WO2011046221A1 - INTERFERON-α MODULATOR - Google Patents

Abstract

Disclosed are: an antisense nucleotide which contains a sequence complementary to IFN-α mRNA, especially a sequence complementary to a base sequence in the SL1 and/or SL2 region of IFN-α mRNA, and which is capable of enhancing the expression of IFN-α; an IFN-α expression enhancer and a prophylactic or therapeutic agent for viral infectious diseases or cancer, each containing the antisense nucleotide; a sense oligonucleotide which contains a sequence complementary to endogenous asRNA for IFN-α mRNA, especially a sequence homologous to a base sequence in the SL1 or SL2 region of IFN-α mRNA, and which is capable of modulating the expression of IFN-α; and an IFN-α expression modulator which contains the sense oligonucleotide.

Description

Interferon-α modulator

The present invention is a result of support from the Japan Science and Technology Agency.
The present invention relates to the use of an antisense nucleotide complementary to mRNA of interferon-α and its interferon-α expression enhancing action, and the use of sense oligonucleotide complementary to antisense RNA and its interferon-α expression regulating action. .

In natural defense against viral infection, innate immunity plays an important role as a rapid defense mechanism in the early stages of infection. Type I interferon (IFN) is a cytokine that plays a central role in antiviral activity in innate immunity. It is secreted transiently by viral infection and acts on surrounding cells to prevent antiviral activity and cytostatic activity. Exhibits various physiological activities. Type I IFNs are divided into IFN-α, -β, -ω, -ε, and -κ. Among these, IFN-α has a group of genes encoding at least 13 functional subtypes on chromosome 9. To do. IFN-α and -β are based on these physiological activities, and plasma-derived preparations and genetically engineered recombinant protein preparations are used for viral infections such as hepatitis B and hepatitis C, tumors, etc. It is used for treatment (or adjuvant therapy) for various diseases.

However, long-term or excessive administration of IFN causes flu-like symptoms (headache, fever, joint pain, muscle pain, loss of appetite, general malaise, nausea / vomiting, etc.), autoimmune diseases, depressive symptoms (insomnia, irritation, etc.) ), Various side effects such as hair loss, thyroid dysfunction, and dementia may occur. For this reason, research is being carried out to protect against viral infection and improve / treat cancer etc. by enhancing IFN production in vivo without administering IFN. However, many substances that enhance IFN production are derived from viruses and pathogens, and many of them have safety problems.

If it is possible to enhance the expression response of type I IFNs such as IFN-α and -β against viral infection, it is considered effective for the prevention and treatment of various viral infections including unknown viruses. Recently, the signal transduction pathway from recognition of viral infection via Toll-like receptor (TLR) to the induction of expression of type I IFN via the kinase cascade has been elucidated. It is not yet fully understood.

Antisense RNA is RNA that has a complementary base sequence to mRNA. Specifically, RNA synthesized using a DNA strand that encodes a sense gene (that is, a non-template strand of mRNA) as a template. It is. Sense-antisense RNA has the ability to form double strands.For example, double-stranded RNA is necessary for RNA interference and double-stranded for degradation of messenger RNA by small RNAs called microRNAs. It is known that RNA is involved. Recent comprehensive cDNA analysis revealed that a large amount of antisense RNA was transcribed (Non-patent Documents 1 and 2). For example, it has been suggested that about 2,500 pairs (Non-patent Document 3) exist in mice and about 2,600 pairs (Non-patent Document 4) exist in humans. It contains a lot of non-translatable antisense RNA.

Although the physiological function of antisense RNA has not been fully elucidated, studies so far have shown that antisense RNA has a variety of physiological functions such as stabilization or destabilization of mRNA and translational suppression from mRNA. It has been found that it belongs to the group of regulatory RNA (Non-patent Document 5).
For example, antisense RNA complementary to endothelial nitric oxide synthase (eNOS) mRNA has been reported to increase in the presence of histone deacetylase inhibitors and decrease eNOS mRNA levels (non-) Patent Document 6). Also in yeast, it is known that antisense RNA of the PHO84 gene suppresses mRNA expression through histone deacetylation (Non-patent Document 7). On the other hand, the present inventors have endogenous antisense RNA (also referred to as “asRNA”) complementary to the 3′-untranslated region (UTR) of inducible nitric oxide synthase (iNOS) mRNA. Antisense RNA hybridizes with iNOS mRNA to stabilize the mRNA, increasing iNOS production and NO synthesis, and sense oligonucleotide complementary to the asRNA binds asRNA to iNOS mRNA It has been reported that the mRNA can be destabilized by inhibiting iNOS and iNOS production and NO synthesis by iNOS can be suppressed ( Patent Documents 1 and 2, Non-Patent Document 8).

However, for the IFN-α gene, it has not been reported so far whether or not endogenous asRNA exists.

WO 2007/142303 pamphlet WO 2007/142304 pamphlet

An object of the present invention is to identify a substance capable of regulating the production of endogenous type I IFN and to provide a novel preventive / therapeutic means for diseases such as viral infections, cancer and autoimmune diseases using the substance. is there. In particular, it is to provide an effective means for preventing and treating viral infections by inducing a persistent type I IFN response to viral infection. In addition, it provides treatment for autoimmune diseases, such as collagen disease and Crohn's disease, where type I IFN production dysregulation caused by an imbalance in the immune system is considered to trigger disease development. That is. Another object of the present invention is to provide a screening system for searching for substances that can regulate the production of endogenous type I IFN.

The present inventors initially focused on the presence of the AU-rich element (ARE) motif, which is thought to be involved in fast turnover of mRNA, similar to iNOS mRNA, in the 3′-UTR of IFN-α mRNA. I thought that endogenous asRNA might be involved in the regulation of IFN-α expression. As a result of strand-specific RT-PCR analysis, the presence of IFN-αasRNA was confirmed (see FIG. 1A). Next, in a preliminary experiment that analyzed changes in the expression of IFN-α mRNA and asRNA after virus infection, IFN-α asRNA was constitutively expressed, but the expression decreased rapidly after the virus infection. A bimodal expression behavior was observed in which the expression level of IFN-α mRNA increased transiently, and the expression of IFN-α い っ た ん mRNA once attenuated and then increased again with the recovery of IFN-α asRNA expression. It was. These results suggested that the regulation of IFN-α expression by IFN-α asRNA was different from that by iRNA-asRNA.

The present inventors have a region (Kimura et al) that forms a secondary structure having two stem loops (SL1 and SL2 (SL2 contains a Bulged SL inside)) important for nuclear export of IFN-α mRNA. ., Medical Mol. Morphol., 43: 145, 2010; see Fig. 2A) and found that deletion of Bulged SL leads to decreased expression of IFN-α mRNA. We thought that IFN-α expression might be regulated by interacting with IFN-α- mRNA targeting the region that forms this secondary structure, not the ARE motif of 3'-UTR. Therefore, a sense oligonucleotide having a sequence of the loop portion of the Bulged SL region was prepared, and the effect of inhibiting the expression of IFN-αasRNA and the effect on the expression of IFN-α were investigated. As a result, the sense oligonucleotide efficiently inhibited the expression of IFN-α asRNA (see FIG. 2B), but the expression of IFN-α asRNA rapidly recovered and increased rapidly after viral infection. The expression of IFN-α mRNA showed the same behavior as that of IFN-α asRNA. On the other hand, in the sense oligonucleotide non-introduced (mock) cells, the expression of IFN-α asRNA and IFN-αRNAmRNA increased more slowly than the introduced cells and then attenuated slowly after virus infection, and after 36 hours after infection The expression of IFN-α mRNA and IFN-α asRNA exceeded the expression level of sense oligonucleotide-introduced cells (see FIGS. 3A and B).

These results suggest that asRNA contributes to the stabilization of IFN-α mRNA and that asRNA knockdown by sense oligonucleotide results in mRNA instability, the present inventors Inhibitors were used to inhibit de novo RNA synthesis, and the effect of IFN-α asRNA knockdown and overexpression on the stability of IFN-α mRNA was examined. As a result, it was confirmed that knockdown of IFN-α asRNA markedly destabilizes IFN-α mRNA (see FIG. 3C), while overexpression of asRNA significantly stabilizes the mRNA ( (See FIG. 5). In addition, Active Hexose Correlated Compound (AHCC), which inhibits iNOS asRNA mRNA stabilization, suppresses constitutive expression of IFN-α asRNA and increased expression due to viral infection, and stabilizes IFN-α mRNA. Inhibited (see FIG. 4). Furthermore, the present inventors have found that the functional center exists in the asRNA region corresponding to the SL2 region (especially the Bulged SL) of the mRNA by overexpressing various fragments of IFN-α asRNA (Fig. 6). This result can be reproduced by introducing an antisense ribooligonucleotide corresponding to the Bulged SL region (see FIG. 7-1), and the action is clearly cytoplasmic (FIG. 7-). 2).
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows.
[1] An antisense nucleotide comprising a sequence complementary to IFN-α mRNA and capable of enhancing IFN-α expression.
[2] The antisense nucleotide according to [1] above, comprising a sequence complementary to the base sequence in the SL1 and / or SL2 region of IFN-α mRNA.
[3] The antisense nucleotide according to the above [2], comprising a sequence complementary to the base sequence in the Bulged SL region of IFN-α mRNA.
[4] An IFN-α expression enhancer comprising the antisense nucleotide according to any one of [1] to [3] above.
[5] The agent described in [4] above, which is used for prevention or treatment of viral infection or cancer.
[6] A sense oligonucleotide comprising a sequence complementary to an endogenous antisense RNA for IFN-α mRNA and capable of regulating the expression of IFN-α.
[7] The sense oligonucleotide according to [6] above, wherein the sequence is a sequence homologous to a base sequence in the SL1 or SL2 region of IFN-α mRNA.
[8] The sense oligonucleotide according to [7] above, wherein the sequence is a sequence homologous to a base sequence in the Bulged SL region.
[9] An IFN-α expression regulator comprising the sense oligonucleotide according to any one of [6] to [8] above.
[10] The agent described in [9] above, which is used for prevention or treatment of abnormal immune response.
[11] A substance for enhancing or suppressing IFN-α expression, characterized by detecting and comparing hybridization of IFN-α mRNA and its endogenous antisense RNA in the presence and absence of a test substance Screening method.
[12] A test substance is brought into contact with a cell expressing IFN-α mRNA and its endogenous antisense RNA, and a change in the amount of mRNA and / or IFN-α protein in the cell is measured. The method according to [11] above.

The antisense nucleotide of the present invention can enhance the production of IFN-α by stabilizing the mRNA in the same manner as endogenous asRNA against IFN-α mRNA. On the other hand, the sense oligonucleotide of the present invention can suppress the overproduction of IFN-α by inhibiting the stabilizing effect of IFN-α asRNA and destabilizing IFN-α mRNA. Furthermore, unlike these IFN-α preparations, these nucleotides can be administered by the patient himself / herself by inhalation and the like, which can reduce the burden on the patient.

(A) IFN-α1 asRNA-derived cDNA (RT1, RT2), various PCR amplification products (PCR1 to 5) from the cDNA, schematic diagrams showing the positions on the IFN-α1 gene, and IFN-α1 asRNA-derived cDNA FIG. 3 is a diagram showing the results of various PCR amplifications (PCR1 to 5) from (RT1, RT2). (B) shows the RT-PCR results for IFN-α1 asRNA using the RNA fraction having a poly A chain and the RNA fraction not having a poly A chain as a template. (C) It is a figure which shows the result of carrying out the comparative analysis of the expression level of IFN- (alpha) 1 (TM) mRNA (S) and IFN- (alpha) 1 (as) RNA (AS) in Namalwa cell 24 hours after Sendai virus (SenV) infection by real-time PCR. (A) IFN-α1α mRNA position responsible for nuclear export (CSS), RNA secondary structure formed by this region (left figure), and deletion mutants of each region of the secondary structure (A: wild It is a figure which shows the expression and localization (right figure) of type mRNA, B: SL1 deletion mutant, C: SL2 deletion mutant, D: Bulged 欠 失 SL deletion mutant. (B) Diagram showing the position of the sense oligonucleotide used for IFN-α1 asRNA knockdown experiments with respect to CSS (upper figure), and the knockdown effect and specificity of IFN-α1 asRNA expression by the sense oligonucleotide (lower figure) It is. (A) shows the results of strand-specific RT-PCR showing the effect of knockdown of IFN-α1 asRNA and IFN-α1 asRNA and IFN-α1 mRNA by sense oligonucleotides. (B) (A) It is the figure which quantified the result shown to (2) by real-time PCR method. (C) IFN-α1 mRNA destabilization by knockdown of IFN-α1 asRNA analyzed under conditions where RNA transcription reaction is inhibited by actinomycin D. (A) shows the results of strand-specific RT-PCR showing the effect of AHCC on the expression of IFN-α1 asRNA and IFN-α1 mRNA. (B) ELISA results showing effects of AHCC on IFN-α protein amount. (A) shows the results of strand-specific RT-PCR showing the effect of overexpression of IFN-α1 / WT asRNA on the expression of IFN-α1 mRNA. (B) (A) It is the figure which quantified the result shown to (2) by real-time PCR method. (C) shows the stabilization of IFN-α1 mRNA by overexpression of IFN-α1 / WT asRNA analyzed under the condition where RNA transcription reaction is inhibited by actinomycin D. (A) It is a figure which shows the result of chain | strand-specific RT-PCR which shows the effect which overexpression of IFN- (alpha) 1 * asRNA and its various fragments exerts on the expression of IFN- (alpha) 1 * mRNA. (B) (A) It is the figure which quantified the result shown to (2) by real-time PCR method. (A) Derived from an antisense ribo-oligonucleotide (asORN) prepared from a functional center sequence in IFN-α1 asRNA determined from the experimental results of Fig. 6 and a sequence forming a base duplex of CSSCRNA secondary structure It is a figure which shows the position with respect to CSS of antisense ribooligonucleotide (ncORN). (B) Quantification of the effects of asORN and ncORN on IFN-α1masRNA and mRNA expression by real-time PCR. (C) Results of analysis of IFN-α1 asRNA and mRNA expression in total RNA, nuclear RNA fraction and cytoplasmic RNA fraction of Namalwa cells 24 hours after SenV infection with asORN or ncORN introduced by strand-specific RT-PCR FIG. (D) (C) It is the figure which quantified the result of (2) by the real-time PCR method.

The present invention provides an IFN-α antisense nucleotide having IFN-α expression enhancing activity. The antisense nucleotide of the present invention may be any nucleotide as long as it contains a sequence complementary to IFN-α mRNA and can enhance the expression of IFN-α protein. Here, the “complementary” sequence is not only a sequence that is completely complementary to mRNA, but also 1 to several (2) as long as it can hybridize with mRNA under physiological conditions of cells and stabilize mRNA. , 3, 4 or 5) may contain base mismatches. Preferably, the sequence complementary to IFN-α mRNA is a stringent condition such as the condition described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999 (for example, 6 × SSC (sodium chloride / sodium citrate) / hybridization at 45 ° C., followed by 0.2 × SSC / 0.1% SDS / 50 to 65 ° C. at least once). It is a sequence that can soy.
Specifically, since the endogenous asRNA for IFN-α mRNA interacts with a thermodynamically unstable portion of the mRNA to stabilize the mRNA, the antisense nucleotide of the present invention comprises IFN- It is preferable to include a sequence complementary to a thermodynamically unstable portion in α mRNA. Examples of the portion that is not thermodynamically stable include a region that is in a single-stranded state when the mRNA has a secondary structure (for example, a region corresponding to the loop portion of the stem-loop structure). The secondary structure of IFN-α mRNA is represented by mfold (see GCG Software; Proc. Natl. Acad. Sci. USA, 86: 7706-10 (1989)) based on the nucleotide sequence information of the mRNA. Such an existing RNA secondary structure prediction program can be used for prediction. There are at least 13 functional subtypes in IFN-α mRNA (eg, Science, 209: 1343-1347 (1980), Gene, 11: 181-186 (1980), Nature, 290: 20-26. (1981), Nature, 313: 698-700 (1985), J. Invest. Dermatol., 83: 128s-136s (1984), J. Interferon Res., 2: 575-585 (1982), J. Interferon Res ., 13: 227-231 (1993), J. Biol. Chem., 268: 12565-12569 (1993), Acta Virol., 38: 101-104 (1994), Biochim. Biophys. Acta, 1264: 363- 368 (1995)), and their base sequence information is readily available. For example, the base sequence of human IFN-α1 mRNA (cDNA) is registered as Accession No. AB445100 in the DDBJ / EMBL / GenBank nucleotide database. The base sequence is shown in SEQ ID NO: 1, and the amino acid sequence of human IFN-α1 deduced from the base sequence is shown in SEQ ID NO: 2, respectively.

Preferably, the antisense nucleotide of the present invention is SL1 of IFN-α mRNA (in the human IFN-α1 mRNA base sequence shown in SEQ ID NO: 1, the base sequence shown by base numbers 229 to 305) and / or It includes a sequence complementary to the base sequence in the SL2 region (in the human IFN-α1 mRNA base sequence shown in SEQ ID NO: 1, the base sequence shown as base numbers 308 to 434). From experiments using various deletion mutants of human IFN-α1 mRNA, two regions represented by base numbers 208 to 452 in the base sequence shown in SEQ ID NO: 1 are formed for nuclear export of the mRNA. It became clear that the nuclear export factor needs to recognize the secondary structure consisting of stem loops (SL1 and SL2). Endogenous asRNA against IFN-α mRNA is considered to inhibit the degradation of mRNA by interacting with the mRNA targeting this nuclear export responsible region (also referred to as CSS), so among the sequences in CSS above A region that meets the above conditions, i.e., a nucleotide containing a sequence complementary to the base sequence of the portion that forms a loop structure in the SL1 or SL2 region when the CSS has a secondary structure, is endogenous IFN-α asRNA. In the same manner, IFN-α mRNA can be stabilized, the mRNA level can be increased, and the production of IFN-α protein can be enhanced. In particular, it includes a sequence complementary to the base sequence in the Bulged SL in the SL2 region (in the human IFN-α1- mRNA base sequence shown in SEQ ID NO: 1, the base sequence shown in base numbers 322 to 352) Antisense nucleotides are more preferred.

The length of the antisense nucleotide of the present invention is not particularly limited, and may include the full length of IFN-α endogenous asRNA or any fragment thereof. However, in terms of sequence specificity, it is complementary to the target sequence. Such a portion contains at least 10 bases or more, preferably about 12 bases or more, more preferably about 15 bases or more. From the viewpoint of ease of administration, etc., those having a base length of 500 bases or less, preferably 300 bases or less, more preferably 150 bases or less are mentioned.

Furthermore, when the antisense nucleotide of the present invention is an oligonucleotide having about 10 to 50 bases, the nucleotide is a sequence that causes a sequence-nonspecific reaction (for example, 5′-CG-3 ′, 5′-GGGG-3 ', 5'-GGGGG-3' and the like are preferably selected, and are preferably selected from those having no similar complementary strand sequence in RNA other than IFN-α mRNA. The absence of a similar complementary strand sequence in other RNAs can be confirmed by performing a homology search against the target mammalian genome sequence using the antisense oligonucleotide candidate sequence as a query. . Here, as homology search means, known nucleic acid homology search software (for example, NCBI BLAST (National Center Biotechnology Information Basic Basic Local Alignment Search Tool) NBLAST and XBLAST programs (version 2.0), FASTA program in GCG software package Etc.) can be used. Moreover, as a genomic DNA data set, for example, all human genome data provided by Celera can be used.

The antisense nucleotide of the present invention is used in various forms depending on the method of introduction into cells. For example, when the antisense nucleotide is an oligonucleotide having about 10 to 50 bases, it may be any one of single-stranded DNA, single-stranded RNA, and DNA / RNA chimera, and it is further added with known modifications. There may be. Here, the “nucleotide” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases.

The nucleotide molecule constituting the antisense nucleotide may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a nuclease or the like, a phosphate residue (phosphate) of each nucleotide constituting the sense oligonucleotide is changed to a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. Can be substituted. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R is, for example, CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN and the like may be substituted). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.
The conformation of the sugar part of RNA is dominated by C2'-endo (S type) and C3'-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target asRNA, BNA (LNA) (Imanishi, an RNA derivative in which the conformation of the sugar moiety is fixed to the N-type by bridging the 2 'oxygen and the 4' carbon. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleicides Nucleic Acids , 22, 1619-21, 2003) can also be preferably used.

The antisense oligonucleotide of the present invention is complementary to a commercially available DNA / RNA synthesizer (Applied Biosystems, Beckman, etc.) based on the IFN-α mRNA (cDNA) sequence. It can be prepared by synthesizing the sequence.

The antisense oligonucleotide of the present invention is provided in a special form such as a liposome or a microsphere, or a hydrophobic substance such as a polycationic substance such as polylysine or a lipid (eg, phospholipid, cholesterol, etc.) is added thereto. It can be provided in the form. Alternatively, the antisense oligonucleotide of the present invention is converted to a peptide having a membrane permeation function (for example, Drosophila-derived Antennapedia homeodomain (AntP), human immunodeficiency virus (HIV) -derived TAT, herpes simplex virus (HSV) -derived Modification with a cell-passing domain such as VP22) can promote the uptake of the oligonucleotide into cells.

On the other hand, when the antisense nucleotide of the present invention is a polynucleotide having a longer base length, introduction of the nucleotide into a cell can be carried out using a gene transfer method known per se. In this case, double-stranded DNA is preferably used as the nucleotide. The antisense nucleotide of the present invention, for example, as described in Examples below, is a cell that expresses endogenous IRNA-α asRNA (for example, a cell that highly expresses endogenous IFN-α asRNA by viral infection). The total RNA is extracted from the RNA, and based on the sequence information of IFN-α mRNA shown in SEQ ID NO: 1, a primer that can amplify an appropriate region of its complementary strand sequence is designed and obtained by performing RT-PCR. can do. The obtained cDNA is inserted into an appropriate expression vector containing a promoter that can function in host animal cells. Examples of expression vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV). , PcDNAI / Neo) or the like.
Examples of the promoter used in the expression vector include EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Molone murine leukemia virus) LTR. HSV-TK (herpes simplex virus thymidine kinase) promoter and the like are used. Of these, EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, SRα promoter and the like are preferable.
In addition to the promoter, the expression vector may optionally contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like. Examples of the selection marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, a puromycin resistance gene, and the like.

The antisense nucleotide of the present invention can enhance the expression of IFN-α mRNA and IFN-α protein by stabilizing IFN-α mRNA. Since IFN-α has various physiological activities such as antiviral action, cell growth inhibitory action, and natural killer cell activation action, the medicament containing the antisense nucleotide of the present invention is an IFN-α expression enhancer. As described above, it can be used for prevention and / or treatment of various diseases. Examples of such diseases include respiratory tract virus infection such as influenza, hepatitis B, hepatitis C (active and inactive), herpes infection (genital herpes, corneal herpesitis, oral herpes, etc.), Viral infections such as warts, AIDS, kidney cancer, renal cell carcinoma, breast cancer, bladder cancer, basal cell carcinoma, head and neck cancer, cervical dysplasia, skin malignant tumor, Kaposi sarcoma, malignant melanoma, infantile hemangioma, Chronic granulomatosis, chronic myelogenous leukemia (CML), adult T-cell leukemia, hairy cell leukemia, hairy cell leukemia, T-cell leukemia virus (HTLV-1) myelopathy, multiple myeloma, lymphoma, etc. Acute sclerosing panencephalitis (SSPE), Sjogren's syndrome, multiple sclerosis (MS), stomatitis, genital warts, intravaginal warts, erythrocytosis, thrombocytosis, mycosis fungoides, sudden hearing loss, senile Discoid macular degeneration, Such as jet disease including but not limited to.

The medicament containing the antisense nucleotide of the present invention has low toxicity, and can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or non-human mammal (eg, mouse, rat, guinea pig, rabbit, sheep, Pigs, cows, cats, dogs, monkeys, etc.) can be administered orally or parenterally (eg, inhalation administration, intravascular administration, subcutaneous administration, transmucosal administration, etc.).
When these nucleic acids are used as preventive / therapeutic agents for the above-mentioned viral infections and cancers, they can be formulated and administered according to a method known per se. That is, the antisense nucleotide of the present invention is inserted alone or in a functional manner into the appropriate expression vector for mammalian cells such as a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector and the like. Then, it can be formulated according to conventional means. The nucleotide can be administered as it is or together with an auxiliary agent for promoting intake by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving the pharmacokinetics, prolonging the half-life, and improving the cellular uptake efficiency, the nucleotide may be formulated (injection) alone or together with a carrier such as liposome and administered intravenously, subcutaneously, etc. .

The antisense nucleotide of the present invention may be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the antisense nucleotide of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.

As a composition for parenteral administration, for example, injections, aerosols, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, etc. May be included. Such an injection can be prepared according to a known method. Aerosol formulations can be placed in compressed acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. Alternatively, it may be formulated as an incompressible pharmaceutical product such as a nebulizer or an atomizer. Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient. Examples of such dosage forms include tablets, pills, capsules, injections (ampoules), aerosols, and suppositories. The antisense nucleotide of the present invention is preferably contained, for example, usually 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.

The dosage of the above-mentioned pharmaceutical containing the antisense nucleotide of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for prevention and / or treatment of adult acute hepatitis C The dosage of the antisense nucleotide of the present invention is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight once a day. It is convenient to administer several times by intravenous injection or inhalation. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

Note that the above-described pharmaceutical composition may contain other drugs as long as an undesirable interaction is not caused by the combination with the antisense nucleotide of the present invention. Other drugs include, for example, antiviral drugs, antitumor drugs, antibacterial drugs, antifungal drugs, antiprotozoal drugs, antibiotics, antiseptic drugs, antiseptic shock drugs, endotoxin antagonists, immunomodulators, non-steroidal drugs Examples include anti-inflammatory drugs, steroid drugs, inflammatory mediator action inhibitors, inflammatory mediator production inhibitors, anti-inflammatory mediator action inhibitors, anti-inflammatory mediator production inhibitors.

An oligonucleotide containing a base sequence complementary to the base sequence of IFN-α's endogenous asRNA (that is, a sense oligonucleotide of IFN-α) inhibits the stabilization of asRNA against IFN-α mRNA. -It can destabilize mRNA and regulate the expression of IFN-α. Therefore, the present invention also provides an IFN-α sense oligonucleotide having IFN-α expression-regulating activity. The sense oligonucleotide of the present invention may be any oligonucleotide as long as it contains a sequence complementary to endogenous asRNA for IFN-α mRNA and can destabilize IFN-α mRNA. Here, the “complementary” sequence is not only a sequence completely complementary to asRNA, but also as long as it can hybridize with asRNA under physiological conditions of cells and inhibit the action of asRNA on mRNA, It may contain one to several (2, 3, 4 or 5) base mismatches. Preferably, the sequence complementary to IFN-α asRNA is stringent conditions such as those described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999 (for example, 6 × SSC (sodium chloride / sodium citrate) / hybridization at 45 ° C., followed by 0.2 × SSC / 0.1% SDS / at least one wash at 50 to 65 ° C.). It is a sequence that can soy.
As described above, the endogenous asRNA for IFN-α mRNA interacts with a thermodynamically unstable portion of the mRNA to destabilize the mRNA, so that the sense oligonucleotide of the present invention comprises IFN-α It preferably contains a sequence that is homologous to a thermodynamically unstable portion in the mRNA. Examples of the portion that is not thermodynamically stable include a region that is in a single-stranded state when the mRNA has a secondary structure (for example, a region corresponding to the loop portion of the stem-loop structure).

Furthermore, the sense oligonucleotide of the present invention does not contain a sequence that causes a non-sequence-specific reaction (for example, 5′-CG-3 ′, 5′-GGGG-3 ′, 5′-GGGGG-3 ′, etc.). It is preferable to select from those having no similar sequence in RNA other than IFN-α mRNA. The absence of similar sequences in other RNAs can be confirmed by methods similar to those described above for antisense oligonucleotides.

Preferably, the sense oligonucleotide of the present invention is SL1 of IFN-α mRNA (in the human IFN-α1 mRNA base sequence shown in SEQ ID NO: 1, the base sequence shown by base numbers 229 to 305) or SL2 region (In the human IFN-α1 mRNA base sequence shown in SEQ ID NO: 1, the base sequence shown by base numbers 308 to 434 is included) a sequence homologous to the base sequence. Among the sequences in the nuclear export responsible region (CSS) of IFN-α1 mRNA, a sequence that is homologous to the base sequence of the portion that forms a loop structure in the SL1 or SL2 region when CSS takes a secondary structure. The sense oligonucleotide containing (see FIG. 2) inhibits the action of the asRNA on IFN-α mRNA by hybridizing to IFN-α asRNA, and regulates the mRNA level, thereby producing IFN-α protein. Can be adjusted. In particular, a sense containing a sequence homologous to the base sequence in the Bulged SL (base sequence 322 to 352 in the human IFN-α1 mRNA base sequence shown in SEQ ID NO: 1) in the SL2 region Oligonucleotides are more preferred.
The “homologous” sequence is not only a sequence that is completely the same as a specific partial nucleotide sequence of IFN-α mRNA, but also an mRNA that hybridizes with endogenous asRNA against the mRNA under physiological conditions of the cell. 1 to several (2, 3, 4 or 5) bases may be different as long as the action of asRNA on can be inhibited.

Examples of more specific sense oligonucleotides include the following (numbers in parentheses indicate corresponding regions in the base sequence shown in SEQ ID NO: 1).
Sequence number 3: 5'-CCAGCAGATCTTCAACCTCT-3 '(base number 319-338)
Sequence number 4: 5'-ATCTTCAACCTCTTTACCAC-3 '(base number 326-345)
Sequence number 5: 5'-GATGAGGACCTCCTAGACAA-3 '(base number 368-387)
Sequence number 6: 5'-GACCTCCTAGACAAATTCTG-3 '(base number 374-393)

The length of the sense oligonucleotide of the present invention is not particularly limited, but from the viewpoint of sequence specificity, the portion complementary to the target sequence in IFN-α asRNA is at least 10 bases, preferably about 12 bases or more. Preferably it contains about 15 bases or more. In addition, in view of ease of synthesis, production cost, ease of administration, and the like, those having a base length of 50 bases or less, preferably 40 bases or less, more preferably 30 bases or less are mentioned.

The sense oligonucleotide of the present invention may be any of single-stranded DNA, single-stranded RNA, and DNA / RNA chimera, and may be those with known modifications. Here, the “nucleotide” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. When the sense oligonucleotide is DNA (ODN), the RNA: DNA hybrid formed by the target asRNA and the sense ODN can be recognized by endogenous RNase H and cause selective degradation of the target asRNA.

The nucleotide molecule constituting the sense oligonucleotide may be natural DNA or RNA. However, in order to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA), the above-mentioned antisense is used. As with nucleotides, various chemical modifications can be included.

Based on the IFN-α mRNA (cDNA) sequence, the sense oligonucleotide of the present invention can be sequenced using a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman, etc.). It can be prepared by synthesis.

The sense oligonucleotide of the present invention is provided in a special form such as a liposome or a microsphere, or a form to which a hydrophobic substance such as a polycationic substance such as polylysine or a lipid (eg, phospholipid, cholesterol, etc.) is added. Can be provided at. Alternatively, the sense oligonucleotide of the present invention is converted into a peptide having a membrane permeation function (for example, Drosophila-derived Antennapedia homeodomain (AntP), human immunodeficiency virus (HIV) -derived TAT, herpes simplex virus (HSV) -derived VP22. And the like can be promoted by incorporating the oligonucleotide into a cell.

When a large amount of IFN-α is produced due to virus infection or the like, non-antigen-specific activation of cytotoxic T cells is induced and tissue is damaged. Dead cells, nucleic acids, nucleic acid-protein complexes, etc. are released from damaged tissues, and cellular and humoral immunity against them is induced, resulting from cytotoxic T cells and antigen-autoantibody complexes. Tissue damage may be further exacerbated. The sense oligonucleotide of the present invention regulates the expression of IFN-α mRNA and IFN-α protein by inhibiting the action of endogenous asRNA on IFN-α mRNA when the host immune response is abnormal In addition, various diseases and conditions caused by overproduction of IFN-α can be prevented and / or treated. Such diseases or conditions include, but are not limited to, psoriasis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease, non-Hodgkin's lymphoma, and the like.

The medicament containing the sense oligonucleotide of the present invention has low toxicity, and can be used as a solution or as a pharmaceutical composition of an appropriate dosage form as a human or non-human mammal (eg, mouse, rat, guinea pig, rabbit, sheep, Pigs, cows, cats, dogs, monkeys, etc.) can be administered orally or parenterally (eg, inhalation administration, intravascular administration, subcutaneous administration, transmucosal administration, etc.).
When these nucleic acids are used as a prophylactic / therapeutic agent for diseases and conditions caused by IFN-α overproduction, they can be formulated and administered in the same manner as the antisense nucleotide of the present invention.

The dosage of the above-mentioned medicament containing the sense oligonucleotide of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., for example, prevention of psoriasis caused by IFN-α overproduction in adults and / or When used for treatment, the sense oligonucleotide of the present invention is usually administered at a dose of about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight. It is convenient to administer by intravenous injection or inhalation once to several times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

The above-described pharmaceutical composition may contain other drugs as long as no undesirable interaction is caused by the combination with the sense oligonucleotide of the present invention. Other drugs include, for example, antiviral drugs, antitumor drugs, antibacterial drugs, antifungal drugs, antiprotozoal drugs, antibiotics, antiseptic drugs, antiseptic shock drugs, endotoxin antagonists, immunomodulators, non-steroidal drugs Examples include anti-inflammatory drugs, steroid drugs, inflammatory mediator action inhibitors, inflammatory mediator production inhibitors, anti-inflammatory mediator action inhibitors, anti-inflammatory mediator production inhibitors.

The present invention also provides a method of screening for a substance that enhances or suppresses the expression of IFN-α by regulating the stabilizing action of endogenous asRNA on IFN-α mRNA. The screening method of the present invention is characterized by detecting the hybridization between IFN-α mRNA and its endogenous asRNA in the presence and absence of a test substance and comparing the degree thereof.
For example, RNA can form a physiological secondary structure by isolating IFN-α mRNA and IFN-α asRNA by a conventional method, solidifying one of them and labeling the other with an appropriate labeling agent. Examples include a method of hybridizing both in the presence and absence of a test substance under the conditions and comparing the amount of label bound to the solid phase under both conditions. Here, as mRNA and asRNA, the full length of each may be used, or the nuclear export responsibility region (CSS; base number of human IFN-α1 mRNA shown in SEQ ID NO: 1) 208 to 452), SL2 region (in the base sequence of human IFN-α1 mRNA shown in SEQ ID NO: 1, the region shown as base numbers 308 to 434) or SL1 region (shown in SEQ ID NO: 1) In the human IFN-α1 mRNA base sequence described above, the region indicated by base numbers 229 to 305), and a fragment containing a sequence complementary to those regions of asRNA may be used.
Examples of the solid phase material include semiconductors such as silicon, inorganic materials such as glass and diamond, films mainly composed of high molecular substances such as polyethylene terephthalate and polypropylene, and the shape of the solid phase includes a slide glass, Examples include, but are not limited to, microwell plates, microbeads, and fiber types. As a method of immobilizing mRNA or asRNA on a solid phase, functional groups such as amino groups, aldehyde groups, SH groups, and biotin are introduced into the RNA in advance, while reacting with the RNA on the solid phase. Functional groups (eg, aldehyde group, amino group, SH group, streptavidin, etc.) are introduced, and the solid phase and RNA are cross-linked by covalent bond between the two functional groups, or the polyanionic RNA is immobilized. Examples of the method include, but are not limited to, a method of immobilizing RNA using polycation coating of a phase and electrostatic bonding. The solid-phase RNA preparation methods include the Affymetrix method, which synthesizes RNA one nucleotide at a time on a substrate (glass, silicon, etc.) using a photolithographic method, the micro spotting method, the inkjet method, and the bubble jet (registered trademark) method. The Stanford method in which RNA prepared in advance is spotted on the substrate using the above method, but considering the base length of the RNA to be used, it is preferable to use the Stanford method or a method combining both.
As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ³² P], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent material, for example, fluorescamine, fluorescein isothiocyanate, Cy3, Cy5 and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, biotin- (strept) avidin can also be used for binding between the probe and the labeling agent.
The test substance may be any known substance or new substance, such as a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound library prepared using combinatorial chemistry techniques, solid phase Examples include random peptide libraries prepared by synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 nM, for example. Examples of the incubation time include about 1 to about 24 hours.
After the RNA on the solid phase is contacted with the labeled RNA (and the test substance) and incubated, the RNA that has not bound to the solid phase is washed away, and the amount of the RNA bound to the solid phase is detected. When the amount of label bound to the solid phase is significantly increased in the presence of the test substance compared to the absence, the test substance is selected as a candidate for an IFN-α mRNA stabilizing substance and hence an IFN-α expression enhancing substance. can do. On the other hand, when the amount of the label bound to the solid phase is significantly reduced in the presence of the test substance compared to the absence, the test substance is treated as an IFN-α mRNA destabilizing substance, and hence an IFN-α expression inhibitory substance. Can be selected as a candidate.

In a preferred embodiment, the test substance is contacted with a cell expressing IFN-α mRNA and IFN-α asRNA, and the change in the amount of mRNA and / or IFN-α protein in the cell is measured. A substance that enhances or suppresses IFN-α expression can be selected directly.
Cells expressing IFN-α mRNA and IFN-α asRNA may be cells that can naturally express both RNAs (eg, leukocytes, lymphoblasts, etc.), and either or both of them may be expressed. It may be a recombinant cell into which the DNA to be expressed is introduced, or a cell infected with a virus. In the case of recombinant cells, examples of host cells include animal cells such as H4IIE-C3 cells, HepG2 cells, 293T cells, HEK293 cells, COS7 cells, 2B4T cells, CHO cells, MCF-7 cells, and H295R cells. it can. DNA encoding IFN-α mRNA and IFN-α asRNA is an expression vector having a promoter that can function in host cells after both RNAs are isolated by conventional methods and converted into double-stranded DNA by reverse transcription reaction, etc. And can be prepared by introducing the vector into a host cell by, for example, calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method or the like.

As the test substance, those described above are used. The contact between the test substance and the cell is, for example, a medium suitable for culturing the cell (for example, a minimum essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM), RPMI1640 Medium, 199 medium, F12 medium, etc.) and various buffers (eg, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetate buffer, etc.) It can be carried out by adding a test substance and incubating the cells for a certain period of time. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 nM, for example. Examples of the incubation time include about 1 to about 48 hours. If necessary, the cells may be infected with the virus during the incubation.

After incubation (or over time), extract RNA from the cells and measure the amount of IFN-α mRNA by RT-PCR, real-time PCR or Northern blot analysis, or collect the culture supernatant and Measure the amount of IFN-α protein by various immunoassays and Western blotting. When the amount of IFN-α mRNA and / or IFN-α protein in cells is significantly increased by the addition of a test substance, the test substance can be selected as a candidate for an IFN-α expression enhancing substance. On the other hand, when the amount of mRNA and / or protein is significantly reduced by the addition of a test substance, the test substance can be selected as a candidate for an IFN-α expression inhibitor.

Alternatively, in cells expressing IFN-α mRNA and IFN-α asRNA by viral infection, production of IFN-α and / or variation in virus titer in the presence or absence of the test substance can be measured. As an index, a substance that enhances or suppresses IFN-α expression can also be screened. When the amount of IFN-α produced is significantly increased in the presence of the test substance compared to the absence of the test substance and / or when the virus growth is significantly suppressed, the test substance is a candidate for an IFN-α expression enhancing substance. Can be selected. On the other hand, when the production amount of IFN-α is significantly reduced in the presence of the test substance compared to the absence, the test substance can be selected as a candidate for an IFN-α expression inhibitor.

The IFN-α expression enhancing substance selected by the above screening method can enhance the expression of IFN-α mRNA and IFN-α protein by enhancing the action of endogenous asRNA on IFN-α mRNA. Therefore, pharmaceuticals containing the above IFN-α expression enhancing substance include, for example, influenza virus infections such as influenza, hepatitis B, hepatitis C (active and inactive), herpes infections (genital herpes, cornea) Herpesitis, oral herpes, etc.), warts, viral infections such as AIDS, kidney cancer, renal cell cancer, breast cancer, bladder cancer, basal cell cancer, head and neck cancer, cervical dysplasia, skin malignant tumor, Kaposi sarcoma, Malignant melanoma, infantile hemangioma, chronic granulomatous disease, chronic myelogenous leukemia (CML), adult T cell leukemia, hairy cell leukemia, hairy cell leukemia, T cell leukemia virus (HTLV-1) myelopathy, multiple bone marrow Cancer such as lymphoma, subacute sclerosing panencephalitis (SSPE), Sjogren's syndrome, multiple sclerosis (MS), stomatitis, genital warts, vaginal warts, erythrocytosis, thrombocytosis, fungal flesh Symptom, sudden deafness, old age Sex discoid macular degeneration, are useful for the prevention and / or treatment of such Behcet's disease.
On the other hand, the IFN-α expression inhibitor selected by the above screening method can suppress the expression of IFN-α mRNA and IFN-α protein by inhibiting the action of endogenous asRNA on IFN-α mRNA. . Therefore, the medicament containing the above IFN-α expression inhibitor is useful for the prevention and / or treatment of, for example, psoriasis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease, and non-Hodgkin's lymphoma.

A medicine containing a substance selected by the above screening method has low toxicity, and can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or non-human mammal (eg, mouse, rat, guinea pig, rabbit). , Sheep, pigs, cattle, cats, dogs, monkeys, etc.) or orally (eg, inhalation administration, intravascular administration, subcutaneous administration, etc.). The pharmaceutical composition used for administration may comprise a selected substance and a pharmacologically acceptable carrier, diluent or excipient.

As a composition for parenteral administration, for example, injections, aerosols, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, etc. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared by, for example, dissolving, suspending or emulsifying a selected IFN-α expression enhancing or suppressing substance in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. Aerosol formulations can be placed in compressed acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. Alternatively, it may be formulated as a non-compressible pharmaceutical product such as a nebulizer or an atomizer. Suppositories used for rectal administration may be prepared by mixing the above IFN-α expression enhancing or suppressing substance with a normal suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient. Examples of such dosage forms include tablets, pills, capsules, injections (ampoules), aerosols, and suppositories. The substance that enhances or suppresses expression of IFN-α is preferably contained in an amount of usually 5 to 500 mg, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

The dose of the above-mentioned pharmaceutical containing the above IFN-α expression enhancing or suppressing substance varies depending on the administration subject, target disease, symptom, administration route, etc., for example, prevention and / or treatment of acute hepatitis C in adults When used in the above, the IFN-α expression-enhancing substance as a single dose is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, It is convenient to administer by intravenous injection once to several times a day. In addition, for example, when used for the prevention and / or treatment of adult psoriasis, the IFN-α expression inhibitor is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight. More preferably, about 0.1 to 5 mg / kg body weight is conveniently administered by intravenous injection once to several times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

Hereinafter, the present invention will be described more specifically with reference to examples. However, these are merely examples and do not limit the scope of the present invention.

(Materials and methods)
1. Cell culture Human B lymphoblast cell line Namalwa cells (ATCC CRL-1432) using RPMI1640 medium (Wako) (R10) containing 10% fetal calf serum (FCS; GIBCO) at 37 ° C, 5% Cultivation was performed under CO ₂ conditions.

2. Preparation of virus for cell infection and measurement of titer Inoculate 200 μl of Sendai virus (SeV) (5,120 HA units / ml) diluted 10,000-fold into the chorioallantoic cavity of embryonated chicken eggs (10 days old) at 35 ° C And then refrigerated overnight at 4 ° C. The titer of SeV in chorioallantoic fluid collected from this infected hatched chicken egg was measured by hemagglutination test using chick erythrocytes on the first day.

3. Construction of antisense RNA (asRNA) expression plasmid Human IFN-α1 gene (WT; nt 1-876 (corresponding to the base number of the base sequence shown in SEQ ID NO: 1)) and 5 'fragment of its protein coding region (ED7; nt 68-487) were excised as a HindIII / XbaI fragment from phuIFN-α1 (Kimura et al., JCS, 117: 2259, 2004) and phuIFN-α1 / ED7 expression vectors, respectively. Also, CSS + fragment (nt: 182-488), CSS fragment (nt: 208-452), SL2 fragment (nt: 308-434), BSL fragment (nt: 322-352) from phuIFN-α1 plasmid DNA, Amplified and prepared as a HindIII / XbaI fragment by PCR reaction. These were inserted into the XbaI / HindIII cloning site of the expression vector pCG-BL using the CMV immediate early region promoter in the antisense direction, and human IFN-α1 / WT asRNA, IFN-α1 / ED7 asRNA, IFN-α1 / CSS + asRNA, IFN-α1 / CSS asRNA, IFN-α1 / SL2 asRNA, IFN-α1 / BSL asRNA, phuIFN-α1R, pIFN-α1 / ED7R, pIFN-α1 / CSS + R, pIFN-α1 / CSSR, pIFN-α1 / SL2R, and pIFN-α1 / BR expression vectors were used.

4. Knockdown (KD) and overexpression of asRNA by DNA transfection and overexpression of asRNA functional center by RNA transfection For knockdown of asRNA, cultured Namalwa cells were transferred to a 6-well plate (Nunc) the day before the experiment. The cells were seeded at 3 × 10 ⁶ cells / 2 ml / well and cultured at 37 ° C. and 5% CO ₂ for 24 hours. Thereafter, the solution was centrifuged at 150 × g for 4 minutes at room temperature, and Namalwa cells corresponding to the required number of wells were collected. After floating this cell pellet in R10 with the number of wells x 1 ml, add 30 μl / 3 x 10 ⁶ cells of MAT-S Immobilizer (IBA; 7-2002-100) and stir every 5 minutes at room temperature. For 15 minutes. Then, using the required number of 6-well plates, dispense 1 ml each of the Namalwa cell suspension with Immobilizer adsorbed to each well in which R10 medium was dispensed to 1 ml per well, and a dedicated magnet plate ( IBA 7-2004-000) at room temperature for 20 minutes. During this period, add 200 μl of RPMI1640 containing 3 μg of sense oligonucleotide (hIFN-α1 / Se1) per 3 x 10 ⁶ cells to 3 μl of MAT-A Reagent (IBA 7-2001-100), mix, and mix at room temperature for 15 The transfection mix was made by incubating for a minute. The Immobilizer-adsorbed Namalwa cells on the magnet plate after a predetermined time were added to this well for each well, allowed to stand for 10 minutes, transferred to a CO ₂ incubator, and cultured at 37 ° C. for 6 hours. However, when investigating the specificity of the asRNA KD effect by sense oligonucleotides, in addition to hIFN-α1 / Se1, hIFN-α1 / Se2, Se3, Se4, NSe1, NSe2 are also used. Although cultured for up to 48 hours, these cells were not infected with the virus.
[Sense oligonucleotide]
hIFN-α1 / Se1: 5'-C * C * A * GCAGATCTTCAACC * T * C * T-3 '(SEQ ID NO: 3)
hIFN-α1 / Se2: 5'-A * T * C * TTCAACCTCTTTAC * C * A * C-3 '(SEQ ID NO: 4)
hIFN-α1 / Se3: 5'-G * A * T * GAGGACCTCCTAGA * C * A * A-3 '(SEQ ID NO: 5)
hIFN-α1 / Se4: 5'-G * A * C * CTCCTAGACAAATT * C * T * G-3 '(SEQ ID NO: 6)

[Negative control sense oligonucleotide]
hIFN-α1 / NSe1: 5'-A * A * T * CTCTCCTTCCTCCT * G * T * C-3 '(SEQ ID NO: 7)
hIFN-α1 / NSe2: 5'-C * C * A * GGAGGAGTTTGATG * G * C * A-3 '(SEQ ID NO: 14)
(* Indicates S nucleotide.)
For overexpression of asRNA, add 2.5 μg of pUC12 to 0.5 μg of the various asRNA expression vectors described in 3 above to the Namalwa cells seeded the day before and cultured for 24 hours. And introduced at 37 ° C. for 20 hours.
The IFN-α1 asRNA functional domain was mapped from the examination of the change in the expression level of IFN-α1 mRNA by introduction of these asRNA expression vectors (see the following result 9, FIG. 6). In order to verify the obtained results, the following antisense ribooligonucleotide having an antisense RNA sequence corresponding to the BSL region in the CSS secondary structure of IFN-α1 mRNA, which is the center of the functional domain, was introduced into Namalwa cells. And cultured at 37 ° C. for 6 hours.
IFNA1 / 346-322ORN: 5'-U (M *) G (M *) U (M *) G (M *) G (M *) U (M *) A (M *) A (M *) A (M *) G (M *) A (M *) G (M *) G (M *) U (M *) U (M *) G (M *) A (M *) A (M *) G (M *) A (M *) U (M *) C (M *) U (M *) G (M *) C (M) -3 ′ (SEQ ID NO: 28)
Negative control: IFNA1 / 224-205 ncORN: 5'-G (M *) A (M *) C (M *) A (M *) G (M *) G (M *) A (M *) G ( M *) G (M *) A (M *) A (M *) G (M *) G (M *) A (M *) G (M *) A (M *) G (M *) A ( M *) U (M *) U (M) -3 ′ (SEQ ID NO: 29)
(*: Nucleotides converted to S, M: converted to 2'-o-Me)

5. SeV infection in Namalwa cells Namalwa cells that knock down or overexpress asRNA expression and overexpress the functional center of asRNA by transfection with the above-mentioned sense oligonucleotide, asRNA expression plasmid or antisense ribooligonucleotide (asRNA-minus, asRNA-overexpressed and asORN) (3 x 10 ⁶ cells per well in 6-well plate) or Namalwa cells (mock) with no introduction of the same number of cells, 150 xg each at room temperature The suspension was centrifuged and washed twice for 4 minutes, and the final sediment was resuspended in 2 ml of RPMI1640. Thereafter, SeV was added at a ratio of 50 HA units / 10 ⁶ cells and infected at room temperature for 1 hour. After completion, the mixture was centrifuged and washed twice again at room temperature at 150 × g for 4 minutes, suspended in R10 medium to 3 × 10 ⁶ cells / 2 ml / well, and seeded again in a 6-well plate. Each infected cell was statically cultured at 37 ° C., collected after 0, 1, 3, 6, 12, 24, 30, 36 and 48 hours, and subjected to the following RNA extraction and purification experiments.
In addition, for the purpose of investigating the effect of asRNA knockdown or overexpression on the stability of the RNA, Actinomycin D (ActD) (Sigma A9415) was applied to 0.75 A for knockdown or overexpression cells after a certain time after SeV infection. Or it added so that it might become 1 microgram / ml, and it used for extraction of the following cell total RNA fraction after progress of predetermined time.

6. Extraction of total RNA fraction or nuclear RNA fraction and cytoplasmic RNA fraction, and purification of RNA using various enzymes
6-a. Extraction of total RNA fraction of cells After the specified time has passed, collect asRNA-minus / infected cells or asRNA-overexpressed / infected cells and mock / infected cells, and centrifuge at 150 xg for 4 minutes at 4 ° C. 1 ml of TRIzol (Invitrogen; 15596-018) was added to each cell sediment. Cells were lysed by pipetting in TRIzol, and left at room temperature for 5 minutes. Thereafter, 0.2 ml of chloroform (Wako; reagent grade, 038-02606) was added, and the mixture was shaken and mixed manually for 15 seconds, and then allowed to stand again at room temperature for 2-3 minutes. Subsequently, the mixture was centrifuged at 12,000 xg for 15 minutes at 4 ° C. Since the sample was separated into three layers by this operation, 400 μl was dispensed from the uppermost colorless aqueous layer (about 600 μl) into a new sterilized 1.5 ml microcentrifuge tube. To this tube, 0.5 ml of isopropyl alcohol (Wako; reagent special grade) was added per 1 ml of TRIzol used, mixed by inverting, left at room temperature for 10 minutes, and then centrifuged again at 12,000 x g for 10 minutes at 4 ° C. The supernatant was aspirated and removed, and 1 ml of 75% ethyl alcohol was added to the RNA precipitate. After centrifugation at 7,500 x g for 5 minutes at 4 ° C, the supernatant was aspirated and removed, 1 ml of 99.5% ethyl alcohol (Wako; reagent grade) was added, and the mixture was again centrifuged at 7,500 x g for 5 minutes at 4 ° C. After aspirating and removing the supernatant, the sediment was air-dried and dissolved in sterile double-distilled water. After adding sterile double-distilled water to this RNA solution to a final volume of 210 μl, add 20 mg / ml glycogen (Roche Applied Science; 901 393) to 1 μl, 7.5 M ammonium acetate (Wako; Molecular Biology) (01820485) 1/2 was added, and after stirring, 2.5 99.5% ethyl alcohol was added, and the mixture was allowed to stand at 4 ° C. for 10 minutes. Thereafter, the mixture was centrifuged at 4 ° C., 15,300 × g for 30 minutes, and the obtained RNA precipitate was washed with 75% and 99.5% ethyl alcohol as described above, air-dried, and 50 μl of TE _8.0 (10 mM Tris · HCl, pH 8.0 / 0.1 mM EDTA) and stored at −20 ° C. until the following enzyme reaction.

6-b. Extraction of nuclear RNA fraction and cytoplasmic RNA fraction AsORN or ncORN was introduced, Namalwa cells were collected 24 hours after infection with SenV, and 490 μl of TNM buffer (10 mM Tris- HCl, pH 7.4 / 10 mM NaCl, 3 mM MgCl ₂ , 0.1 mM EDTA, 0.4 units RNase inhibitor, 1 mM DTT). Thereafter, 10 μl of 10% NP-40 was added (final concentration 0.2%), and pipetting was performed using a tip tip, followed by ice cooling for 5 minutes. Subsequently, centrifugation was performed at 4 ° C. at 500 × g for 5 minutes, and 1.2 ml of TRIzol was added to 400 μl of the supernatant to obtain a sample for cytoplasmic RNA fractionation. Subsequently, centrifugation was performed at 800 × g for 1 minute at 4 ° C. using 400 μl of TNM buffer, and this was repeated three times. TRIzol 0.7 ml was added to the final nuclear sediment to prepare a sample for nuclear RNA fractionation. Thereafter, the cytoplasmic RNA fraction and the nuclear RNA fraction were extracted as described in 6-a.

6-c.Protease digestion Add 20 μg of Proteinase K (PCR grade; Roche Applied Science, 03115 887 001) to the above-mentioned crudely extracted total cell RNA, cytoplasm and nuclear RNA, and add Tris / SDS buffer (100 mM Tris-HCl, pH 7.4 / 50 mM EDTA, 2.5% SDS) In 100 μl, the protein component derived from Namalwa cells mixed at 37 ° C. for 15 minutes was digested. Then add 1 volume of RNase-free TE _8.0 saturated phenol (Nippon Gene, 319-90093): chloroform: isoamyl alcohol (Wako; reagent grade; 135-12015) = 25: 24: 1 mixture (PCIAA), 4 ° C And centrifuged at 15,300 xg for 2 minutes. Add 1 volume of isoamyl alcohol = 24: 1 mixture (CIAA) to the RNA solution in the separated aqueous layer, centrifuge in the same manner, and salt out the total cellular RNA in the aqueous layer in the same manner as in 6-a. Then, the RNA precipitate was dissolved in 50 μl of TE _8.0 .

6-d. DNase I digestion To this total cell RNA solution, add 2 units of TURBO DNase attached to TURBO DNA-free ^TM Kit (Applied Biosystems, AM1907), and mix in 100 μl of attached buffer at 37 ° C for 30 minutes. Digested genomic DNA. Thereafter, 2 units of the same enzyme were further added. After 60 minutes in total, 10 μl of the attached DNase Inactivation Reagent was added, and after sufficient stirring, the mixture was allowed to stand at room temperature for 2 minutes to stop the enzyme reaction. Thereafter, the mixture was centrifuged at 9,100 × g for 2 minutes at 4 ° C., and the total cell RNA in the supernatant was salted out in the same manner as in 6-a. RNA concentrations from mock / infected cells and asRNA-minus / infected cells or asRNA-overexpressed / infected cells were dissolved in 60 μl and 30 μl TE _8.0 , respectively, and the RNA concentration was determined by measuring absorbance at 260, 280 and 320 nm. In addition, the degree of mixing of phenol and protein components was measured. The A260 / A280 ratio of the purified RNA preparation was always 1.95-2.05, and the A320 value was 0.006 or less.

6-e. Fractionation of purified RNA based on the presence or absence of poly-A chain Using the PolyATract mRNA isolation system (Promega), the DNase I described above was used as an index for adsorption to biotinylated oligo (dT) primers and streptavidin-labeled magnetic particles. Among the treated RNA preparations, an mRNA fraction having a poly A chain and an RNA fraction not having a poly A chain were fractionated from total cell RNA derived from mock / infected cells (24 hours).

7. Reverse transcription and PCR
7-a. Reverse transcription reaction 14 μl final for each 2 μg of asRNA-minus / infected or asRNA-overexpressed / infected and mock / infected total cellular RNA purified in the previous section, or poly A strand positive or negative RNA fraction Sterile double-distilled water was added so that the total amount was 2 μg by adding 1973.8 ng of Escherichia coli tRNA to 26.2 ng of the same RNA. Subsequently, 2 μM positive strand specific hIFN-α1 / R2 primer is used for reverse transcription of IFN-α1 mRNA, and 2 μM negative strand specific for reverse transcription of IFN-α1 or IFN-α1 / ED7 asRNA. 1 μl each of hIFN-α1 / 5UF1 primer, hIFN-α1 / F1 primer or hIFN-α1 / F1A primer was added, incubated at 70 ° C. for 10 minutes, and left at 4 ° C. 5 μl reverse transcriptase buffer (Toyobo, TRT-1B), 25 μl 100 mM dNTPs, 1.2 μl sterile double-distilled water, RNase inhibitor (RNase OUT; invitrogen, 10777) -019) 0.3 μl and reverse transcriptase (ReverTra Ace; Toyobo, TRT-101) 1 μl were added, respectively, and the reverse transcription reaction proceeded at 47 ° C. for 30 minutes. Thereafter, the mixture was kept at 70 ° C. for 15 minutes to inactivate the enzyme and then kept at 4 ° C. Next, 1 μl of RNaseH (TakaraBio, 2150A) diluted to 5 units / μl with the above-mentioned reverse transcriptase buffer was added, and the template RNA was digested at 37 ° C. for 20 minutes. Salted out. The obtained cDNA precipitate was dissolved in 40 μl of mRNA-derived and 20 μl of asRNA-derived TE _8.0 .
[Reverse transcription primer]
Positive strand specific primer (for reverse transcription of IFN-α1 mRNA)
hIFN-α1 / R2: 5'-GGATCTCATGATTTCTGCTCTGAC-3 '(SEQ ID NO: 8)
Minus strand specific primer (for reverse transcription of IFN-α1 asRNA)
hIFN-α1 / 5UF1: 5'-GAACCTAGAGCCCAAGGTTCAGAG-3 '(SEQ ID NO: 9)
hIFN-α1 / F1: 5'-TGGTGCTCAGCTGCAAGTCAAGC-3 '(SEQ ID NO: 10)
hIFN-α1 / F1A: 5'-CTCTCTGGGCTGTGATCTCCCTG-3 '(SEQ ID NO: 11)
Mutant IFN-α1 asRNA-specific primer bglR1: 5'-ACAACACCCTGAAAACTTTGC-3 '(SEQ ID NO: 12)
Random primer for reverse transcription of 18S rRNA nonadeoxyribonucleotide mixture; pd (N) 9 (Takara, 3802)

7-b. PCR and Agarose gel electrophoresis For each 2 μl of the cDNA solution obtained in 7-a, 29.6 μl of sterile double-distilled water and 4 μl of 10-fold concentration of Gene taq buffer (Nippon Gene, 314-02873) Then, 3.2 μl of 2.5 mM dNTPs and 0.4 μl of 100 μM Forwardprimer and Reverse primer were added. Next, 0.2 μl of anti-Taq high (Toyobo, TCP-101) and 0.2 μl of taq polymerase (Gene taq; Nippon Gene, 314-02873) are mixed and incubated at room temperature for 5 minutes. After being added to each sample, PCR was performed using DNA Engine PTC-0200G (Bio-Rad) as follows.
1 cycle: 95 ℃, 1 minute
22-38 cycles: 95 ° C, 15 seconds / 72 ° C, 1 minute (The primer annealing temperature is gradually decreased from 95 ° C to 0.3 ° C every cycle)
1 cycle: 72 ℃, 30 seconds [Primer used]
For cDNA derived from IFN-α1 mRNA:
IFN-α1 / F2B: 5′-CTCTACCAGCAGCTGAATGACTT-3 ′ (SEQ ID NO: 13)
IFN-α1 / R2: 5'-GGATCTCATGATTTCTGCTCTGAC-3 '(SEQ ID NO: 8)

For cDNA derived from asRNA:
IFN-α1 / 5UF1B: 5′-ATCTCAGCAAGCCCAGAAGTATC-3 ′ (SEQ ID NO: 15)
IFN-α1 / R0: 5′-GAGATTCTGCTCATTTGTGCCAG-3 ′ (SEQ ID NO: 16)

IFN-α1 / F1B: 5′-CTGGGCTGTGATCTCCCTGAGAC-3 ′ (SEQ ID NO: 17)
IFN-α1 / R1: 5′-AGAGATGGCTGGAGCCTTCTG-3 ′ (SEQ ID NO: 18)

IFN-α1 / 3UF1: 5′-TGAAAACAATTCTTATTGACTCATAC-3 ′ (SEQ ID NO: 19)
IFN-α1 / 3UR3: 5′-CAGTGTAAAGGTGCACATGACG-3 ′ (SEQ ID NO: 20)

IFN-α1 / DF1: 5′-GGAACTTCCTGTATGTGTTCATTC-3 ′ (SEQ ID NO: 21)
IFN-α1 / DR2: 5'-ATACAACCTGGTTTAGAGAAAGGTC-3 '(SEQ ID NO: 22)

IFN-α1 / DF2: 5′-GACCTTTCTCTAAACCAGGTTGTAT-3 ′ (SEQ ID NO: 23)
IFN-α1 / DR3: 5'-GCTCCTTCTTCTCATAGTATTTAGG-3 '(SEQ ID NO: 24)

For simultaneous comparison and quantification of IFN-α1 mRNA and asRNA-derived cDNAs (by quantifying the amount of each cDNA using the following both primer pairs, comparing both cDNAs, and depending on the difference in the primer pair used in the quantification results Make sure there is no discrepancy between the results):
IFN-α1 / F2B: 5′-CTCTACCAGCAGCTGAATGACTT-3 ′ (SEQ ID NO: 13)
IFN-α1 / R2: 5'-GGATCTCATGATTTCTGCTCTGAC-3 '(SEQ ID NO: 8)

IFN-α1 / F1B: 5′-CTGGGCTGTGATCTCCCTGAGAC-3 ′ (SEQ ID NO: 17)
IFN-α1 / R1: 5′-AGAGATGGCTGGAGCCTTCTG-3 ′ (SEQ ID NO: 18)

For cDNA derived from mutant IFN-α1 asRNA:
RseF1: 5′-GTAAAGAGGTTGAAGATCTGC-3 ′ (SEQ ID NO: 25)
bglR1: 5'-ACAACACCCTGAAAACTTTGC-3 '(SEQ ID NO: 12)

For cDNA derived from 18S rRNA:
18S-F: 5'-CTTAGAGGGACAAGTGGCG-3 '(SEQ ID NO: 26)
18S-R: 5'-ACGCTGAGCCAGTCAGTGTA-3 '(SEQ ID NO: 27)
18S rRNA-derived cDNA amplified as an internal standard for assaying the concentration of each purified RNA sample is amplified by 22 cycles, IFN-α1 mRNA-derived cDNA is amplified by 35 + α cycles, and asRNA-derived cDNA is amplified by 37 + α cycles. The product was analyzed by electrophoresis with 3% agarose (Agarose S; Nippon Gene, 312-01193).
In addition, derived from each cell of mock / infection, asRNA-minus / infection and asRNA-overexpressed / infection used for reverse transcription reaction for the purpose of detecting amplified signal derived from genomic DNA mixed in mRNA fraction or asRNA fraction PCR was performed in the same manner using 100 ng or 200 ng of total cellular RNA.
The amount of each cDNA was quantified by real-time PCR. For the amplification and quantitative analysis of cDNA, THUNDERBIRD ^™ SYBR (registered trademark) qPCR Mix (Toyobo, QPS-201) and Chromo 4 real-time analysis system (Bio-Rad) were used. The obtained C _T values of IFN-[alpha] 1 mRNA and IFN-[alpha] 1 asRNA is 2 by ^-AACT method, after normalization using the C _T value of 18S rRNA, amount relative to negative control sample (Relative IFN-α1 mRNA expression Or expressed as Relative IFN-α1 asRNA expression).

8. Determination of the effect of Active Hexose correlated Compound (AHCC) on IFN-α1 mRNA and asRNA expression Namalwa cells cultured in the presence of 0.5 mg / ml AHCC (Amino Up Chemical) were infected with SeV as described in 5 above. It was. Cells were collected at 0, 12, and 24 hours after infection, and the total cellular RNA fraction was extracted as described in 6 above. Using this RNA fraction, the effect of AHCC on the expression of IFN-α1 asRNA and IFN-α1 mRNA was compared with that of cells not added with AHCC.

9. Quantification of hIFN-α protein by ELISA In order to investigate the effect of fluctuations in asRNA expression on IFN-α protein expression, the above-mentioned Namalawa cell culture in which IFN-α1 mRNA expression was induced by infection with SenV The supernatant was collected, and the IFN-α protein released in the supernatant was quantified using a human interferon-α ELISA kit (PBL Biomedical Laboratories, # 41100-4). Specifically, a 0-500 pg / ml solution prepared by appropriately diluting the attached human purified IFN-α and a dilution of the culture supernatant collected at each time were used for the anti-IFN-α specific After adding a certain amount to a 96-well plate on which the antibody was adsorbed, a color reaction was carried out using the attached reagent, and the absorbance at 450 nm was measured. The amount of IFN-α protein secreted into the culture supernatant was calculated from the absorbance of the collected sample at each time using a calibration curve prepared from the measurement result of the absorbance of IFN-α protein at a known concentration.

(result)
1. Identification of the presence of endogenous asRNA derived from the human IFN-α1 gene (IFNA1) Using the total cellular RNA fraction collected after 24 hours from the above mock / infected cells, the negative strand specific primers 5UF1 and F1 Used to reverse transcribe cDNA from IFN-α1 asRNA, respectively. The respective cDNAs are named RT1 (starting from nt: 2) and RT2 (starting from nt: 102) and are shown in FIG. 1A. Subsequently, as a PCR primer pair, 5UF1B (nt: 32-54; corresponding to the 5 ′ untranslated region) / R0 (nt: 210-188; corresponding to the protein coding region), F1B (nt: 131-153; Corresponds to protein coding region) / R1 (nt: 301-281; corresponds to protein coding region), 3UF1 (nt: 652-677; corresponds to 3 'untranslated region) / 3UR3 (nt: 839-818; DF1 (corresponding to nt: 40-63 downstream region from IFNA1 gene polyA chain addition site) / DR2 (nt: 188-164 downstream region from IFNA1 gene polyA chain addition site) DF2 (corresponds to the downstream region of NF: 1644-188 from the IFNA1 gene poly A chain addition site) / DR3 (corresponds to nt: 326-302 downstream region of the IFNA1 gene poly A chain addition site), RT1 and RT2 cDNAs were amplified. Each PCR product is named PCR1, PCR2, PCR3, PCR4, PCR5 from the 5 ′ side, and the corresponding position on the IFNA1 gene is shown in FIG. 1A. From the PCR results presented, RT1 cDNA was amplified in all other primer pairs except DF2 / DR3 located in the most downstream. In contrast, RT2 cDNA reverse-transcribed from nt: 102 is amplified in PCR1 (5UF1B / R0) located upstream from the start point of reverse transcription in addition to PCR5 amplified with the DF2 / DR3 primer pair. Could not be confirmed (FIG. 1A). From the above results, IFN-α1 asRNA starts transcription from the 188 base to 326 base downstream region from the polyA addition site (nt: 877) of the same gene, and reaches at least nt: 2 on the IFNA1 gene. It was shown to cover the region containing the full length of α1 mRNA.
Furthermore, RT-PCR results for IFN-α1 asRNA using a poly A chain positive mRNA fraction revealed that poly A chain was present in the RNA (FIG. 1B). Subsequently, the induced IFN-α1 mRNA and IFN-α1 asRNA were comparatively quantified by real-time PCR in Namalwa cells after 24 hours of SenV infection. As a result, the amount of IFN-α1 asRNA was expressed only in an amount corresponding to 3-3.7% of the expression of IFN-α1 mRNA at the peak of expression induction by SenV infection (24 hours, see FIG. 3A). Also, the quantitative values of both were not affected by the difference in the primer pairs used.

2. RNA secondary structure that regulates nuclear export and expression of IFN-α1 mRNA Based on previous studies, IFN-α1 mRNA that has been transcribed and processed in the nucleus is transported on the same mRNA. It was clarified that the RNA secondary structure consisting of two stem loops (SL1, SL2) formed by the responsible region (CSS; nt: 208-452) needs to be recognized by nuclear exporter (Kimura et al. al., Medical Mol. Morphol., 43: 145, 2010). Among them, the SL2 structure is essential, and the mRNA mutant (IFN-α1 mRNA / ΔSL2) from which this stem loop region was deleted was not transported out of the nucleus and was retained in the nucleus (Fig. 2A, panel C). . On the other hand, deletion of the SL1 region did not affect the nuclear export of the mutant mRNA (IFN-α1 mRNA / ΔSL1) (FIG. 2A, panel B). Interestingly, when the Bulged SL (nt: 322-352) region in the SL2 region was deleted, a decrease in the expression of mutant mRNA (IFN-α1 mRNA / ΔBSL) was observed (Fig. 2A, panel D). This suggests that this region may be involved in the regulation of IFN-α1 mRNA transcription and / or stability.

3. 1. Inhibition of asRNA expression by sense oligonucleotide for IFN-α1 mRNA export responsible region and examination of its specificity Based on the results of (Fig. 2A, panel D), the purpose of investigating the interaction of the bulged SL structure in the CSS secondary structure with asRNA and the possible involvement of asRNA in the regulation of IFN-α1 mRNA expression A sense oligonucleotide (FIG. 2B; Se1, Se2) homologous to the gene sequence to be formed was prepared and introduced into Namalwa cells. In addition, sense oligonucleotides Se3 and Se4 were prepared and similarly introduced into Namalwa cells for the purpose of examining the interaction between IFN-α1 asRNA and other stem loop structures constituting the SL2 region. FIG. 2B shows the analysis result of IFN-α1 asRNA expression 6 to 48 hours after introduction. Inhibition of asRNA expression (knockdown, KD) was observed only in cells into which sense oligonucleotides Se1 and Se2 prepared for the loop structure of the Bulged SL region were introduced, compared to before introduction (0 hour). The effect lasted 48 hours in Se1-introduced cells (FIG. 2B, Se1), but in the case of Se2, it was first confirmed 24 hours after introduction and had already disappeared after 48 hours (FIG. 2B, Se2). On the other hand, when a sense oligonucleotide (Figure 2B, NSe1, NSe2) prepared from the gene sequence that forms the RNA double-stranded structure of the base and SL1 region in the CSS secondary structure is introduced, IFN-α1 asRNA expression No inhibitory effect was observed (FIG. 2B, Nse1, Nse2).

4). Effects of asRNA knockdown on IFN-α1 mRNA expression IFN-α1 asRNA and mRNA signals expressed in SenV-infected Namalwa (asRNA-minus / infected) cells transfected with Se1 and in uninfected (mock / infected) cells Were analyzed by chain-specific RT-PCR over time after infection. The results are shown in FIG. 3A, and the quantification results by Realtime PCR are shown in FIG. 3B.
In mock / infected cells, IFN-α1 asRNA was already observed before virus infection (FIG. 3A, KD (−) / IFN-α1 asRNA, 0hr; FIG. 3B, IFN-α1 asRNA, KD (−), 0hr). ), Constitutive expression in Namalwa cells was confirmed. The expression of this asRNA increased up to 24 hours after SenV infection (FIG. 3A, KD (−) / IFN-α1 asRNA), and reached about 5 times the expression level of 0 hour (FIG. 3B, IFN-α1 asRNA). , KD (-)) gradually decreased. On the other hand, in asRNA-minus / infected cells, it was confirmed that the expression of asRNA was significantly inhibited by the introduction of Se1 sense oligonucleotide, but its expression recovered rapidly by infection, and 6 hours after infection. After the expression level of the control cell was exceeded, expression was about 23 times that of the 0 hour expression after 24 hours, and then rapidly attenuated (FIG. 3A, KD (+) / IFN-α1 asRNA; FIG. 3B, IFN-α1). asRNA, KD (+)).
In Mock / infected cells, IFN-α1 mRNA, as well as asRNA, was confirmed to have a low level of constitutive expression before infection, but its expression level increased rapidly with viral infection, and 12 hours later, the 35 hours of 0 hours A double level was reached but then gradually decreased (FIG. 3A, KD (−) / IFN-α1 mRNA; FIG. 3B, IFN-α1 mRNA, KD (−)). On the other hand, in asRNA-minus / infected cells, mRNA expression increased up to 24 hours after infection, and then rapidly decreased after reaching a level about 76 times that of mock / infected cells over 0 hours. The decrease was rapid in asRNA-minus / infected cells and was already below the mock / infected cell expression level 36 hours after infection (FIG. 3A, KD (+) / IFN-α1 mRNA; FIG. 3B, IFN-α1). mRNA, KD (+)).

5. Effect of mRNA destabilization by knockdown of IFN-α1 asRNA The above 4 (Fig. 3A, B) experimental results suggested that mRNA was stabilized as a mechanism of IFN-α1 mRNA expression regulation by asRNA Therefore, we considered that the half-life of IFN-α1 mRNA in asRNA-minus / infected cells was compared with that in mock / infected cells. For this purpose, Actinomycin D, an RNA transcription inhibitor, was added to both cells 12 hours after SenV infection. After 30 minutes and 1 to 4 hours, IFN-α1 asRNA knockdown resulted in degradation of IFN-α1 mRNA. The effects on
In asRNA-minus / infected cells, the degradation kinetics of IFN-α1 mRNA was approximated by a degradation curve represented by the approximate expression y = −0.369log (x) +0.489 (FIG. 3C, KD (+)). On the other hand, the approximate expression for the degradation kinetics of IFN-α1 mRNA in mock / infected cells was y = −0.097x + 0.996. Therefore, from the change in the expression level of IFN-α1 mRNA obtained 0.5-4 hours after the addition of actinomycin D, the half-life was calculated to be 5.11 hours in mock / infected cells. For minus / infected cells, it was significantly reduced to 0.93 hours. That is, the time required for IFN-α1 mRNA to be halved by asRNA knockdown was reduced to almost 18%, and the remarkable instability was apparent.

6). Effect of AHCC on IFN-α1 asRNA and IFN-α1 mRNA expression To examine the effect of AHCC on IFN-α1 asRNA and IFN-α1 mRNA expression, the cells were cultured in a culture medium containing 0.5 mg / ml AHCC. Namalwa cells were infected with SeV, and the expression kinetics of the induced IFN-α1 asRNA and the same mRNA were compared with those in control cells. As a result, AHCC strongly inhibited constitutive expression of IFN-α1 asRNA in Namalwa cells (FIG. 4A; AHCC (+) / IFN-α1 asRNA, 0 hr), but also over time induced by viral infection. The increase in the expression level was also suppressed (FIG. 4A; AHCC (+) / AHCC (−), IFN-α1 asRNA / 12-24hr). The inhibitory effect by AHCC was also observed in the expression of IFN-α1 mRNA, and the expression level decreased in comparison with AHCC (−) cells in the entire elapsed time of 0-24 hours after SenV infection (FIG. 4A). AHCC (+) / AHCC (-), IFN-α1 mRNA / 0-24hr). As a result, the amount of IFN-α1 protein released into the culture supernatant in AHCC (+) cells decreased to 18.6% of AHCC (−) cells at 12 hours after virus infection and 75% at 24 hours ( FIG. 4B).

7). IFN-α1 / WT overexpression of IFN-α1 mRNA by IFN-α1 / WT asRNA overexpression In order to further verify the stabilization effect of IFN-α1 asRNA by IFN-α1 asRNA, which is considered from the result of 5 above (Fig. 3C) Using SenV-infected Namalwa (Overexpression (+) / infected) cells pre-introduced with an α1 / WT asRNA expression plasmid and untransfected (Overexpression (-) / infected) cells, IFN-α1 asRNA induced by viral infection The mRNA signal was compared over time. The results are shown in FIG. 5A and the quantification results by Realtime PCR are shown in FIG. 5B. In Overexpression (-) / infected cells, the expression of IFN-α1 asRNA was the same as that described above (FIG. 3A, KD (−) / IFN-α1 asRNA; FIG. 3B, IFN-α1 asRNA, KD (−); FIG. 4A, AHCC ( (−) / IFN-α1 asRNA), it continuously increased until 24 hours after SenV infection (FIG. 5A, Overexpression (−) / IFN-α1 asRNA). On the other hand, in Overexpression (+) / infected cells, asRNA expression exceeded the 24-hour level in Overexpression (-) cells before virus infection, and this expression level was maintained until 24 hours after infection (FIG. 5A, Overexpression ( +) / IFN-α1 asRNA).
The expression of IFN-α1 mRNA is the same as that in Overexpression (−) / infected cells (FIG. 3A, KD (−) / IFN-α1 mRNA; FIG. 3B, IFN-α1 mRNA, KD (−); FIG. 4A, AHCC). Similar to (-) / IFN-α1 mRNA), it increased rapidly with viral infection and gradually decreased after reaching the peak after 12 hours (Fig. 5A, Overexpression (-) / IFN-α1 mRNA; Fig. 5B, Overexpression ( -)). In contrast, when IFN-α1 / WT asRNA was overexpressed, IFN-α1 mRNA expression had already reached 15 times the level in Overexpression (-) cells at the start of infection. Although the relative ratio decreased, the expression level continued to increase until 24 hours after infection, reaching a level approximately twice the expression level in Overexpression (-) / infected cells (Fig. 5B, Overexpression (+) / (- )).

8). Stabilizing effect of IFN-α1 mRNA by overexpression of IFN-α1 / WTasRNA The expression level of IFN-α1 mRNA by overexpression of IFN-α1 / WT asRNA observed in the experiment of 7 above (FIG. 5A, B) In order to know the mechanism of the increase, we considered comparing and examining the half-life of IFN-α1 mRNA in Overexpression (+) / infected cells and Overexpression (-) / infected cells. For this purpose, Actinomycin D was added to both cells 6 hours after SenV infection, and the effects of overexpression of IFN-α1 / WT asRNA on the degradation of IFN-α1 mRNA after 30 minutes and 1 to 4 hours. Analyzed.
In Overexpression (+) / infected cells, the degradation kinetics of IFN-α1 mRNA was approximated by a degradation curve represented by the approximate expression y = −0.067x + 1.059 (FIG. 5C, Overexpression (+)). On the other hand, the approximate expression for the degradation kinetics of IFN-α1 mRNA in Overexpression (−) / infected cells was y = −0.182x + 1.081 (FIG. 5C, Overexpression (−)). Therefore, when calculating the half-life from the change in the expression level of IFN-α1 mRNA obtained 0.5-4 hours after the addition of Actinomycin D, the half-life in Overexpression (-) / infected cells is 2.76 hours, In Overexpression (+) / infected cells, it was significantly extended to 8.12 hours. That is, the overexpression of asRNA extended the life span of IFN-α1 mRNA by about 3 times, and its remarkable stabilizing effect was clear.

9. Determination of the functional center of the same asRNA that stabilizes IFN-α1 mRNA The mRNA stabilization action of IFN-α1 asRNA demonstrated in 5 (Fig. 3C) and 8 (Fig. 5C) originates from any site on asRNA For the purpose of knowing this, focusing on the antisense sequence (Fig. 6A / BSL) of SL2 region and BSL structure (Fig. 2) that have been reported to contribute to stabilization in the CSS secondary structure of IFN-α1 mRNA, The nucleotide sequences were sequentially extended in the 5 ′ and 3 ′ directions to prepare plasmid vectors that express various mutant IFN-α1 asRNAs shown in FIG. 6A. FIG. 6A / IFN-α1 mRNA and FIG. 6B show the results of measuring the expression level of IFN-α1 mRNA 24 hours later after infecting Senmalwa cells into which each mutant asRNA expression vector was introduced. The expression level of IFN-α1 mRNA in Mock / infected cells (FIGS. 6A / C) increased 2.3 times due to co-expression of WT asRNA as in FIG. When the antisense RNA of the SL2 region of CSS was co-expressed, the expression level was 3.1 times that of the control (FIG. 6A / IFN-α1 mRNA; FIG. 6B).

10. Increased expression of IFN-α1 mRNA by antisense ribooligonucleotide with IFN-α1 asRNA functional center sequence and determination of intracellular site of expression of the functional center mapping results of IFN-α1 asRNA shown in Fig. 6 In order to achieve this, an antisense ribooligonucleotide (IFNA1 / 346-322ORN; asORN in FIG. 7A) consisting of an antisense strand sequence corresponding to the BSL region in the IFN-α1 mRNA CSS secondary structure was prepared, and its IFN-a1 The mRNA expression regulation effect was analyzed. As a negative control, an antisense ribooligonucleotide derived from a sequence that forms the base duplex of the CSS RNA secondary structure used as the negative control (Nse1) in the knockdown experiment of asRNA expression (3, Fig. 2 above) (IFNA1 / 224-205 ncORN; ncORN in FIG. 7A) was used. As a result, both antisense ribooligonucleotides did not affect the expression of IFN-α1 asRNA, and their expression kinetics and expression level were the same as those of mock transfection / SeV-infected cells (FIG. 7B; IFN-α1). asRNA). In contrast, the expression of IFN-α1 mRNA increases only when an antisense ribooligonucleotide (IFNA1 / 346-322ORN) having a functional center sequence is introduced (FIG. 7B; IFN-α1 mRNA, asORN), and infection About 5 times the amount of IFNA1 / 224-205 ncORN introduced (Figure 7B; IFN-α1 mRNA, ncORN) and about 3 times the amount of mock transfection (Figure 7B; IFN-α1 mRNA, mock Tfx) Was expressed. Although the relative ratio decreases with the progress of infection, its expression level continues to increase until 24 hours after infection, and finally about twice the amount of IFNA1 / 224-205 ncORN-transfected cells and mock-transfected cells were expressed ( FIG. 7B). In order to determine the intracellular expression site of this effect of increasing the amount of IFN-a1 mRNA by asORN, NamORwa cells infected with asORN and infected with SenV were collected after 24 hours, and their nuclear RNA fraction and cytoplasmic RNA fraction were recovered. The image was analyzed. As a result, only in the cytoplasm of the cells into which asORN had been introduced, the expression level of IFN-α1 mRNA was increased about 2-fold compared to ncORN-introduced cells and mock cells (FIG. 7C / D, IFN-α1 mRNA, Total / Cytoplasmic asORN). Even in the same cells, such a change in the expression level was not observed in the nucleus (FIG. 7C / D, IFN-α1 mRNA, Nuclear asORN / ncORN / mock). In addition, the expression level of IFN-α1 asRNA was not changed in both the nucleus and cytoplasm regardless of whether the cells were transfected with asORN or ncORN (Fig. 7C / D, IFN-α1 asRNA, Total / Cytoplasmic / Nuclear asORN / ncORN / mock).

From the above results, IFN-α1 asRNA is post-transcriptionally via the functional center existing at nt: 434-308 (and Bnt region nt: 346-322) on the IFNA1 gene, and in the cytoplasm, IFN-α1 It was shown to stabilize mRNA and regulate its expression. This fact strongly suggests the possibility of controlling the expression of IFN-α1 mRNA by administration of Se1 sense oligonucleotide that knocks down the expression of asRNA and antisense ribooligonucleotide derived from the asRNA functional center. -Expected to lead to the development of alpha protein expression modulators.

While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.
The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were specified. .
This application is based on Japanese Patent Application Nos. 2009-239801 and 2010-107544 filed in Japan on October 16, 2009 and May 7, 2010, respectively, the contents of which are incorporated herein by reference. Is included.

The antisense nucleotide of the present invention is useful as an IFN-α expression enhancer for the prevention and / or treatment of viral infections and cancers. On the other hand, the sense oligonucleotide of the present invention is useful as an IFN-α expression regulator for the prevention and / or treatment of diseases and conditions caused by overproduction of IFN-α.

Claims (12)

1. An antisense nucleotide comprising a sequence complementary to interferon-α mRNA and capable of enhancing the expression of interferon-α.
2. The antisense nucleotide according to claim 1, comprising a sequence complementary to the base sequence in the SL1 and / or SL2 region of interferon-α mRNA.
3. The antisense nucleotide according to claim 2, comprising a sequence complementary to the base sequence in the Bulged SL region of interferon-αM mRNA.
4. An interferon-α expression enhancer comprising the antisense nucleotide according to any one of claims 1 to 3.
5. The agent according to claim 4, which is used for prevention or treatment of viral infection or cancer.
6. A sense oligonucleotide containing a sequence complementary to an endogenous antisense RNA for interferon-α mRNA and capable of regulating the expression of interferon-α.
7. The sense oligonucleotide according to claim 6, wherein the sequence is a sequence homologous to a base sequence in the SL1 or SL2 region of interferon-α mRNA.
8. The sense oligonucleotide according to claim 7, wherein the sequence is a sequence homologous to the base sequence in the Bulged SL region.
9. An interferon-α expression regulator comprising the sense oligonucleotide according to any one of claims 6 to 8.
10. The agent according to claim 9, which is used for prevention or treatment of immune response abnormality.
11. A screening method for a substance that enhances or suppresses interferon-α expression, comprising detecting and comparing the hybridization between interferon-α mRNA and its endogenous antisense RNA in the presence and absence of a test substance.
12. A test substance is brought into contact with a cell expressing interferon-α mRNA and endogenous antisense RNA thereto, and a change in the amount of mRNA and / or interferon-α protein in the cell is measured. Item 12. The method according to Item 11.

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