related factors" of the gastrointestinal tract of mice in Example 2 and Control (Comparative Example 2).

[0198] Test Example 2-1) HE-stained image (tissue structure analysis)

[0199] (Protocol) Tissue sections were prepared from each mouse in different parts of the digestive tract (stomach, duodenum, jejunum, terminal ileum, rectum), and the results of HE staining and analysis performed according to standard methods are shown in Figure 4-A (scale bar: 100 μm).

[0200] (Results) Figure 4-A shows that the mice in Example 2 exhibited clear bleeding, particularly in the terminal ileum, compared to the mice in Comparative Example 2.

[0201] (Discussion) This is likely because the terminal ileum, in particular, is a tissue that is susceptible to inflammation and microvascular blockage, due to the concentration of lymphatic tissue that is easily affected by systemic inflammatory responses, its rich blood flow, and its high blood flow dependence. In other words, the above result that "the ileum, in particular, was damaged" suggests that the mice of the present invention (Example 2) are highly likely to have developed "DIC," which presents with systemic microthrombus formation causing impaired blood flow.

[0202] Test Example 2-2) Comparison of Gastrointestinal Appearance (Appearance Analysis) (Protocol) Control mouse (Comparative Example 2) and CUL3 ECKO Figure 4-B shows the results of visually observing the external appearance (presence or absence of bleeding) of the digestive tract of a mouse (Example 2).

[0203] Furthermore, the tissue extracted from the area indicated by the arrowhead in the leftmost diagram corresponds to the intestinal tissue in the center diagram. The rightmost diagram is a magnified view of the area enclosed by the frame in the center diagram.

[0204] Figure 4-C shows the number (percentage) of mice that experienced intestinal bleeding in each group.

[0205] (Results) As can be seen from Figure 4-B, CUL3 ECKO Gastrointestinal bleeding was observed only in mice (see the arrowhead in the figure on the far right). From Figures 4-B and 4-C, it was confirmed that the mice in Example 2 had a significantly higher incidence of terminal ileum bleeding compared to the mice in Comparative Example 2.

[0206] (Discussion) The results of this test example 2-2, like those of the test example 2-1 above, suggest that the mice of the present invention (Example 2) are highly likely to have developed "DIC".

[0207] Example 2-3) Volcano Plot: Analysis of inflammation-related factors

[0208] (Protocol) Of the total RNA extracted from terminal ileum tissue, the RNA Integrity Number (RIN) was measured using a bioanalyzer (Agilent Technologies). Only samples with RIN values ​​within the acceptable range (> 7.0) were used for sequencing to obtain data for the volcano plot. The obtained raw fast-q data was acquired and processed using CLC Genomics Workbench (QIAGEN) to create count data reflecting gene expression levels. The data was analyzed using RNA-seqChef software.

[0209] A volcanic plot is a type of graph used in biological research, particularly in gene expression analysis. It can simultaneously display the magnitude of the change (Fold Change) on the X-axis and the statistical significance (p-value) on the Y-axis. The volcanic plot on the left shows genes with decreased expression, while the volcanic plot on the right shows genes with increased expression.

[0210] The results of the above analysis are shown in Figure 4-D.

[0211] (Results) As can be seen from Figure 4-D, the expression of genes that are generally known to increase during inflammation (TNF, TGFβ1, AGT, IL6, etc.) was indeed elevated in this experiment as well.

[0212] Furthermore, a different gene expression analysis (box plot) showed that CUL3 ECKO In mice, other "inflammation-related factors (Ccl2, Cd14)" were also clearly increased compared to control mice. Furthermore, it was confirmed that the expression levels of not only "inflammatory factors" but also "blood coagulation-related factors (C3, F10)" were clearly increased (not shown).

[0213] (Discussion) The above results indicate that CUL3 ECKO This study supports the precursors of "bleeding" (inflammation) and "thrombosis" in the mouse digestive tract (terminal ileum) by examining the expression levels of their respective related factors (genes).

[0214] (Overall consideration of Test Examples 2-1 to 2-3) Combining the results shown in Figures 4-A to 4-D above, the CUL3 of the present invention ECKO It has been strongly suggested that mice can serve as a model for conditions such as DIC and multiple organ failure, which involve bleeding and thrombosis.

[0215] [Test Example 3: CUL3] ECKO [Tissue analysis of mice]

[0216] The EGFP gene was incorporated by crossing with reporter mice, resulting in "CUL3 ECKO The results of HE staining and GFP immunostaining performed on kidney tissue sections of "(ROSA26EGFP) mice" (Example 4) and "CUL3 (wt / wt) (ROSA26EGFP) mice" (Comparative Example 4: Control), as shown in Test Examples 3-1 and 3-2, are shown in Figures 5-A and 5-B.

[0217] Test Example 3-1) HE staining and GFP immunohistochemistry in kidney tissue

[0218] (Protocol) Following standard procedures, HE staining and GFP immunostaining were performed on kidney tissue from mice in Comparative Example 4 and Example 4. The results of observing the changes in "tissue structure" and "distribution and morphology of GFP-positive cells" in each tissue are shown in Figure 5-A.

[0219] In each image, the left shows HE staining, and the right shows GFP immunostaining.

[0220] The following reagents were used for EGFP immunostaining.

[0221] EGFP detection: (Primary antibody) Anti-GFP mouse mAb: Nakalai, GF200 (Secondary antibody) HRP-labeled anti-mouse IgG pAb: Promega, W402B

[0222] (Results) Since the changes in tissue structure in Example 4 were difficult to discern from Figure 5-A, further verification was performed using higher magnification images (see Test Example 3-2 below).

[0223] Test Example 3-2) High-magnification imaging verification of HE staining and GFP immunostaining in kidney tissue

[0224] (Protocol) For the mice in Example 4, kidney tissue was stained with HE in the same manner as in Test Example 3-1, and images were created at a higher magnification than in Test Example 3-1. The results of the detailed examination are shown in Figure 5-B.

[0225] The inserted image is a magnified view of the area enclosed by the frame in the original diagram.

[0226] (Results) The following was found from Figure 5-B: HE staining confirmed the presence of thrombi in the kidney tissue.

[0227] (Discussion of Test Examples 3-1 and 3-2) It was confirmed that thrombus formation in kidney tissue is indeed caused solely by the suppression of CUL3 protein expression.

[0228] [Test Example 4: CUL3] ECKO [Confirmation of fibrin deposition (thrombus formation) in the kidneys] CUL3 ECKO Focusing on the glomerular (kidney) structure of the mice in Example 2 and Control (Comparative Example 2), and in order to perform a more detailed histological analysis, three types of staining—PAS staining, PTAH staining, and MSB staining—were performed to confirm fibrin, as these are considered to have superior specificity and reliability compared to immunohistochemistry and other methods.

[0229] (Protocol) For glomerular tissue sections from each mouse, the results of "PAS staining," "PTAH staining," and "MSB staining" were performed according to standard procedures, and the number of glomeruli with fibrin deposition is shown in Figure 6-A.

[0230] Furthermore, the following can be observed with each staining method: PAS staining: Confirmation of visualization of basement membrane structure (red-purple) PTAH staining: Detection of fibrin (blue) MSB staining: Detection of thrombus and fibrin (red)

[0231] (Results) Figure 6-A: PAS staining revealed that the basement membrane, which normally exists along the capillaries within the glomerulus, was enlarged (thickening of the glomerular basement membrane) in conjunction with vascular hypertrophy. PTAH staining and MSB staining confirmed the presence of thrombi within the glomerulus.

[0232] Figure 6-B: According to Figure 6-B, which quantifies the amount of thrombus in Figure 6-A above, CUL3 ECKO In the mice (Example 2), the number of thrombi was significantly increased compared to the control mice (Comparative Example 2) (P = 0.0011). The number of thrombi is the average value for 4 mice in each group. The number of thrombi is expressed as the number of glomeruli containing fibrin deposition.

[0233] (Discussion) Based on the results of verification using three types of staining, "PAS staining," "PTAH staining," and "MSB staining," which are more reliable, CUL3 ECKO The mice exhibit a tendency towards "bleeding," as shown in other test cases, while simultaneously developing seemingly contradictory "thrombi." This suggests that the CUL3 of the present invention is involved. ECKO This strongly suggests that mice can serve as a model for conditions such as DIC and multiple organ failure, which involve bleeding and thrombosis.

[0234] [Test Example 5: CUL3] ECKO [Time course histological analysis of fibrin deposition (thrombus formation) by CUL3 knockout in vascular endothelial cells was confirmed by time course observation of various tissues in the intestine and kidney.]

[0235] Test Example 5-1) Analysis of intestinal tissue section images according to the number of days after TMX injection (HE staining)

[0236] (Protocol) CUL3 at each time point after tamoxifen (TMX) injection ECKO Figure 7-A shows HE-stained images of intestinal tissue from a mouse (Example 2).

[0237] (Results) HE staining revealed that tissue damage and inflammation progressed from day 0 to day 11 after TMX administration. Scale bar = 200 μm

[0238] Example 5-2) Image analysis of kidney tissue sections according to the number of days after TMX injection (PTAH staining, MSB staining)

[0239] (Protocol) CUL3 ECKO Figure 7-B shows images of tissue sections stained with "PTAH staining" or "MSB staining" at various time points after TMX injection of kidney sections from mice (Example 2).

[0240] (Results) The number of fibrin thrombi increased over time, and by day 11, widespread deposition was observed. Scale bar = 200 μm

[0241] Test Example 5-3) CUL3 ECKO Confirmation of the time-dependent changes in the constant of glomeruli (thrombi) with fibrin deposition in the kidneys of mice (Example 2).

[0242] (Protocol) Figure 7-C shows the number of glomeruli (thrombi) with fibrin deposition in the kidney at each day, corresponding to Figure 7-B (percentage of the total).

[0243] The ratio was calculated by manually counting glomeruli with fibrin deposition and identifying the number of glomeruli containing thrombi out of 100 total glomeruli per sample under a light microscope.

[0244] (Results) The number of glomeruli (thrombi) with fibrin deposition increased over time, and was found to increase particularly rapidly from day 9 to day 11.

[0245] (Overall discussion of Test Examples 5-1 to 5-3) In the histological analysis over time, CUL3 was observed 9 to 11 days after TMX injection. ECKO It was revealed that there was an increase in "inflammation (a precursor to bleeding)" in the mouse intestines and "thrombosis" in the glomeruli (kidneys).

[0246] Considering this together with the results of the survival rate confirmation test (time of death) described later, CUL3 ECKO This strongly suggests that the cause of death was DIC or multiple organ failure, accompanied by bleeding and thrombosis.

[0247] [Test Example 6: CUL3 based on hematological parameters] ECKO[Confirmation of ectopic blood coagulation in this case]

[0248] CUL3 ECKO We investigated whether there were any changes in the parameters indicating "ectopic blood coagulation" in mice (Example 2) (n = 4) and control mice (Comparative Example 2) (n = 4).

[0249] "Ectopic coagulation" is known as one of the important diagnostic markers for "DIC," which is blood coagulation that occurs in abnormal locations other than within normal blood vessels. However, by comprehensively evaluating several blood flow parameters such as platelet count (PLT), it may be possible to predict blood coagulation in abnormal locations (ectopic coagulation) that does not remain confined to blood vessels.

[0250] (Protocol) The results of measuring various hematological parameters in the blood of each mouse according to the standard method described below are shown in Figure 8.

[0251] White blood cell (WBC) count: Measured using an automated hematology counter by optical or electrical methods. Red blood cell (RBC) count: Measured using an automated hematology counter and counted the number of red blood cells. Hemoglobin (HGB): Hemoglobin concentration was measured by measuring absorbance using a chemical reaction. Hematocrit (HCT): The volume percentage of red blood cells in the blood was measured using centrifugation. Mean corpuscular volume (MCV): The volume of red blood cells was measured and its average value was calculated. Mean corpuscular hemoglobin (MCH): Calculated by dividing the hemoglobin amount by the number of red blood cells. Mean corpuscular hemoglobin concentration (MCHC): Calculated by dividing the hemoglobin concentration by the volume of red blood cells. Platelet count (PLT): The number of platelets was counted using an automated hematology counter. Red blood cell distribution width (RDW): The distribution of red blood cell sizes was measured and its width was calculated. Platelet Crit (PCT): Calculated by dividing the volume of platelets by the total blood volume. Mean Platelet Volume (MPV): The volume of platelets was measured and its average value was calculated. Platelet Distribution Width (PDW): The distribution of platelet sizes was measured and its width was calculated.

[0252] The results are shown as the median values ​​of each measurement for Example 2 (n = 4) and Comparative Example 2 (n = 4) at the time of sacrifice.

[0253] (result)

[0254] As can be seen from Figure 8, the CUL3 of Example 2 ECKO Compared to Comparative Example 2, the mice showed a significant decrease in RBC, HCT, PLT, and PCT, and an increase in MPV, but WBC, HGB, MCV, MCH, MCHC, RDW, and PDW remained unchanged.

[0255] Generally, a decrease in RBC and HCT is associated with anemia or a tendency towards major bleeding. Similarly, a significant decrease in PLT and PCT also suggests a condition where major bleeding or chronic bleeding is occurring or at high risk.

[0256] On the other hand, an increase in MPV means that new, larger-than-normal platelets are being produced due to the rapid loss of platelets.

[0257] (Consideration)

[0258] The decrease in "red blood cell-related parameters (RBC, HCT)" shown in this study suggests a correlation with the "bleeding" phenomenon actually observed in other study examples. Furthermore, the extreme decrease in "platelet-related parameters (PLT, PCT)" shown in this study example (approximately 10% of that in Comparative Example 2) strongly suggests the occurrence of "ectopic blood coagulation," supports the thrombus formation phenomenon actually observed in other study examples, and supports the very characteristic symptom of "DIC" described in the [Background Technology] above: "(1) Platelets and coagulation factors that constitute the thrombus are consumed in large quantities and become deficient."

[0259] [Test Example 7: CUL3] ECKO [Survival rate and body temperature in mice (vascular endothelial CUL3 knockout mice)] (Protocol) CUL3 ECKO The results of periodic observations of the "survival rate" and "body temperature" of mice (Example 2) and control mice (Comparative Example 2) are shown in Figure 9-A (survival rate) and Figure 9-B (body temperature), respectively.

[0260] The number of mice used for each experiment is as follows:

[0261] CUL3 ECKOMice (Example 2): Male mice (n = 31), female mice (n = 8), total (n = 39)

[0262] Control mice (Comparative Example 2): Male mice (n = 36), Female mice (n = 13), Total (n = 49)

[0263] (Results) Figure 9-A (Survival Rate Graph): Figure 9-A is a graph (Kaplan-Meier survival curve) showing that all of the "disease model mice" of the present invention died on day 11 after TMX injection.

[0264] Log-rank test, P < 0.0001.

[0265] Furthermore, in several similar trials, it was confirmed that all participants in the group in which CUL3 was knocked out died within 10 to 12 days.

[0266] Figure 9-B (Graph of Body Temperature Decline): Figure 9-B shows that when the body temperature of the "disease model mouse" of the present invention was measured periodically after TMX injection, the body temperature rapidly decreased from the 11th day after injection.

[0267] All mice tested showed a significant decrease in body temperature, indicating hypothermia. Data are presented as mean ± SEM (standard error).

[0268] (Discussion) Thus, the temporal changes in Figure 9-A (survival rate graph) and Figure 9-B (body temperature decrease graph) are almost identical, and it was confirmed that all mice died along with a decrease in body temperature within a very short period of 10 to 12 days after TMX injection. Considering the occurrence of "bleeding" and "thrombosis" demonstrated in each of the above-mentioned test cases, and the result of 100% mortality in this test case, CUL3 ECKO It is believed that the mice developed "DIC" followed by "multiple organ failure" and ultimately died. This is because, for example, the amount of "bleeding" confirmed in the other test cases mentioned above was not enough to cause death on its own. Therefore, it is more reasonable to conclude that the cause of death in this test case was not simply massive bleeding, but rather the development of "DIC" or "multiple organ failure" (a fatal disease) due to "bleeding" or "thrombosis," which have a poor prognosis and high mortality rate.

[0269] [Test Example 8: CUL3] ECKO [Confirmation of the effects on blood vessels in each tissue] (Protocol) CUL3 ECKO Tissue sections of the small intestine, brain, liver, kidney, lung, heart, etc., were prepared from each mouse in (Example 2) and Control (Comparative Example 2), and the following analyses were performed according to standard procedures.

[0270] (Results) i) Analysis of RNA-seq data from small intestinal tissue revealed that CUL3 ECKO In the small intestine of the mice in Example 2, the expression of the "HIF-1α (high-proxia-inducible factor 1α)" gene, which is expressed to alleviate hypoxia, was significantly increased compared to the mice in Control (Comparative Example 2) (not shown).

[0271] ii) CUL3 ECKO In the kidneys of the mice in Example 2, an increase in the protein level of "HIF-1α" was also observed compared to the control (Comparative Example 2) mice (not shown).

[0272] Furthermore, the expression levels of the "HIF-1α" gene and protein were measured using conventional methods.

[0273] iii) Measurement of vascular density in each tissue: To investigate the cause of elevated HIF-1α levels, CUL3 was modified by incorporating the EGFP gene into the ROSA26 region. ECKO GFP immunostaining was performed on various tissues such as the brain, liver, kidney, lungs, and heart of each mouse in (Example 4) and Control (Comparative Example 4). From the obtained immunostained images, "vascular density" was quantified using a standard method with image analysis software, and CUL3 ECKO A decrease in vascular density was observed in the brain, liver, kidneys, lungs, and heart of mice, but the decrease was most pronounced in the kidneys (Figure 10).

[0274] Furthermore, all mice used in the analysis died within 11 days of receiving TMX injection.

[0275] This suggests that the decrease in vascular density is leading to inhibited angiogenesis due to a lack of oxygen and nutrients, i.e., "multiple organ failure."

[0276] (Discussion) Based on the results of i) through iii) above, CUL3 ECKO The study suggested that HIF-1α was expressed in various tissues of the mice as a result of blood deficiency caused by bleeding, leading to insufficient oxygen supply to the tissues. This confirmed that the very characteristic symptoms of "DIC" described in the [Background Technology] section above were occurring not only in the gastrointestinal tract but also in various tissues and organs throughout the body, where "organs that do not receive the necessary oxygen and nutrients suffer damage, posing a serious threat to life."

[0277] [Test Example 9: CUL3] ECKO [Confirmation of the cause of death in mice] (Protocol) CUL3 ECKO In each of the mice in Example 2 and Control (Comparative Example 2), we observed for increased pleural effusion and weight loss.

[0278] (Results) The mice in Example 2 all died approximately 10 to 12 days after TMX administration, similar to Figure 9-A in Test Example 7. However, observation of the deceased mice revealed an abnormal amount of pleural fluid in the pleural cavity, and their body weight was clearly lower compared to the mice in Comparative Example 2 (not shown).

[0279] (Discussion) The above results support the mechanisms of action of multiple organ failure, such as the fact that "when hypercoagulation occurs, blood flow is inhibited, and sufficient oxygen and nutrient supply to the lungs, heart, kidneys, and other tissues is impaired, resulting in damage to the blood vessel walls in each organ and leakage of plasma components into the interstitial space (pleura) of the lungs, which accumulates as pleural effusion," or the mechanism of action of DIC, such as "when vascular permeability increases, plasma components leak into the pleural cavity, resulting in the accumulation of pleural effusion."

[0280] [Consideration of Test Examples 1 to 9] As can be seen from the various test examples described above, the present invention's CUL3 ECKOThe mice not only experienced "bleeding" and "thrombus formation," but also "weight loss," "decreased body temperature," and "pleural effusion," and all died within a short period of time. Moreover, the temporal changes in most of these symptoms were consistent with the survival curve, in which all mice died within 10 to 12 days of CUL3 knockout by TMX administration. This clearly indicates that the cause of death in the disease model animals of the present invention was not simply "bleeding" or "thrombus formation," but rather the subsequent development of serious diseases (lethal diseases such as "DIC" and / or "multiple organ failure").

[0281] Furthermore, in the "disease model animals" of the present invention, the detection of "D-dimer," one of the diagnostic criteria for the onset of DIC, was not observed. This is thought to be because all the "disease model animals" died before the thrombus could be broken down into "D-dimer" by the body's defense reaction (secondary fibrinolysis by plasmin, etc.). In other words, the symptoms of the "disease model animals" of the present invention are thought to be close to the symptoms of acute diseases in which secondary fibrinolysis by plasmin, etc., cannot keep up, particularly among "diseases exhibiting excessive intravascular coagulation symptoms" such as "DIC" and "multiple organ failure." Therefore, it is considered to be particularly useful for "elucidating and researching the pathological mechanisms" and "screening preventive and / or therapeutic agents" for these symptoms.

[0282] By using the "disease model animals" of the present invention, it is possible to accelerate pathological research and the development of preventive and / or therapeutic drugs for "serious diseases (lethal diseases) that exhibit excessive intravascular coagulation symptoms," such as "DIC" or "multiple organ failure," which have been difficult to develop until now.

Claims

1. A model animal for a disease exhibiting excessive intravascular coagulation symptoms, characterized by suppression of CUL3 protein expression or function specifically in vascular endothelial cells.

2. The disease model animal according to claim 1, characterized in that the suppression means is gene knockout.

3. The disease model animal according to claim 1, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is disseminated intravascular coagulation (DIC) or multiple organ failure.

4. An animal for creating a disease model exhibiting excessive intravascular coagulation symptoms, characterized by incorporating a system that can induce the suppression of CUL3 protein expression or function in a time-specific and vascular endothelial cell-specific manner.

5. The disease model animal according to claim 4, characterized in that the inhibitory induction system is a genetically modified system using a recombinase.

6. The animal for creating a disease model according to claim 5, characterized in that the recombinase is a Cre enzyme.

7. The animal for creating a disease model according to claim 4, characterized by using a promoter that is specifically expressed in vascular endothelial cells.

8. The disease model animal according to claim 7, characterized in that the promoter specifically expressed in vascular endothelial cells is the promoter of the VE cadherin gene (Cdh5).

9. The disease model animal according to claim 4, characterized in that the time-specific induction is induced by the administration of an estrogen receptor antagonist corresponding to a mutant estrogen receptor.

10. The animal for creating a disease model according to claim 9, characterized in that the estrogen receptor antagonist is tamoxifen.

11. The animal for creating a disease model according to claim 4, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is DIC or multiple organ failure.

12. A method for producing animals for creating disease models exhibiting excessive intravascular coagulation symptoms, characterized by comprising the step of i) below: i) A step of mating animals A) and B) below: A) An animal into which a time-specific inducible recombinase gene and a vascular endothelial cell-specific expression promoter gene have been incorporated B) An animal in which at least a portion of the CUL3 gene has been genetically modified so that it is sandwiched between recombinase recognition sequences 13. A method for producing a disease model animal according to claim 1, characterized by comprising the steps of i) and ii) below: i) A step of mating animals A) and B) below; ii) A step of inducing time-specific induction in the animals obtained in i); A) An animal into which a time-specific induction type recombinase gene and a vascular endothelial cell-specific expression promoter gene have been incorporated; B) An animal in which at least a portion of the CUL3 gene has been genetically modified so that it is sandwiched between recombinase recognition sequences.

14. A method for screening agents for the prevention and / or treatment of diseases exhibiting excessive intravascular coagulation symptoms, characterized by comprising the following steps (1) to (3). (1) A step of administering a candidate substance to a disease model animal according to claim 1 or to an animal for creating a disease model according to claim 4. (2) A step of inducing time-specific induction if the animal administered in (1) is an animal for creating a disease model. (3) A step of comparing the phenotype of each animal before and after administration of the candidate substance, or between a non-administered group and an administered group.

15. The screening method according to claim 14, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is DIC or multiple organ failure.

16. A preventive and / or therapeutic agent for diseases exhibiting excessive intravascular coagulation symptoms, characterized by containing the CUL3 protein and / or its gene.

17. The preventive and / or therapeutic agent according to claim 16, characterized in that the disease exhibiting excessive intravascular coagulation symptoms is DIC or multiple organ failure.

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