JP2005312304A - New interferon control factor activated polypeptide and nucleic acid encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an IFN inducer by finding a substance to directly activate IRF-3 (Interferon Regulatory Factor) and to obtain a nucleic acid encoding the IFN inducer. <P>SOLUTION: An RIG-I (Retinoic acid-inducible gene-I) variant for promoting production of interferon is obtained by constructionally activating an IRF group. The MDA-5 (Melanoma Differentiation Antigen-5) variant for promoting production of interferon is obtained by constructionally activating an IRF group. The nucleic acid encodes the RIG-I variant or the MDA-5 variant. The polypeptide functioning as the IFN inducer and the nucleic acid encoding the polypeptide are obtained for the first time by finding a substance to directly activate the IRF-3. Since administration of the polypeptide to an organism induces the production of IFNs in vivo, the peptide is useful for treating cancers, virus diseases or autoimmune diseases. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel interferon regulator activation polypeptide, a nucleic acid encoding the same, and uses thereof.

  Interferon (IFN) is a cytokine and was discovered in 1957 as an in vivo factor that suppresses influenza virus growth. Subsequent studies have revealed that it has various physiological activities. At the time of discovery, it became clear that it had anti-cancer effects in addition to anti-viral effects, and as its effectiveness became clear in animal experiments, research to apply IFN to the treatment of viral diseases and cancer Became active. This could be done by producing IFN in vitro and administering it to the patient (outpatient method), or by producing IFN in the patient's body (endogenous method).

  Regarding the internalization system, various IFN inducers (inducers) have been studied, but no inducer that can be administered to humans has been found. On the other hand, with regard to the outpatient method, the application of the results of examination of the endogenous method and the development of genetic engineering can establish a method for stably producing large amounts of IFN, and it is possible to use IFN as a therapeutic agent It was.

  Currently, IFN is used as an antiviral drug, anticancer drug or immunomodulator. In particular, IFN-α and -β classified as type I IFN are used worldwide as anti-human hepatitis C virus drugs or anti-cancer drugs.

  As a result of the examination of the endogenous method, viral infection and double-stranded RNA were found as methods that can produce IFN with a certain degree of efficiency in the examination at the cellular level. On the other hand, many substances have been studied as an inducer at the animal level, but the system is too complex for its mechanism, many indirect actions are possible, and it cannot be understood uniformly, There are currently no inducers in clinical use. However, for the reasons described above, the clinical application of inducers is considered extremely useful.

  The production mechanism of IFN at the time of virus infection is shown in FIG. Based on studies to date, IFN is produced by viral infection. Primary production activation and produced IFN stimulate the cells in an autocrine manner, resulting in secondary production that explosively enhances IFN production. This can be divided into bioactivation (Non-patent Document 1). However, stimulation with IFN alone cannot activate IFN production. This indicates that in addition to IFN stimulation, activation of some cell signaling system by virus stimulation is necessary. Therefore, clarifying this primary production activation mechanism is considered to enable the development of inducers.

  From the analysis of IFN production mechanism, IRF-1, 2, 3, 4, 7, ICSBP (IRF-8) and ISGF3g (IRF-9) were found as important cell signaling factors for IFN production. (Non-patent document 1) (IRF is an Interferon Regulatory Factor. Among these factors, we have identified IRF-3 as the most important factor for the mechanism of activation of secondary production. However, overexpression of IRF-3 failed to enhance IFN production, ie, IFR-3 activation was required for increased IFN production rather than a quantitative increase in IRF-3. It was made clear (Non-Patent Document 2).

  Therefore, a substance that directly activates IRF-3 was considered as an inducer that efficiently produces IFN. This method has a simple production mechanism and is considered to be clinically applicable.

  If locally endogenous IRF-3 can be artificially activated, systemic high-dose administration of IFN is known to have several side effects. Production / action can be induced. In addition to the activation of the IFN gene, IRF-3 is known to directly and strongly activate the gene group induced by IFN, and this direct effect is also expected.

Takaoka, A et al, Cancer Sci., (2003) 94 (5) p.405-411 Yoneyama, M et al, EMBO J., (1998) 17 (4) p.1087-1095

  The object of the present invention is to find a substance that directly activates IRF-3 and provide it as an IFN inducer. The activator is a signal molecule originally functioning in a cell, a variant thereof, or a natural or artificial compound that can activate the signal molecule. Furthermore, an object of the present invention is to provide a nucleic acid encoding the IFN inducer. Furthermore, the object of the present invention is to use them for pharmaceutical use, a method for producing interferon using them, a method for predicting the therapeutic effect of interferon using them as an index, and a screening method for interferon inducers or anticancer agents using them as an index. Is to provide.

  As a result of intensive research, the inventors of the present application have found that a polypeptide encoded by a retinoic acid-inducible gene-I (RIG-I) (polypeptide is also referred to as RIG-I) and a melanoma differentiation antigen. -5 (Melanoma Differentiation Antigen-5, MDA-5) mutants lacking each C-terminal region constitutively activate the IRF group, thereby enhancing interferon production. It discovered that it functions as said IFN inducer, and confirmed it experimentally, and completed this invention.

That is, the present invention provides the following.
(1) A RIG-I mutant capable of enhancing interferon production by constitutively activating the IRF group.
(2) An MDA-5 mutant capable of enhancing interferon production by constitutively activating the IRF group.
(3) The mutant according to (1) or (2), wherein the mutant has a deletion or mutation in a region homologous to the RNA helicase.
(4) The mutant according to any one of (1) to (3), wherein the binding ability to double-stranded RNA is reduced or not at all.
(5) The variant according to (1), which has the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more identity with the sequence.
(6) 1 to several amino acids in the amino acid sequence shown in SEQ ID NO: 2 or the amino acids constituting the sequence are substituted and / or deleted and / or one to several in the amino acid sequence The variant according to (1), which has an amino acid sequence in which one amino acid is inserted and / or added.
(7) The variant according to (2), which has the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 80% or more identity with the sequence.
(8) 1 to several amino acids in the amino acid sequence shown in SEQ ID NO: 4 or the amino acids constituting the sequence are substituted and / or deleted and / or one to several in the amino acid sequence The variant according to (2), which has an amino acid sequence in which one amino acid is inserted and / or added.
(9) A nucleic acid encoding the polypeptide according to any one of claims 1 to 8.
(10) The nucleic acid according to (9), which is cDNA or RNA.
(11) (a) the base sequence shown in SEQ ID NO: 1 or a part thereof, (b) the base sequence that hybridizes with the sequence (a) under stringent conditions, or (c) 1 to 10 in the sequence (a) A nucleic acid encoding a mutant according to (1), wherein the nucleotide sequence has one base deleted, substituted and / or added.
(12) The nucleic acid according to (11), which has the base sequence shown in SEQ ID NO: 1.
(13) (a) the base sequence shown in SEQ ID NO: 3 or a part thereof, (b) the base sequence that hybridizes with the sequence (a) under stringent conditions, or (c) 1 to 10 in the sequence (a) A nucleic acid encoding a mutant according to (2), wherein the nucleotide sequence has one base deleted, substituted and / or added.
(14) The nucleic acid according to (13), which has the base sequence shown in SEQ ID NO: 3.
(15) An expression cassette comprising the nucleic acid according to any one of (9) to (14), a promoter that enables expression of the nucleic acid, and a transcription termination signal.
(16) A vector comprising the nucleic acid according to any one of (9) to (14) or the cassette according to (15).
(17) The vector according to (16), which is a plasmid vector.
(18) The vector according to (16), which is a viral vector.
(19) The vector according to (18), wherein the viral vector is derived from an adenovirus, a retrovirus, an AAV virus or a herpes virus.
(20) A defective recombinant retrovirus whose genome comprises the nucleic acid according to any one of (9) to (14) or the cassette according to (15).
(21) A defective recombinant adenovirus whose genome comprises the nucleic acid according to any one of (9) to (14) or the cassette according to (15).
(22) A pharmaceutical composition comprising at least one of the nucleic acids according to (9) to (14).
(23) A pharmaceutical composition comprising at least one expression cassette according to (15).
(24) A pharmaceutical composition comprising at least one vector according to any one of (16) to (19).
(25) A pharmaceutical composition comprising at least one variant according to (1) to (8).
(26) The pharmaceutical composition according to any one of (22) to (25), as a cancer therapeutic agent or a viral disease therapeutic agent.
(27) A host cell is transformed with the nucleic acid according to any one of (9) to (14) or the vector according to any one of (16) to (19), and an interferon group is added to the transformed host cell. A method for producing an interferon group, comprising: recovering the produced interferon group.
(28) Interferon obtained by allowing a test substance to act on a cell and quantifying the RIG-I variant or mRNA thereof described in (1) or the MDA-5 variant or mRNA thereof described in (2) in the cell Of predicting the therapeutic effect of
(29) A screening method for an interferon inducer or anticancer agent using as an index the activation of the RIG-I mutant according to (1) and / or the MDA-5 mutant according to claim 2.

  According to the present invention, a substance that directly activates IRF-3 was found, and a polypeptide that functions as an IFN inducer and a nucleic acid encoding the same were provided for the first time. Since administration of the polypeptide of the present invention to a living body induces IFN production in the living body, the polypeptide of the present invention is useful for cancer treatment, viral disease treatment, autoimmune disease treatment, and the like. In addition, by administering a vector containing the nucleic acid of the present invention to a living body, the polypeptide of the present invention can be produced endogenously in vivo, and also cancer treatment, viral disease treatment, autoimmune disease treatment, etc. Useful for. In addition, IFN production by the cells can be enhanced by administering the polypeptide of the present invention to an IFN-producing cell in vitro or by transforming the cell with a vector containing the nucleic acid of the present invention. Furthermore, it is possible to predict the therapeutic effect of interferon and to screen for an interferon-inducing substance or an anticancer agent using the amount of the polypeptide of the present invention or mRNA encoding the polypeptide as an index.

1. RIG-I and MDA5 mutants related to the present invention
Retinoic acid-inducible gene-I (RIG-I) was identified as a protein induced by retinoic acid, and Melanoma Differentiation Antigen-5 (MDA-5) was identified as a gene expressed during melanoma differentiation. Both are characteristic proteins with a caspase recruitment domain (CARD) on the N-terminal side of the protein and a helicase domain behind it. Of these proteins, it has been clarified in the present invention that deletion of the helicase domain can constitutively enhance IFN production. This suggests that RIG-I and MDA-5 mutants that have lost or reduced helicase function by mutating or deleting the Helicase domain constitutively enhance IFN production. . Accordingly, the present invention includes RIG-I and MDA-5 mutants that lack or reduce helicase function and nucleic acids that encode those mutants.

  A preferred example of the RIG-I mutant polypeptide is a polypeptide having an amino acid sequence consisting of 229 amino acids shown in SEQ ID NO: 2. This peptide is a polypeptide having a 1st to 229th amino acid sequence from the N-terminus of natural RIG-I. In general, a polypeptide having physiological activity is a variant in which a small number of amino acids constituting the amino acid sequence are substituted or deleted, or a small number of amino acids are inserted (hereinafter referred to as the above natural mutation). It is widely known that even if an amino acid constituting the body is substituted and / or deleted and / or a small number of amino acids are inserted, it is referred to as a “modified body”. Are known. A modified form of the RIG-I mutant having the amino acid sequence represented by SEQ ID NO: 1 that can enhance interferon production by constitutively activating the IRF group is within the scope of the present invention. included. Such a modified product preferably has an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and in particular, substitution or deletion. The number of amino acids lost and / or inserted is preferably 1 to several. In addition, the identity of amino acid sequences can be easily performed using BLAST which is software widely used in this field. BLAST is available to anyone at the NCBI (National Center for Biotechnology Information) homepage, and can be easily checked for identity using default parameters. The 20 amino acids constituting the natural protein are neutral amino acids having low side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), and neutral amino acids having hydrophilic side chains (Asn , Gln, Thr, Ser, Tyr Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) Can be grouped. When obtaining a modified product in which a small number of amino acids are substituted, the physiological activity of the polypeptide often does not change as long as the substitution is within each of these groups. In addition, since a fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or the modified form of the polypeptide normally maintains the physiological activity of these polypeptides, such a fusion protein is It can also be used.

  A preferred example of the MDA-5 variant polypeptide is a polypeptide having an amino acid sequence consisting of 294 amino acids shown in SEQ ID NO: 4. This peptide is a polypeptide having an amino acid sequence of 1 to 294 from the N-terminus of natural MDA-5. In general, a polypeptide having physiological activity is a physiologically active polypeptide even if it is a modified form in which a small number of amino acids constituting the amino acid sequence are substituted or deleted, or a small number of amino acids are inserted. It is widely known that activity may be maintained. A modified form of the MDA-5 variant having the amino acid sequence represented by SEQ ID NO: 1 that can enhance interferon production by constitutively activating the IRF group is within the scope of the present invention. included. Such a modified product preferably has an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4, and in particular, substitution or deletion. The number of amino acids lost and / or inserted is preferably 1 to several. In addition, the identity of amino acid sequences can be easily performed using BLAST which is software widely used in this field. BLAST is available to anyone at the NCBI (National Center for Biotechnology Information) homepage, and can be easily checked for identity using default parameters. The 20 amino acids constituting the natural protein are neutral amino acids having low side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), and neutral amino acids having hydrophilic side chains (Asn , Gln, Thr, Ser, Tyr Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) Can be grouped. When obtaining a modified product in which a small number of amino acids are substituted, the physiological activity of the polypeptide often does not change as long as the substitution is within each of these groups. In addition, since the polypeptide having the amino acid sequence shown in SEQ ID NO: 4 and the fusion protein containing the above-mentioned mutants and modifications of the polypeptide usually maintain the physiological activity of these polypeptides, Fusion proteins can also be used.

  In addition, nucleic acids encoding the above-described RIG-1 mutant or MDA-5 mutant and the above-described further modifications are also included in the scope of the present invention. An example of the base sequence of a nucleic acid encoding a RIG-1 variant having the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1. An example of the base sequence of a nucleic acid encoding an MDA-5 variant having the amino acid sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 1. Nucleic acids that hybridize with these nucleic acids under stringent conditions and that encode the above mutants or modifications are also included in the nucleic acids of the present invention. Individually, stringent conditions are conditions in which a reaction is performed at 50 to 65 ° C. using a general hybridization solution such as 5 × Denhardt's reagent, 6 × SSC, 0.5% SDS, or 0.1% SDS. In addition, a nucleic acid that is a nucleotide sequence in which 1 to 10 bases are deleted, substituted, and / or added in the nucleotide sequence of SEQ ID NO: 1 or 3, and that encodes the above-mentioned mutant or modified substance, is also within the scope of the present invention include.

2. Methods for expressing RIG-I and MDA-5 mutant nucleic acids according to the present invention The described nucleic acids can be used as inducers for IFN. It can be used with a viral infection treatment, a cancer treatment, or an autoimmune disease treatment. Therefore, the present invention provides the following: It also relates to any expression cassette comprising the described nucleic acid, a promoter allowing its expression and a transcription termination signal. The promoter is selected from promoters that function in mammalian, preferably human cells. Thus, a promoter can be any promoter or inducible sequence that stimulates or represses transcription of a gene specifically or non-specifically, inductively or non-inductively, strongly or weakly. Specific examples include eukaryotic or viral promoter sequences. This may be, for example, a promoter sequence derived from the genome of the target cell. Among eukaryotic promoters, promoters that express constitutive genes (HPRT, PGK, actin, tubulin gene, EF-1 gene, etc.), intermediate filament promoters (GFAP, desmin, vimentin, neurofilament, Promoters such as keratin genes), therapeutic gene promoters (eg, promoters such as MDR, CFTR, factor VIII, apo AI gene), tissue specific (pyruvate kinase, villin, fatty acid binding intestinal protein, smooth muscle α-actin gene) Or a promoter that responds to a stimulus (promoter such as a steroid hormone receptor gene or a retinoic acid receptor gene) can be used. The promoter may also be a promoter sequence derived from a viral genome, such as an adenovirus EIA and MLP gene promoter, an early promoter of CMV, or a promoter such as RSV or LTR. In addition, these promoter portions can be modified by the addition of activating sequences, regulatory sequences, or sequences that allow tissue specific or tissue dominant expression.

  Vectors for introducing foreign genes into mammals and mammalian cells are well known in this field, and various vectors are commercially available. In the present invention, the nucleic acid of the present invention can be inserted into the cloning sites of these commercially available vectors to obtain the vector of the present invention.

3. Use of RIG-I and MDA-5 mutant nucleic acids according to the present invention as therapeutic agents
It can be used as a therapeutic drug for diseases with therapeutic effects of IFN. For example, it can be used for treatment of viral infection, cancer treatment, or autoimmune disease treatment. In a particularly preferred embodiment of the invention, the nucleic acid or cassette is introduced into the vector. The vector to be used is a chemical one (liposome, nanoparticle, peptide complex, lipid or cationic polymer, etc.), a virus-derived one (retrovirus, adenovirus, herpes virus, AAV, etc.), or a plasmid-derived one. possible. The use of viral vectors is based on the natural transfection characteristics of the virus. Thus, for example, adenoviruses, herpes viruses, retroviruses and adeno-associated viruses can be required. The vector of the present invention may be a non-viral substance capable of promoting nucleic acid introduction into eukaryotic cells and expression therein. Chemical or biochemical synthetic or natural vectors are useful natural virus substitutes, especially for convenience and safety reasons and because there are no theoretical restrictions on the size of the DNA to be transfected. is there.

  The nucleic acid or vector used in the present invention may be formulated to be applied to topical administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, subcutaneous administration, intraocular administration, transdermal administration, and the like. The nucleic acid or vector is used in injectable form. Thus, particularly when injected directly into the site to be treated, it can be mixed with any pharmaceutically acceptable vehicle to form an injection solution. This may in particular be a sterile solution, an isotonic solution, or a dry composition capable of forming an injectable solution by the addition of sterile water or physiological saline as the case may be. The present invention relates to all pharmaceuticals including at least one of the aforementioned nucleic acids or vectors.

  The dose is appropriately set according to the purpose of administration, symptoms and the like, and is not particularly limited. However, when administering a polypeptide, the amount of the active ingredient is usually 0.1 mg / day orally for adults per day. In the case of parenteral administration, the dose is about 0.01 μg to 10 mg. Moreover, when inject | pouring into a solid cancer tissue etc., it is about 0.1-10 mg per 100 g tissue normally. In addition, when the vector of the present invention is administered to induce IFN production endogenously, the dose is usually 0.1 mg to 10 mg per kg body weight of the animal for intramuscular, intraperitoneal, intravenous administration, etc. In the case of intradermal administration using a gene gun, it is usually about 0.01 mg to 1 mg per kg body weight of the animal.

4). INDUSTRIAL APPLICATION METHOD OF IFN PRODUCTION BY RIG-I AND MDA-5 VARIANT NUCLEIC ACID INDUCED BY THE INVENTION It is possible to establish cell lines that produce IFN in large quantities. The cells are not limited as long as they are established cells. Examples thereof include, but are not limited to, COS cells, CHO cells, fibroblasts and the like used to produce proteinaceous pharmaceuticals.

5). Prediction of IFN treatment effect by quantification of RIG-I and MDA-5 mutants related to the present invention In the present invention, there is a correlation between the cell sensitivity of IFN-β and the expression level of RIG-I mutant and MDA-5 mutant. I found it. That is, the expression level of RIG-I and MDA-5 mutants is higher in cells where IFN-β action is more likely to appear. This is an important finding for predicting IFN treatment effect and toxicity. In other words, monitoring the therapeutic effect and toxicity by detecting the expression level of RIG-I and MDA-5 variant at the lesion site and the expression level of RIG-I and MDA-5 variant at the non-lesion site Thus, it is possible to prevent the development of unnecessary toxicity. The expression level can be measured by a conventional method such as a method for measuring the mRNA level of each mutant or an immunoassay using an antibody specific for each mutant. Since RIG-I and MDA-5 that do not have a C-terminal deletion do not have an IRF activation effect, when using the mRNA level as an indicator, complete RIG-I or MDA-5 Only the mRNA of RIG-I mutant or MDA-5 mutant is quantified by subtracting the amount of mRNA coding for. Similarly, when using an antibody that reacts with the RIG-I mutant or MDA-5 mutant, complete RIG-I or MDA can be obtained by using monoclonal antibodies whose epitope is the C-terminal region of these mutants. RIG-I mutant and MDA-5 mutant can be measured separately from -5.

  Hereinafter, the present invention will be described in more detail with reference to examples. These examples are illustrative of the invention and are not intended to limit the scope of the invention.

Identification of activators of IRF-3
The human cancer cell line K562 cells are known to lack endogenous type I IFN genes such as α and β types. We found that even when these cells were infected with Newcastle disease virus (NDV), IRF-3, a transcription factor essential for inducing expression of type I IFN, was not activated. However, activation of IRF-3 was observed when these cells were pretreated with IFN and then infected with NDV. Therefore, it was considered that the protein encoded by the gene newly induced by IFN treatment is essential for the activation of IRF-3 by the virus. Expression cloning was performed for the purpose of searching for this unknown gene.

1. Identification of RIG-I variants
Treat K562 cells with 1000 units / ml IFN-β (Toray Industries, Inc.) for 10 hours, extract total RNA using ISOGEN (Nippon Gene), and then prepare mRNA using Oligotex-dT30 mRNA Purification kit (TAKARA) did. CDNA was synthesized from the obtained mRMA using SMART cDNA Library Construction kit (Clontech), and inserted into a mammalian cell expression vector pEFBOSDori to prepare an expression cDNA library. The library was transformed into an E. coli host to obtain a clone. After culturing 20 clones together, plasmid DNA was extracted. Such a pool was cultured in 4992 (theoretical total number of clones: 4992 × 20 = 99,840). Identification of an activator of IRF-3 was carried out by transfecting mouse L929 cells with DNA from each pool and two types of reporter genes simultaneously using lipofectamine (Invitrogen). The reporter is a firefly luciferase gene (p-55C1BLuc) having 8 repeats of the IRF binding sequence upstream of the promoter sequence, and a Renilla luciferase gene (ptkRL) having a herpes virus-derived thymidine kinase gene promoter. The former aims to evaluate gene activation by the IRF family including IRF-3, and the latter aims to correct the efficiency of DNA transfection. At 24 hours after transfection, the cells were stimulated with polyI: C by DEAE dextran method and 50 microM Tilorone, and each luciferase activity was measured using Dual-Luciferase Assay System (Promega) after 6 hours. After correcting the firefly luciferase activity with the Renilla luciferase activity, repeat the assay multiple times for the highly active pool, obtain each clone retrospectively from the DNA for the reproducible pool, adjust the DNA, and perform the same as in the primary screening The assay was repeated. After confirming the reproducibility of the clone with high activity again, the base sequence of the cDNA insert was determined. One of the obtained clones, B-H12, has the highest luciferase activity and, as a result of sequencing, encodes the N-terminal part of Retinoic Acid Inducible Gene-I (RIG-I) There was found. In this clone, the translation starting from ATG to the 284th amino acid was encoded, and then 22 amino acids derived from the vector were added. As a result of detailed analysis, it was found that the vector-derived amino acid sequence is irrelevant to the activity, and that the above-described activation occurs if there is at least a portion from the start codon to the 229th (RIG-I 1-229). Next, to confirm whether full-length RIG-I has the same activity as RIG-I 1-229, human 293 cells were combined with RIG-I 1-229 and full-length RIG-I (RIG-I FL). Overexpressed. The full length of RIG-I was cloned using the PCR method. SEQ ID NO: 5 (5′-aat tca tgt cca tag tag-3a), SEQ ID NO: 6 (5′-ctc att ccc tgt agc tga ag-3 ′) and SEQ ID NO: using the cDNA used above as a template 7 (5'-cga ttt act aag gtg gac atg-3 ') as a primer, full length RIG-I is a combination of SEQ ID NOs: 5 and 6, and RIG-I1-229 is a combination of SEQ ID NOs: 5 and 7 Ex- PCR was performed using Taq (TAKAR) to obtain cDNA encoding RIG-I full length and RIG-I1-229. This cDNA was inserted into pEFBOSDori to construct expression vectors pEFBOS-RIG-I and pEFBOS-RIG-I1-229. PEFBOS-RIG-I and pEFBOS-RIG-I1-229 were introduced into human 293 cells, and the concentration of IFN-β in the supernatant of the culture solution expressing RIG-I and RIG-I 1-229 was measured by ELISA. Measured by the method (Toray Industries, Inc.). As a result, increased production of IFN-β was confirmed only in the culture supernatant in which RIG-I1-229 was expressed (FIG. 2). Therefore, it was shown that RIG-I 1-229 is an inducer that constitutively activates IRF-3 to enhance IFN production.

2． Identification of MDA-5 mutants After searching the human genome database, we found MDA-5 as a gene with relatively high homology to the RIG-I gene. MDA-5, like RIG-1, was found to have a portion of about 200 amino acids homologous to RIG-I on the N-terminal side of the helicase motif. Therefore, MDA-5 and the N-terminal side of MDA-5 (MDA-5-294) were actually expressed and confirmed. MDA-5 and MDA-5-294 were cloned using the RT-PCR method. SEQ ID NO: 8 (5'-tct tca atc aag tgc taa tc-3 '), SEQ ID NO: 8 (5'- tct tca atc aag tgc taa tc-3') The full-length MDA-5 is a combination of SEQ ID NOs: 8 and 9, and MDA-5-294 is a combination of SEQ ID NOs: 8 and 10, using the number 10 (5'-cca tcg att cac tct tca tct gaa tca ctt c-3 ') as a primer. PCR was performed using Ex-Taq (TAKARA) in combination to obtain MDA-5 and MDA-5-294 cDNA. This cDNA was inserted into pEFBOSDori to construct expression vectors pEFBOS-MDA-5 and pEFBOS-MDA-5-294. Introducing pEFBOS-MDA-5 and pEFBOS-MDA-5-294 into human 293 cells, and measuring the concentration of IFN-β in the supernatant of the culture medium expressing MDA-5 and MDA-5-294 by ELISA (Toray Industries, Inc.) As a result, increased production of IFN-β was confirmed only in the culture supernatant in which MDA-5-294 was expressed (FIG. 3).

  Therefore, it was shown that MDA-5-294 is an inducer that constitutively activates IRF-3 and enhances IFN production in the same manner as RIG-I 1-229.

Antiviral effects of RIG-I and MDA5 mutants
PEFBOS-RIG-I1-229 or pEFBOS-MDA-5-294 was introduced into HeLa cells to express RIG-I1-229 or MDA-5-294 in the cells. This was infected with NDV, and the effect of cell damage caused by viral infection of cells was evaluated. As a result, in the cells in which RIG-I1-229 or MDA-5-294 was expressed in the cells, no cell damage was observed, and virus resistance to NDV was shown. That is, it was shown that RIG-I1-229 or MDA-5-294 exhibits an antiviral action.

Cell growth inhibition by RIG-I and MDA5 mutants
Introducing pEFBOS, pEFBOS-RIG-I, pEFBOS-RIG-I1-229, pEFBOS-MDA-5 or pEFBOS-MDA-5-294 into 293 cells, and then introducing RIG-I, RIG-I1-229, MDA-5 or MDA -5-294 was expressed in cells. The proliferation of cells 3 days after gene transfer was evaluated using MTS (Promega). The proliferation of cells expressing RIG-I1-229 or MDA-5-294 in the cells was compared with the control group (pEFBOS introduction). And it was significantly suppressed. On the other hand, even when RIG-I or MDA-5 was introduced, cell proliferation was not suppressed. From this result, it was revealed that RIG-I1-229 and MDA-5-294 have a cell growth inhibitory action.

Enhanced production of IFN-β by polymerization of RIG-I and MDA5 mutants
HeLa cells were transfected with a construct in which FKBP (FK binding protein) was repeated three times or a construct in which RIG-I1-229 was fused thereto to obtain a clone that was stably expressed. The cells were treated with AP20187, a drug that dimerizes FKBP, for 10 nM for 17 hr, and IRF-3 activation was examined by Native PAGE / Western blotting. As a result, it was found that strong activation of IRF-3 occurs when the fusion of FKBP and RIG-I1-229 is multimerized. At this time, IFN-β activity was confirmed in the cell supernatant (FIG. 4). In addition, when these cells were further cultured for 2 days, cell proliferation was inhibited only in cells in which IRF-3 activation was observed. Therefore, it was found that suppression of cell proliferation was induced by artificially activating the RIG-I mutant. As a result, it was shown that IRF-3 was activated and IFN-β was induced by polymerizing RIG-I and MDA5 mutants without requiring any virus or double-stranded RNA. In addition, by screening for a drug that activates RIG-I, a new IFN inducer or anticancer drug can be screened.

It is a figure explaining the IFN production mechanism at the time of virus infection. It is a figure which compares and shows the production amount of IFN- (beta) by RIG-I and the RIG-I mutant of this invention. It is a figure which compares and shows the production amount of IFN- (beta) by MDA-5 and the MDA-5 variant of this invention. It is a figure which shows activation of IRF-3 by superposition | polymerization of a RIG-I mutant.

Claims (29)

  A RIG-I mutant capable of enhancing interferon production by constitutively activating the IRF group.   An MDA-5 variant that can enhance interferon production by constitutively activating the IRF group.   The mutant according to claim 1 or 2, which has a deletion or mutation in a region homologous to RNA helicase.   The mutant according to any one of claims 1 to 3, wherein the binding ability to double-stranded RNA is reduced or not at all.   The variant according to claim 1, which has the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more identity with said sequence.   Of the amino acid sequence shown in SEQ ID NO: 2 or the amino acids constituting the sequence, one to several amino acids are substituted and / or deleted and / or one to several amino acids in the amino acid sequence The variant according to claim 1, which has an amino acid sequence inserted and / or added.   The variant according to claim 2, which has the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 80% or more identity with the sequence.   Of the amino acid sequence shown in SEQ ID NO: 4 or the amino acids constituting the sequence, one to several amino acids are substituted and / or deleted, and / or one to several amino acids in the amino acid sequence The variant according to claim 2, which has an amino acid sequence inserted and / or added.   A nucleic acid encoding the polypeptide according to any one of claims 1 to 8.   The nucleic acid according to claim 9, wherein the nucleic acid is cDNA or RNA. (A) the base sequence shown in SEQ ID NO: 1 or a part thereof, (b) the base sequence that hybridizes with the sequence (a) under stringent conditions, or (c) 1 to 10 bases in the sequence (a) A nucleic acid encoding a variant according to claim 1, wherein is a nucleotide sequence deleted, substituted and / or added.   The nucleic acid according to claim 11, which has the base sequence shown in SEQ ID NO: 1. (A) the base sequence shown in SEQ ID NO: 3 or a part thereof, (b) the base sequence that hybridizes with the sequence (a) under stringent conditions, or (c) 1 to 10 bases in the sequence (a) A nucleic acid encoding a variant according to claim 2, wherein is a nucleotide sequence deleted, substituted and / or added.   The nucleic acid of Claim 13 which has a base sequence shown to sequence number 3.   An expression cassette comprising the nucleic acid according to any one of claims 9 to 14, a promoter enabling expression of the nucleic acid, and a transcription termination signal.   A vector comprising the nucleic acid according to any one of claims 9 to 14 or the cassette according to claim 15.   The vector according to claim 16, which is a plasmid vector.   The vector according to claim 16, which is a viral vector.   The vector according to claim 18, characterized in that the viral vector is derived from an adenovirus, a retrovirus, an AAV virus or a herpes virus.   A defective recombinant retrovirus whose genome comprises the nucleic acid according to any one of claims 9 to 14 or the cassette according to claim 15.   A defective recombinant adenovirus whose genome comprises the nucleic acid according to any one of claims 9 to 14 or the cassette according to claim 15.   15. A pharmaceutical composition comprising at least one nucleic acid according to claim 9-14.   A pharmaceutical composition comprising at least one expression cassette according to claim 15.   A pharmaceutical composition comprising at least one vector according to any one of claims 16 to 19.   A pharmaceutical composition comprising at least one variant according to claim 1.   26. The pharmaceutical composition according to any one of claims 22 to 25 as a cancer therapeutic agent or a viral disease therapeutic agent.   A host cell is transformed with the nucleic acid according to any one of claims 9 to 14 or the vector according to any one of claims 16 to 19, and the transformed host cell produces an interferon group to produce A method for producing an interferon group, comprising recovering the produced interferon group.   A therapeutic effect of interferon by allowing a test substance to act on a cell and quantifying the RIG-I mutant or mRNA thereof according to claim 1 or the MDA-5 mutant or mRNA according to claim 2 in the cell. How to predict. A screening method for an interferon inducer or an anticancer agent using as an index the activation of the RIG-I mutant according to claim 1 and / or the MDA-5 mutant according to claim 2.

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