JPH09121867A - Antiinfluenza virus antisense oligonucleotide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject oligonucleotide, capable of suppressing the protein expression of A type, B type and C type influenza viruses and variant strains thereof and useful as an antiviral agent for violently variable influenza viruses. SOLUTION: This oligonucleotide comprises a base sequence complementary to the base sequence of a target region containing a translation initiation codon AUG of a PB2 gene or an NP gene of the influenza viruses and has, e.g. a base sequence of formula I or II. The oligonucleotide can be synthesized according to a well-known method such as a usual phosphodiester method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to anti-influenza virus antisense oligonucleotides.

[0002]

2. Description of the Related Art Influenza virus is still a worldwide epidemic, and many people are caught up in the epidemic due to airborne infection. Considering its infectiousness, if a new subtype emerges, the scale of the epidemic will increase, not to mention the acquired immunodeficiency syndrome (AIDS).

Influenza virus belongs to the family Orthomyxoviridae and is a single-stranded RNA virus having a minus strand, and its gene consists of 8 segments. Those eight
Among the proteins encoded by segmental genes, hemagglutinin (HA) and neuraminidase (NA) are two types of spikes on the surface of virus particles, which protrude from the envelope. Also, a type of membrane protein (M) (M
There is a gene segment encoding 2). Two types of glycoproteins embedded in a host-derived lipid bilayer membrane (envelope) are present on the surface of virus particles in a spike shape. Furthermore, another type (M1) of the membrane protein (M) exists, and the gene segment encoding the membrane protein (M1) is also included in the viral gene. A nucleoprotein complex (RNP) exists in the center of the virus, which is a gene RNA, three RNA polymerase subunits (PB1, PB2, and PA), and a nucleoprotein (NP).
Consists of Each of these proteins (PB1, PB2,
Gene segments encoding PA and PA, and NP) are also included in the viral gene. From the eighth segment, a nonstructural protein (NS) is made.

Influenza virus is classified into A type and B type depending on the serotypes of nucleoprotein NP and membrane protein M2.
And C type. In addition, in influenza virus, three proteins PB1 and PB
2 and PA are subunits that compose RNA polymerase, and the amino acid sequence of this RNA polymerase is well conserved in influenza virus types A, B and C, and their mutants. Then, RNA polymerase catalyzes an RNA synthesis reaction, a poly A addition reaction, a cap restriction reaction, and the like. In particular, PB1 is involved in RNA synthesis activity, and PB2 is
It recognizes the cap structure of the mRNA of the host cell and cleaves it. PA acts on the transcription elongation reaction. In addition, NP is a polynucleotide binding protein that is non-specific to the nucleotide sequence,
The RNA of the NP-RNA complex forms a double-stranded or helical structure and is important for the viral transcription reaction. And in particular, PB2, PB1, PA, NP, and NS are each synthesized early in infection.

Among influenza viruses, influenza virus type A causes a pandemic with a large-scale change in antigenicity. In addition, type A is the most malignant in terms of strong infectivity. As an antiviral agent against this type A,
Amandadine or rimandadine are known, but they are not sufficient in that they cannot counter the emergence of resistant strains and show strong side effects, and drugs that exhibit satisfactory antiviral effects are still being developed. Absent. In addition, treatment with an inactive vaccine has also been attempted, but due to the difference between the inactive vaccine and the antibody produced by natural infection, not only the number of vaccine recipients tends to decrease, but also antibody production persistence is maintained. In that respect, it is not sufficiently effective to prevent the epidemic completely. In general, since inactive vaccines have a short duration of effect, it has been desired to develop a vaccine of an attenuated virus, but none has reached the stage of practical use. A major problem is that significant viral antigen mutations are one of the causes of delay in vaccine response.

On the other hand, with the recent development of gene analysis technology, it is possible to easily analyze a mutant gene sequence. Therefore, as an antiviral agent for influenza virus with severe mutation, an antisense oligonucleotide method targeting a gene, For example, the antisense DNA method is most suitable. What is the antisense oligonucleotide method?
It is a technique of inhibiting transcription, splicing, or translation of a target gene at the DNA level or mRNA level by using an oligonucleotide having a base sequence complementary to the target gene. This technique can specifically suppress the expression of viral proteins with a mechanism of action completely different from that of pharmaceuticals [S. T. Crooke, Therape
utic Applications of Olig
onlinetides, Springer-Ver
lag, (1995)].

[0007] Further, in the antisense oligonucleotide method, in addition to inhibiting the above-mentioned transcription, splicing, nuclear membrane permeation, or translation, the antisense oligonucleotide binds to the mRNA to give m
It is also known that the specific degradation of RNA is promoted. The potential of antisense oligonucleotide technology as a drug with high biological specificity has been proved by many in vitro experiments targeting oncogenes or various viruses. There is.

[0008]

DISCLOSURE OF THE INVENTION The present inventor examined effective target regions in viral genes in order to develop effective antisense technology against influenza viruses with severe mutations. Specifically, from the region containing the translation initiation codon AUG of the PB2 gene, PB1 gene, PA gene, and NP gene most involved in transcription and replication,
Surprisingly, as a result of conducting research on a region that can strongly inhibit translation initiation as compared with other regions by selecting regions up to the hairpin loop and targeting them with antisense oligonucleotides, Also, the 5'-side AUG of the PB1 gene, which was said to have an effect in the past,
Antisense oligonucleotides complementary to the translation initiation region [Proc. Natl. Acad. Sci. USA,
87, 3430-3434 (1990)], and that antisense oligonucleotides complementary to the loop-forming region of the PB1 gene have no effect, on the other hand, PB2 gene Alternatively, an antisense oligonucleotide having a base sequence complementary to the base sequence of the region containing the translation initiation codon AUG of the NP gene extremely strongly inhibits the translation start as compared with the case where another region is used as the target region. Found.
The present invention is based on such findings, and an object of the present invention is to antisense oligonucleotides capable of extremely effectively suppressing protein expression of influenza A, B and C influenza viruses, and mutants thereof. To provide.

[0009]

[Means for Solving the Problems] The above objects are according to the present invention: PB2 gene of influenza virus or NP
It can be achieved by an oligonucleotide characterized in that it comprises a base sequence complementary to the base sequence of the target region containing the translation initiation codon AUG of the gene.
In the present specification, the “influenza virus” means
Includes A, B and C influenza viruses, and variants thereof. Therefore, the PB2 gene of influenza virus means each influenza virus of A type, B type and C type, and each PB2 gene (plus chain) of their mutants, and similarly, the NP gene of influenza virus is A type. , B-type and C-type influenza viruses, and PB2 genes (plus strands) of their mutants.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The oligonucleotide of the present invention comprises
A region containing the translation initiation codon AUG of the influenza virus PB2 gene or NP gene is used as a target region, and includes a base sequence complementary to the base sequence of the target region. The target region is not particularly limited as long as it contains the translation initiation codon AUG, and the translation initiation codon AUG
Can include regions upstream and / or downstream of. The number of bases of the oligonucleotide of the present invention is not particularly limited, but it is preferably at least the number of bases capable of specifically hybridizing to the target region, and the oligonucleotide of the present invention can penetrate cell membranes or nuclear membranes. The number of bases is preferably equal to or less than that.

The number of bases capable of specifically hybridizing to the target region is preferably 15 bases or more, more preferably 20 bases or more. Further, in order to ensure membrane permeability, it is preferably 30 bases or less, more preferably 28 bases or less.
Base or less. Therefore, the oligonucleotide of the present invention is preferably 15 to 30 bases, more preferably 20 to 30 bases.
It consists of 28 bases. The oligonucleotide of the present invention includes
As long as it can specifically bind to the target region (for example, mRNA) to form a duplex, it is not necessary to continuously include a base sequence complementary to the target region, and non-complementary base 1
Alternatively, more than one may be deleted, inserted and / or substituted at one or more places. However, an oligonucleotide that continuously contains a base sequence complementary to the target region is preferable.

The specific base sequence of the oligonucleotide of the present invention can be appropriately determined according to the target influenza virus type. For example, when targeting a certain mutant strain of influenza virus A, first, the nucleotide sequence of the region containing the translation initiation codon AUG of the PB2 gene and / or the NP gene of the mutant strain is analyzed. Subsequently, the target sequence region and the number of bases are determined from the viewpoint of stable duplex formation, and an antisense oligonucleotide can be synthesized by a known method.

In the oligonucleotide of the present invention, the internucleotide bond between each nucleoside is independently a phosphodiester bond or a modified phosphodiester bond. Examples of the modified phosphodiester bond include non-crosslinked oxygen atom 2 of phosphodiester bond.
A phosphoroamido in which one of two non-bridging oxygen atoms of a phosphodiester bond is replaced with an amino group or a substituted amino group. Phosphorothioate type bond in which one oxygen atom is replaced by sulfur atom among two non-bridging oxygen atoms of date type bond and phosphodiester bond,
Or a phosphorodithioate-type bond in which two oxygen atoms of the two non-bridging oxygen atoms of the phosphodiester bond are replaced with two sulfur atoms, and one or more of them are nucleosides. It can be introduced at one or more points in the bond between. From the viewpoint of nucleotide sequence specificity, double-stranded stability, antinuclease resistance, cell membrane permeability, low cytotoxicity and moderate metabolism, and a simple preparation method, it is preferable that the modified phosphodiester bond is a modified phosphodiester bond. From the viewpoint of good stability in vivo, a phosphorothioate type bond is particularly preferable. Furthermore,
It is particularly preferred that half or more (particularly all) bonds between nucleosides are modified phosphodiester bonds (particularly phosphorothioate type bonds).

Various known methods can also be used to improve the membrane permeability of the oligonucleotides according to the invention. For example, a method of embedding in liposome [M. M
ozuno et al. , Cancer Res. ,
50, 7826 (1991)], a method of introducing cholesterol to the end of the oligomer [B. Oberhauer
and E. Wagner, Nucleic Aci
ds Res. , 20, 533 (1993)], transferrin-poly-L-lysine complex [G. Citr
o, Proc. Natl. Acad. Sci. USA,
89, 7031 (1992)],
Hepatocytes expressing galactose receptor,
A method for specifically incorporating an oligomer into a cancer cell expressing a transferrin receptor, or a cationic lipid, for example, lipofectin is allowed to interact with the oligomer and added to a target cell so that the oligomer is incorporated into the cell and further to the nucleus. To increase anti-sense effect [M. Chiany et al. , J. et al.
Biol. Chem. , 266, 18162 (199
1)]

The oligonucleotide of the present invention is complementary to each base constituting the base sequence of the target gene of influenza virus, that is, A (adenine), G (guanine), C (cytosine), and U (uracil). As a base, T (thymine) or U (uracil), C, G, and A can be used, respectively. When the oligonucleotide of the present invention is RNA, A that constitutes the nucleotide sequence of the target gene of influenza virus
U is used as a base complementary to.

The oligonucleotides of the present invention can be bound to deoxynucleosides, ribonucleosides, and / or / as long as they can specifically bind to a target region to form a duplex.
Or their modified ribonucleosides such as 2'-O
It can be formed from modified ribonucleosides. As the modified ribonucleoside, 2′-O-methylribonucleoside is preferable from the viewpoint of the strength of binding with the target nucleotide sequence. Therefore, the oligonucleotide of the present invention comprises an oligoribonucleotide (RNA) consisting of a ribonucleoside and / or a modified ribonucleoside, an oligodeoxyribonucleotide (DNA) consisting of only a deoxyribonucleoside, or a ribonucleoside (and / or a modified ribonucleoside). It can be a chimeric oligoribo / deoxyribonucleotide (RNA / DNA) consisting of both deoxyribonucleosides.

Examples of the oligonucleotide of the present invention include influenza virus A / PR / 8/34 [P
B1: G. Winter and S.M. Fields,
Nucleic Acids Res. 10 2135
(1982); PB2: G. Winter and
S. Fields, Cell, 28, 203 (198
2); NP: G. Winter and S.M. Field
ds, Viology 114, 423 (1981)]
An antisense oligonucleotide containing a nucleotide sequence complementary to the nucleotide sequence of the region containing the translation initiation codon AUG of the PB2 gene and / or the NP gene can be mentioned. This antisense oligonucleotide
It is particularly effective against influenza virus type A, but also against influenza virus type B and influenza virus type C.

The antisense oligonucleotide containing a nucleotide sequence complementary to the nucleotide sequence of the region containing the translation initiation codon AUG of the PB2 gene is as follows: And complementary at least 15 bases (preferably 15 to
(30 bases). Further, an antisense oligonucleotide containing a base sequence complementary to the base sequence of the region containing the translation initiation codon AUG of the NP gene, complementary to the base sequence represented by the sequence of SEQ ID NO: 27 in the sequence listing. An oligonucleotide containing at least 15 bases (preferably 15 to 30 bases) out of such base sequences can be mentioned.

Specifically, the antisense oligonucleotide of the present invention is composed of 20 bases containing the translation initiation codon AUG of the PB2 gene, the internucleotide bond is a phosphodiester bond, and SEQ ID NO: 1 in the sequence listing. An oligonucleotide having a base sequence represented by the sequence No .; an oligonucleotide having a phosphorothioate type internucleotide bond in the oligonucleotide and having a base sequence represented by the sequence No. 2 in the sequence listing; translation initiation of the NP gene An oligonucleotide having 20 bases containing a codon AUG, an internucleotide bond being a phosphodiester bond, and having a base sequence represented by the sequence of SEQ ID NO: 19 in the sequence listing; the internucleotide bond in the oligonucleotide is phosphorothio An over preparative, of SEQ ID NO: 2
An oligonucleotide having a base sequence represented by the sequence 0 can be mentioned, and these four kinds of oligonucleotides are particularly effective against influenza virus type A, respectively. Regarding influenza virus type B and influenza virus type C, as described above, oligonucleotides complementary to each are effective against influenza virus type B and influenza virus type C.

The oligonucleotide of the present invention can be synthesized by a known method. Except for a site for introducing a 2′-O-methylribonucleotide or a phosphorothioate bond, for example, a DNA / RNA automatic method by a usual phosphodiester method or a phosphotriester method, for example, an H-phosphonate method, or a phosphoramidite method is used. It can be synthesized using a synthesizer. Oligonucleotides having 2'-O-methylribonucleotides include, for example, 5'-dimethoxytrityl-2'-O-methylribonucleoside-3 '-[(2-cyanoethyl)-(N,
N-diisopropyl)]-DNA / RNA by phosphoramidite method using phosphoramidite unit
It can be synthesized using an automatic synthesizer. Oligonucleotides having phosphorothioate linkages can be synthesized, for example, by adding 15% N, N instead of water / iodine / pyridine solution, which is an oxidizing agent used in ordinary polynucleotide synthesis.
N, N ', N'-tetraethylthiolamm disulfide /
It can be synthesized using an acetonitrile solution.

The antiviral effect of the oligonucleotide of the present invention can be evaluated by, for example, the plasmid pOUMS101 [Pro
c. Natl. Acad. Sci. USA, vol. 8
8, pp. 5369-5373 (1991)], and mouse breast cancer cell line C127 clonal cells (hereinafter referred to as 76 cells) [Y. Nakamura, K .; Odaan
d S. Nakada, J .; Biochem. , 11
0,395 (1991)], and can be confirmed by the expression level of chloramphenicol acetyltransferase (hereinafter referred to as CAT) in the 76 cells.

The above-mentioned plasmid pOUMS101 is a DNA fragment obtained by replacing the protein coding region of the eighth segment of influenza virus PR8 with the CAT gene (the 5'upstream region of the 8th segment of the protein coding region).
A plasmid prepared by inserting a base sequence consisting of 3 base pairs and a base sequence consisting of 12 base pairs of the 3'downstream region of the protein coding region of the eighth segment) into the downstream of the phage T7 RNA polymerase promoter. . In addition, 76 cells are cells in which expression of RNA polymerase (PB1, PB2, and PA) of influenza virus type A (A / PR / 8/34) and NP protein is induced by addition of steroid hormone dexamethasone. is there.

Specifically, the plasmid pOUMS101
CAT with T7 RNA polymerase
Negative strand RNA containing a gene (hereinafter sometimes referred to as vRNA) is synthesized in vitro. Separately, by adding dexamethasone, PB1, P
Induced expression of B2, PA, and NP proteins 76
The oligonucleotide to be inspected is added to the cells, and when the oligonucleotide is taken up into the cells, the vRNA previously synthesized is added to 76 cells and allowed to be taken up into the cells. Tested oligonucleotide was PB2 in 76 cells
When it does not bind to the gene or the mRNA of the NP gene, vRNA is replicated and CAT is expressed by PB1, PB2, and PA, their respective RNA polymerases and NP proteins. On the other hand, when the test oligonucleotide was in the 76 cells, the mRNA of PB2 gene or NP gene
When bound to NA, PB1, PB2, and PA,
Since the production of each RNA polymerase and NP protein is controlled, and as a result, the replication of vRNA is suppressed, the expression level of CAT is also suppressed. Therefore, 76
The antiviral effect of the oligonucleotide to be tested can be evaluated by measuring the intracellular CAT expression level.

[0024]

The action of the antisense oligonucleotide of the present invention is not limited to the following reasoning. However, when the antisense oligonucleotide is present in a host cell, for example, the duplicated + cRNA has an antisense action of the present invention. By binding the sense oligonucleotide, cRN
It is considered that A can be used as a template to prevent the synthesis of offspring viral RNA. Further, when the mRNA transcribed from -vRNA is released into the cytoplasm from the nucleus of the host cell, it is considered that the antisense oligonucleotide of the present invention binds to the mRNA and blocks translation into a protein. Therefore, the oligonucleotide of the present invention can be used as a therapeutic agent for influenza due to its antiviral effect against influenza virus.

[0025]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1 Oligonucleotide Design For use in the following examples, P
The B2 gene, the PB1 gene, the PA gene, and the NP gene were selected, and further, the translation initiation region and the loop formation region were selected for the PB2 gene and the PB1 gene, and only the translation initiation region was selected for the PA gene and the NP gene. The nucleotide sequences of the translation start region and loop formation region of the PB2 gene are shown in FIG. 1, and the nucleotide sequences of the translation start region and loop formation region of the PB1 gene are shown in FIG. The nucleotide sequence of the translation initiation region of the PA gene is shown in FIG. 3, and the nucleotide sequence of the translation initiation region of the NP gene is shown in FIG.

Specifically, as the translation initiation region of the PB2 gene, the 18th to 37th base sequences underlined in FIG. 1 (base sequence represented by the sequence of SEQ ID NO: 22 in the sequence listing) are selected. As a loop forming region of the PB2 gene,
The 1279th to 1298th base sequences (base sequence represented by the sequence of SEQ ID NO: 23 in the sequence listing) indicated by double underline in FIG. 1 were selected. As the translation initiation region of the PB1 gene, the 15th to 34th base sequences underlined in FIG. 2 (base sequence represented by the sequence of SEQ ID NO: 24 in the sequence listing)
2 as a loop forming region of the PB1 gene.
The 1279th to 1298th base sequences (base sequence represented by the sequence of SEQ ID NO: 25 in the sequence listing) indicated by double underline are selected. As the translation initiation region of the PA gene, the 15th to 34th base sequences underlined in FIG. 3 (the base sequence represented by the sequence of SEQ ID NO: 26 in the sequence listing) were selected. Then, as a translation initiation region of the NP gene, as shown in FIG.
The 36th to 55th base sequences (base sequence represented by the sequence of SEQ ID NO: 27 in the sequence listing) underlined with are selected. In addition, in FIGS. 1 to 4, *** on the AUG
Indicates that the AUG is the translation initiation codon, and the horizontal line (−) in the base sequence indicates the omitted base sequence.

The base sequences around the selected base sequence are also shown in FIGS. Specifically, in FIG. 1, the base sequence selected as the translation initiation region of the PB2 gene and the 1st to 50th base sequences around it (the base sequence represented by the sequence of SEQ ID NO: 28 in the sequence listing) ) And the nucleotide sequence selected as the loop forming region of the PB2 gene and its surrounding 1269th to 1st
The 302nd base sequence (base sequence represented by the sequence of SEQ ID NO: 29 in the sequence listing) is shown. In addition, in FIG. 2, the base sequence selected as the translation start region of the PB1 gene and the 1st to 49th base sequences (base sequence represented by the sequence of SEQ ID NO: 30 in the sequence listing) and PB1 around the base sequence. The base sequence selected as a loop forming region of a gene and the 1269th to 1308th base sequences around the base sequence (base sequence represented by the sequence of SEQ ID NO: 31 in the sequence listing) are shown. In addition, in FIG. 3, the above-mentioned base sequence selected as the translation initiation region of the PA gene and the 1st to 54th nucleotides around the base sequence are selected.
The th base sequence (the base sequence represented by the sequence of SEQ ID NO: 32 in the sequence listing) is shown. In addition, in FIG. 4, the base sequence selected as the translation initiation region of the NP gene and the 1st to 70th base sequences in the vicinity thereof (SEQ ID NO: 33 in the sequence listing).
The base sequence represented by the sequence

Regarding the translation initiation region of the PB2 gene,
Antisense oligonucleotides, sense oligonucleotides, and random oligonucleotides were designed.
In the present specification, the term "random oligonucleotide" means that the number of bases (A, G, C, and T) constituting the random oligonucleotide is the number of bases of each base constituting the corresponding antisense oligonucleotide. And has a base sequence designed so as not to form a double strand with any part of the entire influenza virus gene. In addition, a loop forming region of the PB2 gene, a translation initiation region of the PB1 gene, a loop forming region of the PB1 gene, P
For the translation initiation region of A gene and the translation initiation region of NP gene, antisense oligonucleotides and random oligonucleotides were designed.

Furthermore, regarding each oligonucleotide in the translation initiation region of the PB2 gene, an oligonucleotide in which all the internucleotide bonds are natural phosphodiester bonds (hereinafter referred to as N-oligo) and all the internucleotides An oligonucleotide whose bond is a phosphorothioate type bond (hereinafter referred to as S-oligo) was designed. Loop forming region of PB2 gene,
Regarding the translation initiation region of the PB1 gene, the loop formation region of the PB1 gene, the translation initiation region of the PA gene, and the translation initiation region of the NP gene, N-oligo and S-oligo are used as antisense oligonucleotides, and random oligonucleotides. As a result, only S-oligo was designed.

Each of the oligonucleotides thus designed is shown in FIGS. Meanings of the symbols shown in the columns of abbreviations in FIGS. 5 to 7 are as follows. "Aug" means that the sequence of the designed DNA is in the translation initiation region of the viral gene, and "loop" means that the sequence of the designed DNA is in the loop forming region of the viral gene. "(N)"
Means that all internucleotide linkages are natural phosphodiester linkages, and “(s)” is
It is meant that all internucleotide linkages are phosphorothioate type linkages. "As" is the design DN
A means antisense to the designed viral gene sequence, and “sen” means design D
NA means that it has a sense with respect to the sequence of the viral gene to be designed, and "ran" means the designed DNA.
Means that the sequence is random with respect to the sequence of the viral gene for which it was designed. Each design D shown in FIGS.
"A", "G", "C", and "T" in the sequence of NA
Represents adenine, guanine, cytosine, and thymine, respectively, and "s" indicates that the bond between each nucleoside is a phosphorothioate type bond. The numbers shown in the columns of the position of the design target shown in FIGS. 5 to 7 are the base numbers indicating the positions in the viral gene shown in FIGS. 1 to 4 of the sequence targeted for the design.

Among the designed DNAs (oligonucleotides) shown in FIGS. 5 to 7, PB2-aug (n) as and PB2-
aug (s) as, NP-aug (n) as, and NP
-Aug (s) as] are oligonucleotides belonging to the present invention, and the other 17 types are comparative oligonucleotides. The number of bases of each oligonucleotide is 20 bases.

Example 2 Synthesis of Oligonucleotide Designed in Example 1 using an unmodified (normal) DNA program and a phosphorothioate type oligonucleotide program by an automatic DNA synthesizer (Model 392: Applied Biosystems). 21 kinds of the prepared oligonucleotides (N-oligo and S-oligo) were synthesized. That is, 1 μmol scale solid-phase column (Kruakem, UK) and DNA synthesis reagent (Kruakem, UK)
And were synthesized by the phosphoramidite method, and then cut out from the column and deprotected according to a conventional method [A. Cholet & E. H. Kawashima
a, Nucleic Acids Res. , 13, 1
529 (1985)]. A 1/500 amount (about 4 μg) of the obtained oligonucleotide was subjected to 20% polyacrylamide gel electrophoresis containing 7 M urea. Electrophoresis
It was carried out at a constant voltage of 150 V for 1.5 hours. After completion of the electrophoresis, methylene blue staining was performed to confirm that each synthetic oligonucleotide had the target chain length.

For each oligonucleotide confirmed to have the target chain length, 1/5 to 1/2 amount (about 1.2 mg) of the previously obtained oligonucleotide was
The sample was again subjected to 20% polyacrylamide gel electrophoresis containing 7M urea (for excision and purification). Electrophoresis is 200
The test was performed at a constant voltage of V for 6 hours. After the electrophoresis was completed, the gel was peeled off from the gel plate, wrapped in a wrap, irradiated with ultraviolet light, and a band having a desired chain length was marked with a marker. The gel inside the mark was cut into a dice with a disinfected cutter knife and collected in a 1.5 ml sample tube. Fill the sample tube with 10 mM Tris-HCl (pH
7.5) / 1 ml of 1 mM-EDTA solution was added, and 3
Vibration at 7 ° C. for 3-12 hours, DN from gel piece to solution
A was extracted. The extract was collected and phenol /
After performing chloroform extraction and removing acrylamide, ethanol precipitation was performed, and each oligonucleotide was purified. The yield of each is 0.3-0.
It was 6 mg. The resulting purified oligonucleotide (about 0.5 μg) was subjected to polyacrylamide gel electrophoresis containing 7 M urea, and after completion of the electrophoresis, ethidium bromide staining was performed. Each oligonucleotide showed one band.

Example 3: CAT protein expression inhibitory activity
Test (1) (1) Evaluation system for antisense oligonucleotide In this example, the 8th strain of influenza virus PR8 strain was used.
A DNA fragment in which the protein coding region of the segment is replaced by the CAT gene (a nucleotide sequence consisting of 13 base pairs of the 5'upstream region of the protein coding region of the eighth segment and a 3'downstream region of the protein coding region of the eighth segment of (Including a base sequence consisting of 12 base pairs), phage T7RNA
The plasmid pOUMS101 [Proc. Na
tl. Acad. Sci. USA, vol. 88, p
p. 5369-5373 (1991)] and dexamethasone for addition of influenza virus type A (A /
PR / 8/34) RNA polymerase (PB1, PB
2 and PA) and NP protein expression-induced mouse breast cancer cell line C127 clonal cells (hereinafter, 76).
Cells) [Y. Nakamura et a
l. , J. et al. Biochem. , 110, 395 (199
1)] was used to confirm the antiviral effect of the oligonucleotide of the present invention. The 76 cells described above are cell lines characterized by being capable of producing a CAT protein by transfection of a negative strand RNA containing the CAT gene, which was synthesized in vitro using the plasmid pOUMS101 as a template. FIG. 8 shows an outline of the operation procedure of this example.

(2) Negative chain R containing CAT gene
Preparation of NA The above plasmid pOUMS101 was prepared by
c. Natl. Acad. Sci. USA, vol. 8
8, pp. It was carried out by a conventional method as described in 5369-5373 (1991). FIG. 8 shows the plasmid pOUM.
The partial structure of S101 is typically shown. In FIG.
“CAT” indicates the CAT gene encoding the CAT protein, “T7” indicates the phage T7 RNA polymerase promoter, and “5′-NS” indicates 5 of the protein non-coding region of the eighth segment of influenza virus PR8 strain. 'Shows a base sequence consisting of 13 base pairs of the upstream region, and "3'-NS" is a base sequence consisting of 12 base pairs of the 3'downstream region of the protein non-coding region of the eighth segment of influenza virus PR8 strain. Indicates
“CAP” indicates the cap structure of mRNA, and “CAT”
"Protein" refers to CAT protein. The plasmid pOUMS101 has an MboII recognition site at a position 3'downstream from the region represented by 3'-NS.

The resulting plasmid pOUMS101 (1
0 μg) was cut with 32 units of the restriction enzyme MboII (Toyobo Co., Ltd.) to form a linear chain. After cutting, the mixture was extracted with phenol / chloroform (the same amount), the upper layer (aqueous layer) was carefully separated, and ethanol precipitation was performed. Then 1 /
Fifteen amounts (about 0.6 μg) were subjected to 0.8% agarose gel electrophoresis (1.5 hours at a constant voltage of 50 V) to confirm cleavage. Obtained linear plasmid pOUMS10
1 (10 μg) as a template, according to the attached instructions,
CAT by T7 RNA polymerase (Toyobo Co., Ltd.)
Negative strand RNA containing the gene (shown as vRNA in Figure 8) was synthesized. Subsequently, the mixture was extracted with phenol / chloroform (the same amount), the upper layer (aqueous layer) was carefully separated, and ethanol precipitation was performed. After that, part (1 /
(100 amount) was subjected to polyacrylamide gel electrophoresis containing 7 M urea to confirm the synthesized product of the target chain length.

(3) Measurement of CAT protein expression inhibitory activity Y. Nakamura et al. , J. et al. Bioch
em. , 110, 395 (1991), the Dulbecco's MEM medium (hereinafter referred to as D-MEM) containing 10% inactivated (inactivated by heat treatment at 56 ° C. for 30 minutes) fetal bovine serum. Refer to)
(Nissui Pharmaceutical Co., Ltd.) The cells were cultured in 5 ml until they became 50-60% confluent. Remove the culture solution to 10 ^-3 M
Medium 3 prepared by diluting a concentration of dexamethasone (Sigma) (shown as Dex in FIG. 8) with D-MEM to a final concentration of 10 ⁻⁶ M (1000-fold dilution)
76 ml of the above 50-60% confluent state
Added to cells. After culturing the cells for 24 hours, the cells were sufficiently washed with phosphate buffered saline [hereinafter referred to as PBS (-)] so that fetal calf serum did not remain. Antisense S-oligo, antisense N-oligo, sense S-oligo, sense N- prepared in Example 2
Oligo or random S-oligo (0.03 μM each)
And N- [1- as a transfection reagent
(2,3-Dioleoyloxy) propyl] -N, N,
N-trimethylammonium methyl sulfate (DO
TAP: Boehringer Mannheim (5 μg / m)
l) and 2.5 ml of serum-free D-MEM (containing HEPES and bovine albumin) were added to 76 cells.

After culturing the cells for an additional 4 hours, the cells were incubated with PB.
It was washed with S (-), and further, the CAT obtained in (2) above.
Negative strand RNA (100 ng) containing gene and DO
Serum-free D-MEM (H containing TAP (5 μg / ml))
EPES and bovine albumin) were added to 76 cells and the cells were cultured for 4 hours. Then, DOTAP was removed, the cells were washed with PBS (-), and then the cells were cultured for 20 hours with D-MEM containing 10% inactivated fetal bovine serum. Then, 76 cells were detached, and after crushing the cells, the amount of total protein recovered was measured by the Bradford method. Furthermore, enzyme-linked immunosorbent assay (CAT-ELIS) using a digoxigenin (DIG) -labeled anti-CAT antibody (Anti-CAT-DIG) (Boehringer Mannheim).
According to A), the amount of CAT protein expressed in cells was measured. That is, the amount of DIG-labeled anti-CAT antibody bound to the CAT protein expressed in 76 cells was measured using a peroxidase-labeled anti-DIG antibody, and the CAT protein expression levels were compared. Table 1 shows the obtained results.

[0039]

[Table 1] Oligonucleotide CAT protein expression inhibitory activity (%) PB2-aug (n) as 46 PB2-aug (s) as 40 PB2-aug (n) sen 18 PB2-aug (s) sen <5 PB2-aug (N) ran 9 PB2-aug (s) ran 9 PB2-loop (n) as 11 PB2-loop (s) as 10 PB2-loop (s) ran <5 PB1-aug (n) as <5 PB1-aug (S) as 14 PB1-aug (s) ran <5 PB1-loop (n) as <5 PB1-loop (s) as 7 PB1-loop (s) ran <5 PA-aug (n) as <5 PA -aug (s) as 18 PA- aug (s) ran <5 NP-aug (n) as 60 NP-aug (s) as 40 NP- ug (s) ran <5

The antisense oligonucleotides of the present invention, PB2-aug (n) as and PB2-aug
(S) as, and NP-aug (n) as and NP-
aug (s) as is significant CAT at 0.03 μM concentration
While the protein expression inhibitory activity (40% or more) was shown, the other comparative oligonucleotides showed 0.0
At a concentration of 3 μM, almost no CAT protein expression inhibitory activity was observed [18% or less]. Since each of the oligonucleotides of the present invention exhibited strong CAT protein expression inhibitory activity, it is considered that they can bind to mRNA, exhibit the function as an antisense nucleotide, and inhibit the expression of influenza virus gene. To be In addition, when the translation initiation region of PB2 was used as the target region and when the loop forming region of PB2 was used as the target sequence, no antisense effect was observed when the PB2 loop forming region was used as the target sequence. It was also found that the translation initiation region containing the AUG sequence which is the initiation codon is the most effective gene sequence position as the target region of PB2.

Example 4: CAT protein expression inhibitory activity
Test (2) Two kinds of antisense oligonucleotides [PB2-aug (s) as and NP-aug (s) as] as oligonucleotides of the present invention, and six kinds of antisense oligonucleotides [PB1 as comparison oligonucleotides]
-Aug (s) as, PA-aug (s) asPB2-
aug (s) ran, PB1-aug (s) ran, P
A-aug (s) ran and NP-aug (s) ra
n] was used, and the same procedure as in Example 3 was repeated except that DOTAP was not used when adding the above oligonucleotide to 76 cells. Table 2 shows the obtained results. At 0.03 μM concentration, DOT was detected with both antisense S-oligo and random S-oligo.
The ability to suppress CAT protein expression was lower than that when the oligonucleotide was administered using AP (Example 3 above). As is clear from this result, the transfection method using DOTAP is a very effective means for enhancing the antisense effect.

[0042]

[Table 2] Oligonucleotide CAT protein expression inhibitory activity (%) PB2-aug (s) as <5 PB2-aug (s) ran <5 PB1-aug (s) as <5 PB1-aug (s) ran <5 PA-aug (s) as <5 PA-aug (s) ran <5 NP-aug (s) as 11 NP-aug (s) ran <5

[0043]

INDUSTRIAL APPLICABILITY The oligonucleotide of the present invention can suppress the protein expression of influenza virus. By using the oligonucleotide of the present invention, an extremely effective therapeutic drug for influenza can be constructed.

[0044]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence TTCTTTCCAT ATTGAATATA 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Location: 1 ． . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence TTCTTTCCAT ATTGAATATA 20

SEQ ID NO: 3 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TATATTCAAT ATGGAAAGAA 20

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Location: 1. . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence TATATTCAAT ATGGAAAGAA 20

SEQ ID NO: 5 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CTTCTTATAT ACGATTATAT 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1. . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Array CTTCTTATAT ACGATTATAT 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence CCCTATTGAC GAAATTCAGA 20

SEQ ID NO: 8 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Location: 1 ． . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Array CCCTATTGAC GAAATTCAGA 20

SEQ ID NO: 9 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1. . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence ATGCTCACAT ACTGAAGTAC 20

SEQ ID NO: 10 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence TGACATCCAT TCAAATGGTT 20

SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Location: 1 ． . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence TGACATCCAT TCAAATGGTT 20

SEQ ID NO: 12 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1. . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence CTTATAAGTC CATTGATGAC 20

SEQ ID NO: 13 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence TCAGGATGGA GACGCCTAAT 20

SEQ ID NO: 14 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Location: 1 ． . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence TCAGGATGGA GACGCCTAAT 20

SEQ ID NO: 15 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Location: 1. . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence GAGCATGAAG AGTTGTACCC 20

SEQ ID NO: 16 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence AATCTTCCAT TTTGGATCAG 20

SEQ ID NO: 17 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Location: 1 ． . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence AATCTTCCAT TTTGGATCAG 20

SEQ ID NO: 18 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1. . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence TCATAAGTTC GACTTTCGTA 20

SEQ ID NO: 19 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence GGGACGCCAT GATTTTGATG 20

SEQ ID NO: 20 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Location: 1 ． . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Sequence GGGACGCCAT GATTTTGATG 20

SEQ ID NO: 21 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1. . 20 Method for determining characteristics: E Other information: The linkage between nucleosides is a phosphorothioate linkage. Array TTAGGTGGAG TCAACGCTGT 20

SEQ ID NO: 22 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence UAUAUUCAAU AUGGAAAGAA 20

SEQ ID NO: 23 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence UCUGAAUUUC GUCAAUAGGG 20

SEQ ID NO: 24 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence AACCAUUUGA AUGGAUGUCA 20

SEQ ID NO: 25 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence AUUAGGCGUC UCCAUCCUGA 20

SEQ ID NO: 26 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence CUGAUCCAAA AUGGAAGAUU 20

SEQ ID NO: 27 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence CAUCAAAAUC AUGGCGUCCC 20

SEQ ID NO: 28 Sequence length: 50 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence AGCGAAAGCA GGUCAAUUAU AUUCAAUAUG GAAAGAAUAA AAGAACUAAG 50

SEQ ID NO: 29 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence UUAGAGGUGA UCUGAAUUUC GUCAAUAGGG CGAA 34

SEQ ID NO: 30 Sequence length: 49 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence AGCGAAAGCA GGCAAACCAU UUGAAUGGAU GUCAAUCCGA CCUUACUUU 49

SEQ ID NO: 31 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence UAAGCACUGU AUUAGGCGUC UCCAUCCUGA AUCUUGGACA 40

SEQ ID NO: 32 Sequence length: 54 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic RNA sequence AGCGAAAGCA GGUACUGAUC CAAAAUGGAA GAUUUUGUGC GACAAUGCUU 50 CAAU 54

SEQ ID NO: 33 Sequence length: 70 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Genomic RNA sequence AGCAAAAGCA GGGUAGAUAA UCACUCACUG AGUGACAUCA AAAUCAUGGC 50 GUCCCAAGGC ACCAAACGGU 70

[0078]

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing a partial nucleotide sequence of an influenza virus PB2 gene.

FIG. 2 is an explanatory diagram showing a partial base sequence of the influenza virus PB1 gene.

FIG. 3 is an explanatory view showing a partial base sequence of influenza virus PA gene.

FIG. 4 is an explanatory view showing a partial base sequence of influenza virus NP gene.

FIG. 5 is an explanatory diagram showing the base sequences of the oligonucleotide of the present invention and the comparative oligonucleotide designed in Example 1.

FIG. 6 is an explanatory view showing a base sequence of a comparative oligonucleotide designed in Example 1.

FIG. 7 is an explanatory diagram showing base sequences of another oligonucleotide of the present invention and a comparative oligonucleotide designed in Example 1.

FIG. 8 is an explanatory view showing the outline of the operation of the CAT protein expression inhibitory activity test conducted in Example 3.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // A61K 31/70 ADY A61K 31/70 ADY (C12N 15/09 ZNA C12R 1:92)

Claims (4)

[Claims] 1. An oligonucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of a target region containing the translation initiation codon AUG of the PB2 gene or the NP gene of influenza virus. 2. The oligonucleotide of claim 1, wherein at least one internucleotide linkage is a phosphorothioate type linkage. 3. The oligonucleotide according to claim 1, which has a nucleotide sequence represented by the sequence of SEQ ID NO: 2 in the sequence listing. 4. The oligonucleotide according to claim 1, which has a base sequence represented by the sequence of SEQ ID NO: 20 in the sequence listing.