WO2007116800A1

WO2007116800A1 - Type i interferon-unresponsive non-human model animal

Info

Publication number: WO2007116800A1
Application number: PCT/JP2007/056818
Authority: WO
Inventors: Shizuo Akira; Hiroki Kato; Osamu Takeuchi
Original assignee: Japan Science And Technology Agency; Osaka University
Priority date: 2006-04-07
Filing date: 2007-03-29
Publication date: 2007-10-18
Also published as: JP2007274988A

Abstract

Disclosed are: a picornavirus-specific type I interferon-unresponsive non-human model animal in which the response of type I interferon to the infection with a virus belonging to the picornavirus family is defected; a method for identification of an anti-picornavirus agent using the type I interferon-unresponsive non-human model animal; and others. A non-human animal in which the entire of a part of an endogenous gene encoding MDA5 (melanoma differentiation associated gene-5) is inactivated by gene mutagenesis such as disruption, deletion, substitution or the like to thereby disrupt the MDA5-expressing function of the gene can be used as the type I interferon-unresponsive non-human model animal.

Description

Type I interferon non-responsive non-human animal model

Technical field

[0001] The present invention relates to a non-human animal in which the function of expressing MDA5 (Melanoma differentiation associated gene-5) has been lost, in which type I interferon response to infection with a virus belonging to the picornavirus family is deleted. Identification of anti-picornavirus agents using non-human model animals that are non-human model animals that do not respond to type I interferons and non-human models that do not respond to type I interferons It relates to methods.

Background art

[0002] Innate immune responses against microbial pathogens such as viruses and bacteria are expressed by the host pattern recognition receptors (PRR) such as Toll-like receptor (TLR) and helicase family members, and pathogen-associated molecular patterns (PAMP). It is caused by recognizing the specific structure of the called invading pathogen (see, for example, Non-Patent Document 1 and Non-Patent Document 2). Host pattern recognition receptors (PRRs) play an essential role in the recognition of molecular patterns specific to viruses, including DNA, single-stranded (ss) RNA, double-stranded (ds) RNA, and glycoproteins. Yes. TLR3 located in the endosomal membrane is known to recognize viral dsRNA in addition to the synthetic dsRNA analog polyinosine polycytidylic acid (poly I: C). In addition, RIG-1 (Retinoic acid-inducible protein-1), which is a cytoplasmic protein, and MDA-5 (Melanoma differentiation associated gene-5), also known as a helicard, have been identified as dsRN A detectors. RIG-I is a DExDZHbox type RNA helicase with two domain (CARD) -like modules that recruit caspases. The helicase domain is thought to be involved in dsRNA recognition, and CARD has been reported to be essential for the initiation of downstream signaling leading to the activity of IRF3, IRF7 and NF-κB (For example, refer nonpatent literature 3). In addition, RIG-I-deficient mice consistently produce type I interferon (IFN) against vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and Sendai virus (SeV) infection. Can be reported (See, for example, Non-Patent Document 4). Furthermore, the activity of IRF3 and NF-κB in response to viral infection was impaired in RIG-I deficient cells. MDA-5 has a structure similar to RIG I and has been reported to be involved in mediating antiviral responses (see, for example, Non-Patent Document 5 and Non-Patent Document 6).

[0003] Although RIG-I and MDA5 are suggested to be involved in the recognition of viral dsRNA, in addition to the functional relationship between these dsRNA detectors in vivo, we are well aware of the functional role of MDA5. Be cunning. Thus, since the clear function of the dsRNA detector has not been elucidated, interferon responses based on these actions could not be suppressed in mammals.

[0004] Non-Patent Document 1: Annu Rev Immunol. 20, 197-216, 2002

Non-Patent Document 2: Annu Rev Immunol. 21,335-376, 2003

Non-Patent Document 3: See Nat Immunol. 5, 730-737, 2004

Non-Patent Document 4: Immunity Vol.23 19-28 July 2005

Non-Patent Document 5: Curr Biol. 12, 838-843, 2002

Non-Patent Document 6: Proc Natl Acad Sci USA. 101, 17264-17269, 2004

Disclosure of the invention

Problems to be solved by the invention

[0005] An object of the present invention is to provide a picornavirus-specific type I interferon-refractory non-human model animal lacking the response of type I interferon to infection with a virus belonging to the picornavirus family, It is to provide a method for identifying an anti-picornavirus agent using a non-human model animal that does not respond to type I interferon.

Means for solving the problem

[0006] The inventors first established MDA5 ^{_ /} -mice in order to investigate the functional role of MDA5 in vivo. These MDA5 ^{_ / _} mouse-derived fibroblasts and rod cells lack a type I interferon response to infection with encephalomyocarditis virus (EMCV) and Tyler virus (Thei ler's virus) belonging to the picornavirus family. Forces that were used Responsiveness to other RNA viruses was normal. Thus, MDA5 is recognized as a receptor that specifically recognizes picornavirus viruses and induces type I interferon production. The present invention has been completed.

[0007] That is, the present invention is (1) inactive by gene mutation such as disruption, deletion, substitution, etc. of all or part of the endogenous gene of non-human animal that codes for MDA5 (Melanoma differentiation associated gene-5). Non-human animals that have lost the function of expressing MDA5 and lack the response of type I interferon to infection with viruses belonging to the picornavirus family. And (2) non-human model animal force model mouse characterized in that it is a model mouse, and (3) an endogenous gene encoded by MDA5 (Melanoma differentiation associated gene— 5) Non-human animals that have lost their ability to express MDA5 to be infected with viruses belonging to the Picornavirus family by destroying all or part of the puppy and making it inactive due to genetic mutations such as deletion and substitution. versus A method of using as a type I interferon non-responsive non-human model animal deficient in a response to type I interferon, or (4) the non-human model animal is a model mouse as described in (3) above Regarding the method.

[0008] In addition, the present invention provides (5) MDA5 (Melanoma differentiation associated gene—inactive by gene mutation such as disruption, deletion, substitution, etc. of all or part of the endogenous gene of a non-human animal that co-operates with 3). Infect non-human animals that have lost the function of expressing MDA5 with a virus belonging to the Picornavirus family and administer the test substance before, after, or at the same time as the virus infection. RANTES or IL-6 production is measured, and if the production of type I interferon, RANTES or IL 6 is observed, the test substance is evaluated as an anti-picornavirus agent. (6) Identification as described in (5) above, characterized by comparing and evaluating (6) the production of type I interferin, RANTES or IL 6 in wild-type animals of the same species as non-human animals Law and, (7) a non-human animal, it relates to a method of identifying the (5) or (6), wherein it is a mouse.

[0009] Further, the present invention provides (8) MDA5 (Melanoma differentiation associated gene—inactive by gene mutation such as disruption, deletion, substitution, etc. of all or part of the endogenous gene of a non-human animal that co-operates with 3). Infect non-human animals that have lost the function of expressing MDA5 with myocarditis virus (EMCV) belonging to the Picornavirus family, before or after EMCV infection. Administer the test substance at the same time, conduct histological analysis of the heart of the non-human animal, and if the suppression of focal necrosis of myocardial cells is observed, evaluate the test substance as an anti-EMCV agent. (9) Compared with the degree of suppression of focal necrosis of cardiomyocytes in wild-type animals of the same species as non-human animals (8) The identification method described in (10), the identification method described in (8) or (9) above, wherein the non-human animal is a mouse, and (11) MD A5 (Melanoma differentiation associated gene-5) Picorna is introduced into cells derived from non-human animals that have been inactivated by genetic mutations such as disruption, deletion, or substitution, and have lost the function of expressing MDA5. Infected with a virus belonging to the virus family and tested before, after, or simultaneously with the virus infection When the amount of type I interferon, RANTES or IL-6 produced in the non-human animal-derived cell is measured and production of type I interferon, RANTES or IL-6 is observed, (12) Type I interferon, RANTES or IL in cells derived from wild-type animals of the same species as non-human animals, characterized by evaluating the test substance as an anti-picornavirus. The identification method according to (11) above, characterized in that it is compared with the production amount of 6, and (13) the non-human animal is a mouse as described in (11) or (12) above And (14) the identification method according to any one of (11) to (13) above, wherein the cell is an embryonic fibroblast or a rod-shaped cell.

Brief Description of Drawings

FIG. 1 a is a diagram showing the structures of a mouse MDA5 gene, a targeting vector, and a predicted disrupted gene. The country indicates coding exon. B; indicates BamHI. Fig. 1b shows the results of Southern blot analysis of offspring from heterozygous intercross. Genomic DNA was extracted from the tail of the mouse, digested with BamHI, separated by electrophoresis, and hybridized with the radiolabeled probe shown in a. Southern blot showed that wild type (+ Z +) mice had a 9.4 kb single band, homozygous (one Z) mice had a 4.6 kb band, and heterozygous (+ Z) mice had both bands. Obtained. Fig. 1c shows the results of Northern plot analysis of peritoneal exudate cells (PEC). Extract wild-type (WT) and MDA5_, PEC total RNA treated with 1000 U / ml IFN—β for 8 hours Northern blot analysis was performed for the expression of MDA5 mRNA. The same membrane was rehybridized with the j8 lactin probe. Fig. 1d shows the results of Western blot analysis of MDA5 expression. WT and MDA5 ^{_ / _} MEF were treated with lOOOUZml of IFN-β for 8 hours, and whole cell lysates were immunoblotted with MDA5 antibody.

A and b in FIG. 2 is intravenously display of the mouse, the 200 ₈ Poly 1: Ji Leave injected the indicated time, generation of IFN-a, IL 6 and IL 12p40 in serum FIG. 3 is a view showing the results of measuring the ELISA with ELISA. Data are mean SD of serum samples. FIG. 2c shows the results of analyzing the surface expression of CD86 by flow cytometry on spleen CDllc ⁺ B220_cDC of mice injected with PBS or poly I: C. FIG. 2d shows the results of stimulation of WT, RIG—I—, and MDA5 ^{_ / _} GMCSF—DC with poly I: C with increasing concentrations for 24 hours. The production of IFN- «, IL-6 and IL-12p40 in the culture supernatant was measured by ELISA. Figure 2e shows the results of treating WT, RIG-1 ^{_ / _} , and MDA5 ^{_ / _} MEF with in vitro transcribed ds RNA of the indicated length that forms a complex with ribofactamine 2000 for 24 hours. FIG. The production of IFN-β in the culture supernatant was measured by ELI SA. The error bars for d and e indicate 3 times S. D.

[FIG. 3] FIG. 3a is a diagram showing the results of infecting WT, RIG—I ^{__ / _} , and MDA5 ^{__ / _} MEF with the indicated minus-strand ss RNA virus. The production of IFN-α in the culture supernatant was measured by E LISA. B and c in FIG. 3 are diagrams showing the results of infecting WT, RIG—I—, and MDA5 MEF with the indicated positive strand ssRNA viruses, JEV (b) and EMCV (c). The production of IFN— | 8 in the culture supernatant was measured by ELISA. FIG. 3 d and e are diagrams showing the results of infecting WT, RIG-1 ^{_ / _} , and MDA5 ^{_ / _} GMCSF-DC with EMCV with gradually increasing moi. The production of IFN-a (d) and IL-6 (e) in the culture supernatant was measured by ELISA. F and g in FIG. 3 temporarily transfect WT and RIG—I—, MDA5 ”^ MEF with a reporter construct that includes the IFN— | 8 promoter and with or without the indicated expression vector, The cells are then infected with EMCV or Se VCm and subjected to luciferase assembly The error bars a, b, c, d, and e indicate the three SDs.

[Figure 4] Figure 4a shows 2 X 10 ⁷ pfu JEV, RIG-1 ^{+ / _} , RIG- I, MDA5 ^{+ / _} and M FIG. 4 shows the results of measuring the production level of IF N-α by ELISA, after injecting intravenously into DA5 ^{_ / _} mice, and collecting serum of mouse force 24 hours after the injection. Bars show average values. FIG. 4b is a graph showing the results of monitoring the survival of display mice (6 weeks old) infected with 2 × 10 ⁷ pfu JEV via vein over 15 days. FIG. 4c shows the results of monitoring the survival of mice (3 weeks old) infected intranasally with 4 × 10 ⁶ pfu VS V over 9 days.

[Fig. 5] Fig. 5a shows that 10 ⁷ pfu of EMCV was intravenously inoculated into MDA5 + and MDA5 ^{__ / _} mice (n = 5), and serum was collected 4 hours after injection, and IFN-α, Rantes and FIG. 6 is a view showing the results of confirming the production level of IL-6 by ELISA. Fig. 5b shows the results of monitoring the survival of display mice (6 weeks old) intraperitoneally infected with 10 fu EMCV every 6 hours for 6 days. Figure 5c shows the result of MDA5 and MDA5 ^{__ / _} mice being infected intraperitoneally with 10 fu EMCV. After 48 hours, the mice were disposed and the virus value in the heart was confirmed by plaque assembly. FIG. Fig. 5d shows the results of histological changes evaluated by hematoxylin and eosin staining on the heart of MDA5 ^{+ / _} and MDA5 ^{__ / _} mice 2 days after infection. . Arrows indicate focal necrosis of cardiomyocytes. Figures 5e and f show the results of an echocardiographic assessment of cardiac function 48 hours after EMCV infectivity. E in Fig. 5 is a translucent view of transthoracic echocardiography in M mode of MDA5 ^{+ / _} and MDA5 ^{__ / _} mice 48 hours after EMCV infectivity. F in Fig. 5 shows the shortening rate (FS) after infection confirmed by echocardiography.

FIG. 6 shows the results of Northern blot analysis of MEFs. WT (wild type), RI G— 1 ^{_ / _} , and MDA5 ^{_ / _} MEFs were infected with SeV V (−) with moi = 10, and the extracted total RNA was subjected to Northern blot analysis, and IFN—β The expression of IP 10 mRNΑ was examined. The 28S and 18S ribosomal RNA bands on the gel stained with ethidium bromide were used as RNA loading controls.

[Fig. 7] Fig. 7 shows that WT (wild type) and MDA5 GMCSF—DCs are infected with the moi-increased Tyler virus (Theiler's virus) or Mengovirus (Mengovirus) for 24 hours, and in the culture supernatant. It is a figure which shows the result of having measured the production amount of IFN-a of ELISA by ELISA. [Fig.8] Fig.8 shows that B220 + and B220-CD1 lc + cells isolated from MyD88 ^{_ / _} , MDA5 ^{_ / _} , and MDA5 ^{_ / _} Flt3L— DCs by MACS were infected with EMCV with moi = 1 for 24 hours FIG. 4 shows the results of measuring the amount of IFN-α produced in the culture supernatant by ELISA.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] As the non-human model animal of the present invention, all or part of the endogenous gene of MDA5-encoding non-human animal is inactivated by gene mutation such as destruction / deletion / substitution, and MDA5 is expressed. If the non-human animal has lost its function to prevent infection with type I interferon against infection with a virus belonging to the picornavirus family, it is not particularly limited. In addition, the method of the present invention includes a non-human animal in which all or part of the endogenous gene encoding MDA5 is inactivated by a gene mutation such as disruption / deletion / replacement and the function of expressing MDA5 is lost. Is used as a non-human model animal that does not respond to type I interferon and lacks the type I interferon response to infection with a virus belonging to the Picornavirus family. There is no particular limitation as long as it is an IFN-α or IFN-β.

[0012] In the present invention, type I interferon unresponsiveness refers to the reactivity of the living body or the cells, tissues or organs of type I interferon to infection (stimulation) with a virus belonging to the picornavirus family. It means that it has been reduced, lost power, or almost lost. Therefore, in the present invention, the type I interferon-nonresponsive human model animal is a type I interferon-producing reactivity of a living body or a cell, tissue or organ constituting the living body against infection with a virus belonging to the picornavirus family. This refers to non-human animals such as mice, rats, and rabbits that have lost or are almost lost. Among these, a type I interferon non-responsive model mouse can be preferably exemplified. The type I interferon-nonresponsive human animal of the present invention is preferably non-responsive to RANTES and IL 6 in addition to type I interferon non-responsiveness.

[0013] The virus belonging to the Picornaviridae family is a single positive chain RN. An RNA virus with the genome of A, which has no envelope, has an icosahedral force psid, and has a small RNA (pico-rna). Viruses belonging to the genus Enterovirus (such as viruses, Coxsackie virus, Echovirus), viruses belonging to the genus Rhinovirus (such as human rhinovirus, Mengo virus), and encephalomyocarditis virus (EMCV) Parechoviruses such as viruses belonging to the genus Cardiovirus, viruses belonging to the genus Aphthovirus such as foot-and-mouth disease virus, hepatouinoleus genus such as hepatitis A virus, and human parechovirus Mention may be made of viruses belonging to the genus.

[0014] Examples of non-human animals that have lost the function of expressing MDA5 of the present invention include MDA5 ^{_ / _} mice and rodents such as MDA5 ^{_ / _} rats that lack MDA5 gene function. be able to. These MDA5 As the non-human animal whose function deficient genes that may be obtained with wild-type littermates capable of birth to come MDA5 ^{_ / _} non-human animal force accurate comparative experiment following the law of Mendel Is preferable. A method for producing such an animal deficient in the function of the MDA5 gene will be described below using MDA5 ^__ mice as an example.

[0015] The MDA5 gene can be screened using a probe derived from the mouse MDA5 gene after a mouse gene library is amplified by PCR or the like. The screened MDA5 gene can be subcloned using a plasmid vector or the like, and can be identified by restriction enzyme mapping and DNA sequencing. Next, all or part of the gene encoding MDA5 is replaced with pMCl neo gene cassette, etc., and diphtheria toxin A fragment (DT-A) gene or simple herpesvirus thymidine kinase ( A target vector is prepared by introducing a gene such as the HSV-tk) gene.

[0016] The produced targeting vector is linearly introduced and introduced into ES cells by the electro-poration method (electroporation) method, and homologous recombination is performed. Select ES cells that have undergone homologous recombination with antibiotics such as G418 and ganciclovir (GANC). In addition, it is preferable to confirm whether the selected ES cells have the desired recombinant ability by the Southern plot method or the like. The confirmed ES cell clone Is microinjected into the blastocyst of the mouse, and the earning blastocyst is returned to the temporary parent mouse to produce a chimeric mouse. When this chimeric mouse is wild-type mice crossed, hetero contact coalescing mouse (F1 mouse; MDA5 ^{+ / _)} can be obtained, also by crossing heterozygous mice to this, MDA5 ^_ of the present invention ^{/ _Mice} can be made. In addition, as a method for confirming whether MDA5 occurs in MDA5 ^{_ / _} mice, for example, RNA is isolated from the mouse obtained by the above method and examined by Northern blotting or the like. There are methods to examine the expression of MDA5 in Western blotting.

[0017] In addition, the produced MDA5 ^{__ / _} mouse force-type interferon non-responsiveness means that, for example, viruses belonging to the picornavirus family can be used for macrophages, mononuclear cells, rod cells, etc. of MDA5 mice. This can be confirmed by infecting immune cells or embryonic fibroblasts in vitro or in vivo, and measuring the amount of type I interferon, IL-6, RANTES, etc. produced in vigorous cells.

[0018] As a method for identifying the anti-picornavirus agent of the present invention, all or part of the endogenous gene of the non-human animal encoding MDA5 is inactivated by gene mutation such as disruption 'deletion' substitution, A non-human animal that has lost the function of expressing MDA5 is infected with a virus belonging to the Picornavirus family, and a test substance is administered before, after, or simultaneously with the virus infection, and the non-human animal type I interferon, RANTES or When the amount of IL-6 production is measured and production of type I interferon, RANTES or IL-6 is observed, a method for evaluating the test substance as an anti-picornavirus agent, or for non-human animals encoding MDA5 All or part of the endogenous gene is inactivated by gene mutations such as deletion or replacement, and cells derived from non-human animals that have lost the function of expressing MDA5 are treated with picornavirus family. Infect the virus belonging to the group 1 and contact the test substance with the cell before, or after the virus infection, and measure the amount of type I interferon, RANTE S or IL-6 produced in the cell derived from the non-human animal. When the production of type I interferon, RANTES or IL 6 is observed, the method is not particularly limited as long as the test substance is evaluated as an anti-picornavirus agent. The method for identifying an anti-picornavirus agent of the present invention Can be used to screen for anti-picornavirus agents. A powerful form of implementation of the identification method of the present invention The state will be described below with an example.

[0019] The type I interferon-unresponsive non-human model animal power of the present invention is obtained by combining immune cells such as rod-shaped cells and embryonic fibroblasts, a test substance, and a virus belonging to the picornavirus family. After culturing and measuring and evaluating the degree of type I interferon production in the cells, or after administering the test substance to the type I interferon non-responsive non-human animal model of the present invention, An identification method for infecting immune cells capable of obtaining human animal power with a virus belonging to the Picornavirus family and measuring the degree of type I interferon production in the immune cells, or the type I interferon non-responsiveness of the present invention After infecting a human model animal with a virus belonging to the picornavirus family in advance, the immune cells capable of obtaining the non-human animal power can be obtained in the presence of the test substance. Culturing and measuring and evaluating the degree of type I interferon production in the immune cells, or infecting the type I interferon-nonresponsive human model animal of the present invention with a virus belonging to the picornavirus family An identification method for administering a test substance at the same time or before and after the infection and measuring the degree of type I interferon production in immune cells obtained by the non-human animal model, and the type I interferon non-response of the present invention A non-human animal model is infected with a virus belonging to the Picorna virus family, and a test substance is administered simultaneously with or before the infection, and the degree of production of type I interferon in the serum obtained from the non-human animal model is measured. Evaluate identification methods. Instead of measuring the level of type I interferon production, the level of RANTES or IL-6 production can also be measured. For evaluation, wild-type animals of the same species as non-human animals, particularly litters of the same litter. It is preferable to compare and evaluate the production of interferon type I, RANTES or IL 6 in /!

[0020] The anti-EMCV agent of the present invention can be identified by destroying or deleting all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation asso dated gene-5). Infect non-human animals that have lost their ability to express MDA5 by infecting with myocarditis virus (EMCV) belonging to the Picornavirus family, and administer the test substance before, after, or simultaneously with EMCV infection. If the histological analysis of the heart of the non-human animal is performed and suppression of focal necrosis of cardiomyocytes is observed, it is not particularly limited as long as it is an identification method for evaluating the test substance as an anti-EMCV agent, The anti-EMCV agent of the present invention Anti-EMCV agents can be screened using standard methods. In the evaluation, it is preferable to compare and evaluate the degree of suppression of focal necrosis of cardiomyocytes in wild-type animals of the same species as non-human animals, particularly litter pups.

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

Example

[0022] [Materials and Methods]

(1) Preparation of MDA5 mouse

The MDA5 gene was isolated from genomic DNA from which ES cell (GSI-I) force was also extracted by PCR. A simple perpes virus driven by the PGK promoter by constructing a targeting vector by substituting the neomycin-resistant gene cassette (neo) for the MDA50RF (including DEXH box) 4.3-kb fragment. Thymidine kinase (HSV-TK) was inserted into the genomic fragment for negative selection. After transfecting the targeting vector into ES cells, double resistant colonies of G418 and gancyclovir were selected, screened by PCR, and further confirmed by Southern blot. Homozygous recombinants were microinjected into female C57BL / 6 mice, and heterozygous F1 progeny were intercrossed to obtain MDA5 ^{_ / _} mice.

[0023] (2) MyD88 ^{_ /} — Mouse, TRIF ^{_ / _} Mouse, TLR3 ^{_ / _} , TRIF Mouse and RIG— I — Mice Production

MyD88 mice, TLR3 ^{_ / _} mice, and TRIF mice were prepared by the method described in the literature Sceience 301, 640-3 (2003). IFN α / β R ^{_ / _} mice were prepared by the method described in Int Immunol 14 1225-31 (2002). Furthermore, RIG-adult mice were produced by crossing with ICR mice.

[0024] (3) Production of RIG—I ^{__ / _} MDA5 ^{__ / _} mice

When a RIG—I heterozygote (RIG—I ^{+ / _} ) mouse and an MDA5 heterozygote (MDA5 ^{+ / _} ) mouse are crossed, their children become RIG—I ^{+ / +} MDA5 ^{+ / +} , RIG— I ^{+ / _} MDA5 ^{+ / +} , RI G— I ^{+ / +} MDA5 ^{+ / _} , RIG— I ^{+ / _} MDA5 ^{+ / _} For this genotype, genomic DNA extracted from the mouse tail should be used, and PCR or Southern blotting should be used. It can be identified more. Among, RIG- by mating the mouse between each other I ^{^{+ / _}} MDA5 ^{+ / _} ^{^{genotype, RIG- I + / + MDA5 +}} / +, RIG- I + / _ MDA5 + / +, RI G — I ^{+ / +} MDA5 ^{+ / _} , RIG— I ^{+ / _} MDA5 ^{+ / _} , RIG— l _MDA5 ^{+ / +} , RIG— I— / ^_ MDA5 ^{+ / _} , RIG— I ^{+ / +} MDA5 ^{_ / _} , RIG— I ^{+ / _} MDA5 ^{__ / _} , RIG— I ^{__ / _} MD A5 ^{__ / _} Among these, RIG—I ^{__ / _} MDA5 ^{__ / _} mice were identified by PCR or Southern plotting.

[0025] (4) Cells, reagents and viruses

RIG—I—, MEF, and bone marrow-derived GMCSF or Flt3L—DC were prepared as described in literature Immunity 23, 19-28 (2005). PDC and cDC were isolated from Flt3L-DC as described above by MACS using anti-B220 and CD1 lc microbeads manufactured by Miltenyi Biotech. Poly (I: C) was purchased from Amersham Biosciences. An anti-MDA5 polyclonal antibody was produced corresponding to amino acids 1005-1019 of mouse MDA5. NCV, VSV, INFNORE ENZYNORES, ANS1, JEV, EMCV, Tyler virus, and Mengovirus were prepared by methods described in the literature. As SeV (V—) and (Cm) mutants, those provided by Dr. A. Kato were used.

[0026] (5) Northern blot

PEC was treated for 8 hours with or without lOOOUZml of IFN-β, and total RNA was extracted with TRIzol reagent (Invitrogen). The RNA was electrophoresed, transferred to a nylon membrane, and then hybridized with the indicated cDNA probe. A 308 bp fragment (777-1084) was used as a probe to detect MDA5 mRNA expression. The same membrane was rehybridized with the β-actin probe.

[0027] (6) Western blot

MEF was treated with lOOOUZml of IFN—j8 for 8 hours. The cells were then lysed in lysis buffer containing 1.0% Nonidet-P40, 150 mM NaCl, 20 mM Tris—Cl (pH 7.5), ImM EDTA and a protease inhibitor cocktail (Roche). Dissolved. The cell lysate was lysed with SDS-PAGE and transferred to a PVDF membrane. Plotting the membrane with the specific antibody of MDA5 protein and visualizing with enhanced fluorescence system (Perkinermer) [0028] (7) Histological analysis

EMCV-infected mouse force Hearts were collected and fixed with 3.7% formaldehyde. Cross sections (5 μm) were cut across the heart and stained with hematoxylin and eosin.

[0029] (8) Plaque Atsey

EMCV infected mouse hearts were homogenized in PBS. Virus titration in PBS containing virus was confirmed with standard plaque assay as described above. The prepared HeLa cells were infected with serial dilutions. DMEM containing 1% low melting point agarose was overlaid on the cells and incubated for 48 hours. Thereafter, plaques were counted.

[0030] (9) Luciferase Atsey

Embryo 11. MEF obtained from wild-type or RIG-I ^{_ / _} MDA5 ^{_ /} -embryo in 5 days with empty beta (control), RIG-I or MDA5 expression vector and reporter containing IFN-β promoter Transfected temporarily with the construct. As an internal control, Renilla luciferase construct was tranfected. Transfected cells were either mock treated (Mock) or infected with EMCV with increasing moi or SVc— (moi = 20) for 24 hours. Cells were lysed and subjected to luciferase assembly using a dual luciferase reporter assembly system (Promega).

[0031] (lO) lFN-a, IL 12p40, IL 6 and measurement of Rantes production

The culture supernatant was collected and analyzed for IFN-a, IL 6 or IL-12p40 production by enzyme-linked immunosorbent assay (ELISA). An ELISA kit for mouse IFN-α was purchased from PBL Biomedical Laboratories, and an ELISA kit for IL-6, IL-12p40, and Rantes was obtained from R & D Systems.

[0032] (11) Flow cytometry

Mice were injected intravenously with 200 μg of poly I: C, and the mice were discarded 24 hours after the injection. As described above, mouse spleen cells were also prepared using collagenase and DNasel. Spleen cells were stained with CD86-FITC (BD Biosciences Pharmingen), B220-PE, and CDlc-A PC, and analyzed with FACS Calibur (BD Bioscience). [0033] (12) Preparation of in vitro transcribed dsRNA

The mouse Lamin A / C cDNA sequence was amplified by PCR, cloned with the pT7 Blue T vector (Novagen) and sequenced. In another PCR reaction using primers tagged with the following T7 sequences, the ends of the 200 and 400 bp DNA sequences corresponding to mouse Lamin A / C were tagged with a T7 RNA polymerase promoter;

T7 Lamin. Forward, TAATACGACTCACTATAGGacttgttggctgcgcaggc T7 Lamin. 200 Reverse, TAATACGACTCACTATAGGccactcgcctcagcatct cat

T7 Lamin. 400 Reno, TAATACGACTCACTATAGGctgttccacctggtcctca tg

The PCT product was gel purified and used for in vitro transcription and production of dsRNA using the T7 RiboMAX ™ Express RNAi System (Promega) as per manufacturer's instructions.

[0034] (13) Echocardiography

2. In mice anesthetized with 5% avertin (8 μ 1 / g) using an ultrasonography (SONOS-5500, equipped with a 15-MHz linear transducer, Philips Medical Systems). Echo examination was performed. The heart was projected as a two-dimensional parasternal short-axis image, and an M-mode echocardiogram of the mid ventricle was recorded at the papillary muscle level. LV heart rate, anterior and posterior wall thickness, and end-diastolic and end-systolic inner diameters were obtained from M-mode images.

[0035] [Result]

To investigate the functional role of MDA5 in vivo, MDA5 ^{_ / _} mice were established and the contribution of MDA5 to virus recognition was analyzed (see Figures la and lb). MDA5 mRNA expression was not detected in MDA5 ^{_ / _} cells, as was the case with MDA5 protein (see Figures lc and Id). In contrast to RIG—mice, many of whom die at the embryonic stage, MDA5 ^{_ / _} mice are born and grow healthy according to Mendelian ratio, and no macroscopic developmental abnormalities are observed until age 24 weeks. I got it. Flow cytometric analysis of CD3, B220, and CD11c in the spleen and lymph nodes shows no significant change in the number of lymphocytes and rods between wild-type and MDA5 ^{__ / _} mice! It was confirmed. [0036] TLR3, RIG-I and MDA5 are believed to be involved in the recognition of poly I: C and the subsequent induction of antiviral responses. However, the in vivo contribution of these molecules in response to dsRNA stimulation has not yet been clarified. Therefore, the inventors next examined the in vivo response to poly I: C in mice deficient in RIG-I, MDA-5, TRIF, or both MDA-5 and TRIF. Surprisingly, administration of poly I: C rapidly induced IFN-a, IL 6 and IL-12 in the serum of both wild-type and RIG-I— and mice, and RIG-I was poly-I in vivo. : It was shown not to be essential for the response via C (see Figure 2a). In sharp contrast, the production of IL-6 and IL-12ρ40, in which MDA5— 'mice do not produce IFN-α in response to poly I: C, was also significantly reduced. In addition, the production of force IL-12p40, which produced normal amounts of IFN-a, was significantly reduced in TRIF mice, and the production of IL-6 was also significantly reduced. Furthermore, MDA5 ^{_ / _} TRIF ^{_ / _} mice did not induce IFN-a, IL-6, and IL-12ρ40 in response to poly I: C (see FIG. 2b). These results indicate that MDA5 is essential for poly I: C-induced IFN-α production and TLR3 signaling is important for IL-12 production, while both MDA5 and TLR3 regulate IL 6 production. It shows that In addition to the induction of site force-in, poly-I: C treatment of wild-type mice also enhanced the surface expression of costimulatory molecules such as CD86 in conventional spleen DC!] (See Figure 2c). Absence of either MDA5 or TRIF did not affect CD86 expression. Double deficiency resulted in inhibition of CD86 up-regulation. This indicates that both the power of both MDA 5 signaling and TLR3 signaling is involved in in vivo poly I: C responsive c DC activity. When bone marrow-derived DC (GMCSF-DC) prepared using GM—CSF is stimulated with poly I: C, wild-type or RIG— 1 ^{_ /} — mouse! /, Either cell is poly I: C In response to this, IFN-a, IL-6 and IL-12p40 are produced in a dose-dependent manner. In contrast, MDA5 GMCSF-DC had a severe reduction in the production of these site forces (see Figure 2d). Therefore, it was confirmed that in the cytoplasm, MDA5, not RIG-I, mainly recognized poly I: C!

[0037] However, inosinic acid is not used in the virus replication process in the natural state! Therefore, the present inventors synthesized long dsRNA by in vitro transcription and lacked MDA5 or RIG-I. IFN response in mouse embryonic fibroblasts (MEF). MEF was stimulated with two dsRNAs (200 bp and 400 bp) of different lengths corresponding to the sequence of mouse Lamin AZC forming a complex with Lipofuxion reagent. Unlike poly I: C, wild-type and MDA5 ^{_ / _} MEF produced equivalent amounts of IFN-β (see Figure 2e). RIG — I ^{__ / _} MEF did not produce any detectable amount of IFN — β. This indicates that it is essential for the detection of RIG-I force S in vitro transcribed dsRNA. In addition, in vitro transcribed dsRNA corresponding to several other sequences induced type I IFN via RIG-I rather than MDA5. Overall, this provides evidence that MDA5 and RIG-I are differentially involved in the detection of poly I: C and in vitro transcribed dsRNA, respectively.

From these findings, the present inventors hypothesized that RIG-I and MDA5 are involved in the detection of different RNA viruses. RNA viruses are classified into negative strand ssRNA virus, plus strand ssRNA virus, and dsRNA virus based on the genome structure. We have previously shown that a series of negative strand RNA viruses are recognized by RIG-I. We first examined the production of IFNα in MDA5 MEF in response to a series of negative strand ssRNA viruses including NDV, Sev, VSV, and influenza virus. Because most wild-type viruses other than NDV do not induce IFN-a in MEF because the IFN response is suppressed by viral proteins (data not shown), we Mutant viruses lacking inhibitory proteins were also used. Vigorous viruses include V protein deficient (V-) or mutated C protein (Cm) SeV, M protein mutant deficient VSV (NCP), and influenza virus deficient in NS 1 protein (A NS I) is included. As shown in Figure 3a, RIG-I ^{_ / _} MEF significantly impaired IFN-α production compared to wild-type cells, but MDA5 was responsible for the production of IFN-α in response to these viruses. Was not indispensable. Paramyxovirus V proteins such as Sev have been shown to cooperate with MDA5 to suppress dsRNA-induced IFN production! /. Both wild type and SeVV (-) induced expression of IP-10 gene and type I IFN in control and MDA5 MEF (Fig. 2a and Fig. 6). RIG-I- and MEF did not respond to both wild type and SeVV (-). This is related to the presence of virus inhibitory proteins This shows that Magna5 does not contribute to SeV recognition. The Japanese encephalitis virus (JEV), a plus-strand ssRNA virus belonging to the flavivirus, also did not require the force MDA5 that required RIG-I to produce IFN-β (Fig. 3b).

[0039] Next, the IFN response of MEF to encephalomyocarditis virus (EMCV), which is a positive strand ssRNA virus belonging to the picornavirus family, was examined. Surprisingly, EMCV-induced IFN-β production was inhibited in MDA5 ^{_ / _} MEF (Fig. 3c). In contrast, wild-type and RIG-I ^{_ / _} MEF produced equivalent amounts of IFN-β, indicating that EMCV is specifically recognized by MDA5 (Figure 3c). . In addition, the production of IFN-α and IL-6 in response to E MCV is inhibited by MDA5 GMCSF-DC, which means that MDA5, which is not RIG-I, is an EMCV in MEF and conventional DC. It is indispensable for the recognition (Figures 3d and 3e). In addition, other viruses belonging to the Picornavirus family, Tyler virus and Mengo virus, also induced IFN-α via MDA5 (Fig. 7).

[0040] Next, the induction of IFN-β in response to exposure to SeVCm or EMCV was completely inhibited, and RIG-I or MDA5 expression vector was transferred to RIG-I-, MDA5 double-deficient MEF. As a result, a reconstruction experiment was performed (Fig. 3f). Local expression of human RIG-I but not MDA5 resulted in SeVCm-responsive IFN- | 8 promoter activity. Conversely, cells expressing human MDA5 but not RIG-I activated the IFN-8 | 8 promoter in a dose-dependent manner in response to EMCV (Fig. 3f). These results indicate that human RIG-I and MDA5 recognize different RNA viruses! /.

[0041] Previous studies have shown that pDC primarily uses the TLR system instead of RIG-I for the recognition of several RNA viruses. MyD88 is an adapter protein involved in signaling of various TLRs except TLR3. pDC highly expresses TLR7 and TLR9, and generates type I IFN in a MyD88-dependent manner upon exposure to the virus. To address whether MDA5 is involved in virus detection in pDC, the present inventors exposed B220 + pDC purified from Flt3L-constructed BM-derived (Flt3L-DC) DC to EMCV, and IFN—α Production was measured. PDC from MyD88 mice but not from MDA5 ^{__ / _} mice showed a severe defect in IFN-α production. This is because RIG-I and MDA5 !, the gap also plays an important role in pDC! / Instant! Show that! /, (Figure 8). Conversely, production of IFN-α in B220—conventional DC purified from Flt3L—DC requires MDA5 instead of MyD88 (FIG. 8). These results indicate that neither MDA5 nor RIG-I is essential for viral induction of IFN-α in pDC.

[0042] In addition, the in vivo role of MDA5 or RIG-I in host defense against viral infection was examined. By mating RIG- mice with force ICR mice that mostly die during the embryonic stage, we have been able to efficiently obtain viable adult mice. Adult RIG— mice showed no macroscopic developmental abnormalities and survived after 20 weeks of age. When mice were infected with JEV, serum IFN-α levels were significantly reduced in RIG- mice compared to littermate control mice. In contrast, Μ mice did not show a major defect in JEV-induced systemic IFN-α production (Figure 4a). Compared to the control mice, it was RIG-I-, a mouse that was more susceptible to JEV infection than the MD A5 mouse, which is consistent with the above findings (Fig. 4b). In addition, RIG-1 ^{_ /} -mice, but not MDA5 ^{_ / _} mice, died of VSV infection, which is consistent with inhibition of the IFN response (Fig. 4c). Thus, recognition of a series of viruses through RIG-I plays an important role in antiviral host defense in vivo.

[0043] Furthermore, mice infected with EMCV as a virus model recognized by MDA5 were examined. In serum of MDA5 mice, induction of IFN-a, RANTES and IL-6 was severely reduced. This indicates that MDA5 is essential for the induction of humoral responses in vivo during EMCV infection (Figure 5a). Therefore, the susceptibility of MDA5 ^{_ / _} mice to EMCV infection was further investigated and compared with that of IFN-a, BR- ', MDA5 "^, RIG ^{_ / _} , and TLR3 ^{_ / _} mice. As shown in Figure 5b, the MDA5 ^{__ / _} and IFN— mice were compared to the control littermate mice.

Susceptibility to CV infection was high. In contrast, the 1¾0-1 or 11 ^ 3 deficiency did not affect the survival of EMCV-infected mice. EMCV is known to selectively infect cardiomyocytes and cause myocarditis. M compared to control mice This markedly increased virus titer in the heart of DA5 ^{_ / _} mice was consistent with increased sensitivity to EMCV (Figure 5c). Histological analysis of the heart 2 days after EMCV infection revealed that focal necrosis of cardiomyocytes had developed in MDA5 ^{_ / _} mice, but the heart of wild-type mice at that time No abnormalities were shown (Fig. 5d). It should be noted that at this point immune cell infiltration was not observed in either wild type or MDA5_ ^/ one heart sections. To assess changes in cardiac function in response to EMCV infection, cardiac work was analyzed by echocardiography two days after infection (Figure 5e). Cardiac contractility, determined by shortening rate (FS), was severely reduced in MDA5 ^{__ / _} mice (wild type 48.2 ± 4.9% vs. MDA5 21.2 ± 5.8%). This suggests that MD A5 ^{__ / _} mice suffered severe heart failure due to virus-induced cardiomyopathy (Figure 5f). Thus, recognition of EMCV via MDA5 is a prerequisite not only to prevent myocardial dysfunction but also to trigger an antiviral response. Overall, these results indicate that RIG-I and MDA5 play an essential role in the recognition of different groups of RNA viruses and the subsequent generation of type I IF N and inflammatory site force-ins! / ヽShow clearly that! /

Industrial applicability

In the present invention, it has been clarified that MDA5 specifically recognizes a virus of the picornavirus family. MDA5 is a helicase, and by regulating this function, it is possible to control infection of viruses belonging to this group, such as poliovirus, Coxsaki virus, rhinovirus that is the cause of cold symptoms. There is sex. In addition, MDA5 ^{_ / _} mice are expected to be used as model mice for human viral infections by mating with gene mutant mice expressing human virus receptors. In addition, drugs that control the activity of MDA5 can be screened using MDA5-deficient (MDA5 ^{__ / _} ) mice and control (wild-type) mice, for example, mated with mice expressing the poliovirus receptor, It can be a model animal susceptible to poliovirus infection. This mouse can be used to screen for new drugs.

Claims

The scope of the claims

[1] A function of expressing MDA5 by inactivating or deactivating all or part of the endogenous gene of human animal encoding MDA5 (Melanoma differentiation associated gene—5) A non-human animal model that is a non-human animal model that is a non-human animal that has lost the response of type I interferon to infection with a virus belonging to the picornavirus family.

[2] The non-human model animal according to claim 1, wherein the non-human model animal is a model mouse.

[3] All or part of endogenous gene encoding MDA5 (Melanoma differentiation associated gene—5) was destroyed or inactivated by gene mutation such as deletion or replacement, and the function of expressing MDA5 was lost. A method of using a non-human animal as a type I interferon-unresponsive non-human model animal that lacks a type I interferon response to infection with a virus belonging to the picornavirus family.

4. The method according to claim 3, wherein the non-human model animal is a model mouse.

[5] A function that inactivates all or part of the endogenous genes of human animals that encode MDA5 (Melanoma differentiation associated gene— 5) and expresses MDA5 by deactivating or substituting the gene. Infecting non-human animals that have lost the virus with a virus belonging to the picornavirus family and administering the test substance before, after, or simultaneously with the virus infection, the amount of type I interferon, RANTES, or IL-6 produced in the non-human animals And when the production of type I interferon, RANTES, or IL-6 is observed, the test substance is evaluated as an anti-picornavirus agent.

[6] The identification method according to [5], wherein the comparison is performed with a production amount of type I interferon, RANTES or IL-6 in a wild-type animal of the same species as the non-human animal.

7. The identification method according to claim 5 or 6, wherein the non-human animal is a mouse.

[8] A function of expressing MDA5 by inactivating or deactivating all or part of the endogenous gene of human animal encoding MDA5 (Melanoma differentiation associated gene—5) The lost non-human animal is infected with myocarditis virus (EMCV) belonging to the picornavirus family, and the test substance is injected before, after, or simultaneously with EMCV infection. When the histological analysis of the heart of the non-human animal is conducted and the suppression of focal necrosis of cardiomyocytes is observed, the test substance is evaluated as an anti-EMCV agent. Identification method.

[9] The identification method according to claim 8, wherein the method is compared with a degree of inhibition of focal necrosis of cardiomyocytes in a wild-type animal of the same species as the non-human animal.

10. The identification method according to claim 8 or 9, wherein the non-human animal is a mouse.

[11] A function that inactivates all or part of the endogenous genes of human animals that encode MDA5 (Melanoma differentiation associated gene— 5), and inactivates them by gene mutations such as deletion and replacement, and expresses MDA5 Infecting a cell belonging to the Picornavirus family with a cell derived from a non-human animal that has lost the amount of the test substance, contacting the test substance with the cell before, during, or simultaneously with the virus infection, the I in the cell derived from the non-human animal The production amount of type I interferon, RANTES or IL-6 is measured, and when the type I interferon, RANTES or IL-6 production is observed, the test substance is evaluated as an anti-picornavirus agent. Method for identifying anti-picornavirus agent.

[12] The identification method according to [11], wherein the comparison is performed with a production amount of type I interferon, RANTES or IL 6 in a cell derived from a wild-type animal of the same species as the non-human animal.

13. The identification method according to claim 11 or 12, wherein the non-human animal is a mouse.

[14] The identification method according to any one of [11] to [13], wherein the cells are embryonic fibroblasts or rod-shaped cells.