WO2017135397A1 - Antisense oligonucleotide for suppressing expression of complement b factor - Google Patents

Abstract

The present invention provides: an antisense oligonucleotide having CFB expression-suppressing activity; a pharmaceutical composition including the antisense oligonucleotide; and a drug that suppresses the expression of CFB including the antisense oligonucleotide, the drug preventing or treating disorders mediated by abnormalities of the complement alternative pathway such as atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranous-proliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapid progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, or autoimmune diseases.

Description

Antisense oligonucleotide that suppresses expression of complement factor B

The present invention relates to an antisense oligonucleotide for use in suppressing the expression of complement factor B or a pharmaceutical composition containing the antisense oligonucleotide.

Complements are a group of proteins in the blood that mediate immune responses. Complement effects include phagocytosis of pathogenic bacteria by phagocytic cells, damage to viruses with outer membranes, and loss of infectivity. Is mentioned. Among complements, C3 is the most abundant in serum, and its action is controlled by being activated by complement factor B (CFB) in the alternative pathway (non-patented) Reference 1).

If C3 continues to be activated due to abnormalities in regulatory factors related to the alternative pathway of the complement or stabilization with autoantibodies to C3 convertase, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria Disease (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), It is known to develop diseases such as systemic lupus erythematosus (SLE), asthma, psoriasis, neuromyelitis optica, myasthenia gravis and autoimmune disease (Non-patent Document 2). Although it can be expected that the above-mentioned diseases can be prevented or treated by specifically inhibiting the action of complement factor B, complement factor B is present in the blood at a relatively high concentration of 300 μg / mL ( Non-patent document 3), it is not easy to continue to inhibit all these complement factor B by, for example, a general antibody drug.

On the other hand, for example, an antisense method is known as a method for suppressing gene expression itself (Patent Document 1). Specifically, oligonucleotides complementary to the target gene mRNA or mRNA precursor (antisense oligonucleotide) are introduced into the target gene mRNA or mRNA precursor in two. It forms a chain and can specifically suppress the expression of the target gene. As a method for suppressing gene expression other than the antisense method, a method using RNA interference (hereinafter referred to as RNAi) is also known. In this method, the expression of the target gene can be specifically suppressed by introducing a double-stranded RNA (siRNA) having the same sequence as the target gene (Patent Document 2).

A part of siRNA sequence targeting human complement factor B gene has been disclosed (Patent Document 3). Moreover, although some antisense sequences targeting the gene have been disclosed (Patent Document 4), the present invention is a sequence different from these sequences.

International Publication No. 98/56905 Pamphlet International Publication No. 2001/75164 Pamphlet International Publication Number 2015/089368 Pamphlet International Publication No. 2015/038939 Pamphlet

An object of the present invention is to provide a novel antisense oligonucleotide capable of suppressing the expression of complement factor B. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease associated with the expression of complement factor B.

The present invention relates to the following (1) to (13).
(1) An antisense oligonucleotide that suppresses the expression of complement factor B, and is capable of hybridizing under stringent conditions to a nucleic acid comprising the target base sequence represented by any of SEQ ID NOs: 53 to 103. 8 Antisense oligonucleotide of ~ 80 bases length.
(2) An antisense oligonucleotide that suppresses the expression of complement factor B and has a length of 8 to 80 bases comprising at least 8 consecutive bases of the antisense base sequence represented by any of SEQ ID NOs: 2 to 52 Antisense oligonucleotides.
(3) An antisense oligonucleotide having a length of 8 to 80 bases comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 53 to 103.
(4) The antisense oligonucleotide according to (3), comprising the base sequence represented by any of SEQ ID NOs: 2-52.
(5) The antisense oligonucleotide according to (4), comprising a sequence in which one to several bases are deleted, substituted, inserted or added in the base sequence represented by any of SEQ ID NOs: 2 to 52 .
(6) An antisense oligonucleotide comprising the base sequence represented by any of SEQ ID NOs: 2-52.
(7) The antisense oligonucleotide according to any one of (1) to (6), wherein the vicinity of the 5 ′ end and / or the vicinity of the 3 ′ end is composed of sugar-modified nucleotides.
(8) An antisense oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 104 to 154 and 155.
(9) An antisense oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 118, 125, 150 and 155.
(10) The antisense oligonucleotide according to any one of (1) to (9), comprising a ligand.
(11) A pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1) to (10).
(12) A method for treating a disorder mediated by an abnormality in the alternative complement pathway, comprising a therapeutically effective amount of the antisense oligonucleotide according to any one of (1) to (10) or (11 ) Comprising administering to a human in need of such treatment.
(13) The disorder is atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma or autoimmune disease, the method according to (12).

It is possible to suppress the expression of complement factor B by administering the antisense oligonucleotide of the present invention or the pharmaceutical composition containing the antisense oligonucleotide. The antisense oligonucleotide of the present invention or the pharmaceutical composition containing the antisense oligonucleotide is useful for the treatment / prevention of a disorder mediated by an abnormality in the alternative complement pathway.

FIG. 1 shows the results of in vivo knockdown in mice administered with an antisense oligonucleotide represented by any of SEQ ID NOs: 118, 125 and 150. The vertical axis shows the relative expression rate of CFB mRNA in each antisense oligonucleotide administration group when the semi-quantitative value of complement factor B (CFB) mRNA in the control group is 1. FIG. 2 shows the results of in vivo knockdown in mice administered with the antisense oligonucleotide represented by SEQ ID NO: 155. The vertical axis shows the relative expression rate of CFB mRNA in the antisense oligonucleotide-administered group when the semi-quantitative value of CFB mRNA in the control group is 1.

1. Antisense oligonucleotide of the present invention The present invention provides an antisense oligonucleotide that suppresses the expression of complement factor B (CFB) (also referred to herein as the “antisense oligonucleotide of the present invention”). .

As used herein, the term “antisense oligonucleotide” refers to a DNA constituting a target gene, an mRNA precursor transcribed from such DNA, and an oligonucleotide complementary to the mRNA. Suppresses the action of DNA, mRNA precursor or mRNA (transcription, post-transcriptional editing, translation, etc.) by forming double-stranded or triple-stranded with target DNA, mRNA precursor or mRNA.

As a gene encoding CFB targeted by the antisense oligonucleotide of the present invention (hereinafter also referred to as CFB gene), a cDNA base sequence corresponding to human CFB full-length mRNA registered as Genbank Accession No. NM_001710 (SEQ ID NO: 1), and naturally occurring variants thereof (see, for example, Hum. Mutat. 31: E1445-E1460 (2010), refsnp No. rs12614, rs641153, etc.) are also antisense of the present invention. It goes without saying that it can be a target gene for oligonucleotides.

As used herein, “complementary” refers to, for example, base pairing between two bases via a slow hydrogen bond, such as the relationship between adenine and thymine or uracil, and the relationship between guanine and cytosine. Means a relationship.
In addition, “complementary” means not only when two nucleotide sequences are completely complementary in a double-stranded region (a region overlapping when both sequences are aligned), but also the entire double-stranded region. As long as it can have a double helix structure, that is, as long as both sequences can hybridize under stringent conditions, a case where 1 to several mismatches exist is also included.

Therefore, the antisense oligonucleotide of the present invention is a nucleic acid molecule capable of hybridizing under stringent conditions to a nucleic acid comprising DNA encoding CFB, an mRNA precursor, or a target base sequence in mRNA.

In the present specification, “stringent conditions” means that the antisense oligonucleotide of the present invention hybridizes to the target base sequence of the CFB gene, but does not hybridize to other sequences or even if it is a target. It means a condition that is significantly less than the amount hybridized to the base sequence and hybridizes only in a negligible amount that is relatively negligible. Such conditions can be easily selected by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, it is hybridized at 42 ° C in 6xSSC (0.9M NaCl, 0.09M trisodium citrate) or 6xSSPE (3M NaCl, 0.2M NaH ₂ PO ₄ , 20mM EDTA · 2Na, pH7.4), and further 42 ° C The conditions for washing with 0.5xSSC are examples of stringent conditions, but are not limited thereto. As a hybridization method, for example, Southern blot hybridization method or the like can be used. Specifically, it should be performed according to the method described in Molecular Cloning: A Laboratory Mannual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (1994) (Wiley-Interscience), etc. Can do.

The antisense oligonucleotide of the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides or molecules having functions equivalent to those of the nucleotides. For example, DNA that is a polymer of deoxyribonucleotides, and DNA ribonucleotides. Examples thereof include RNA as a combination, chimeric nucleic acids composed of DNA and RNA, and nucleotide polymers in which at least one nucleotide of these nucleic acids is substituted with a molecule having a function equivalent to that of the nucleotide. In addition, uracil (U) in RNA can be uniquely read as thymine (T) in DNA.

Examples of molecules having functions equivalent to nucleotides include nucleotide derivatives. The nucleotide derivative may be any molecule as long as it is a molecule in which nucleotides have been modified. For example, in order to improve nuclease resistance or stabilize the molecule compared to DNA or RNA, In order to increase affinity, cell permeability, or visualization, deoxyribonucleotides or molecules modified with ribonucleotides are preferably used.

Examples of the molecule in which the nucleotide is modified include a sugar moiety-modified nucleotide, a phosphodiester bond-modified nucleotide, a base-modified nucleotide, and a nucleotide in which two or more of the sugar moiety, phosphodiester bond and base are modified.

As the sugar-modified nucleotide, any or all of the chemical structure of the sugar of the nucleotide may be modified or substituted with any substituent, or may be substituted with any atom. '-Modified nucleotides are preferably used.

Examples of 2′-modified nucleotides include ribose 2′-OH groups from OR, R, R′OR, SH, SR, NH ₂ , NHR, NR ₂ , N ₃ , CN, F, Cl, Br, and I. 2 ′ substituted with a substituent selected from the group consisting of: R is alkyl or aryl, preferably alkyl having 1 to 6 carbons, and R ′ is alkylene, preferably alkylene having 1 to 6 carbons -Modified nucleotides are included, preferably 2'-modified nucleotides substituted with F or methoxy groups. In addition, 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- [2- (N, N-Dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) ethoxy group, and 2'-substituted with a substituent selected from the group consisting of 2-cyanoethoxy group Examples include modified nucleotides. More preferred examples include 2′-modified nucleotides substituted with a substituent selected from the group consisting of a methoxy group and a 2- (methoxy) ethoxy group.

In addition, as the sugar moiety-modified nucleotide, a crosslinked structure-type artificial nucleic acid (BNA) having two circular structures by introducing a crosslinked structure into the sugar moiety is also preferably used. Specifically, a locked artificial nucleic acid (LNA) (Tetrahedron Letters, 38, 8735, (1997) and Tetrahedron, in which a 2 ′ oxygen atom and a 4 ′ carbon atom are bridged via methylene 54, 3607, (1998)], Ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)], Constrained Ethyl (cEt) [The Journal Organic gan Chemistry 75, 1569 (2010)], Amido-Bridged Nucleic Acid (AmNA) [Chem Bio Chem 13, 2513 (2012)] and 2'-O, 4'-C-Spirocyclopropylene bridged nucleic acid (scpBNA) [Chem. Commun., 51 , 9737 (2015)].

Furthermore, peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], peptide ribonucleic acid (2001) PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like can also be mentioned as sugar-modified nucleotides.

As the phosphodiester bond-modified nucleotide, any or all of the chemical structure of the nucleotide phosphodiester bond modified or substituted with any substituent or any atom may be used. For example, a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, a phosphate Examples include nucleotides in which a diester bond is substituted with a phosphoramidate bond, and preferably nucleotides in which a phosphodiester bond is substituted with a phosphorothioate bond.

The base-modified nucleotide may be any nucleotide as long as it is part or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent, or substituted with an arbitrary atom. In which oxygen atoms are substituted with sulfur atoms, hydrogen atoms are substituted with alkyl groups having 1 to 6 carbon atoms, halogen groups, methyl groups are hydrogen, hydroxymethyl, alkyl groups with 2 to 6 carbon atoms And those in which an amino group is substituted with an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, a hydroxy group, or the like. In addition, it is one of the preferable embodiments of the present invention to use 5-methylcytosine (5-mC) as a base-modified nucleotide instead of cytosine (C).

Examples of nucleotide derivatives include nucleotide derivatives modified with at least one of nucleotide or sugar moiety, phosphodiester bond or base, ligands such as lipids such as cholesterol, fatty acids, tocopherols, retinoids, N-acetylgalactosamine (GalNAc) , Sugars such as galactose (Gal) and mannose (Man), full antibodies, fragment antibodies such as Fab, scFv, VHH, proteins such as low density lipoprotein (LDL), human serum albumin, RGD, NGR, R9, CPP, etc. Peptides, small molecules such as phenazine, phenanthridine, anthraquinone, folic acid, synthetic polymers such as synthetic polyamino acids, dyes such as nucleic acid aptamer, acridine, fluorescein, rhodamine, coumarin, Cy3 series, Alexa series, black hole quencher Such as fluorescence There may be mentioned those obtained by adding another chemical substance such as a group directly or via a linker. Specifically, polyamine addition nucleotide derivatives, cholesterol addition nucleotide derivatives, steroid addition nucleotide derivatives, GalNAc addition nucleotide derivatives, bile acid addition nucleotide derivatives, fatty acid addition nucleotide derivatives, vitamin addition nucleotide derivatives, Cy5 addition nucleotide derivatives, Cy3 addition nucleotide derivatives, Examples include 6-FAM-added nucleotide derivatives and biotin-added nucleotide derivatives, and preferably include GalNAc-added nucleotide derivatives. These can be modified at the 5 'end, 3' end and / or inside the sequence by reacting a modifying agent capable of reacting on the solid phase during the extension reaction on the solid phase. It can also be obtained by previously synthesizing and purifying nucleic acids into which a functional group such as an amino group, mercapto group, azide group or triple bond has been introduced, and allowing a modifier to act on them.

The nucleotide derivative may form a crosslinked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, or a structure obtained by combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.

The antisense oligonucleotide of the present invention includes those in which some or all atoms in the molecule are substituted with atoms (isotopes) having different mass numbers.

The antisense oligonucleotide of the present invention may be introduced into the form of a hairpin oligomer or circular oligomer as long as it is a nucleic acid that hybridizes under stringent conditions to the target base sequence of the CFB gene. It may contain structural elements such as loops.

The length of the antisense oligonucleotide of the present invention is 8 to 80 bases, preferably 8 to 30 bases. For example, it can be 8-20 bases, 10-20 bases, 13-20 bases, 13-16 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases.

Specific examples of the target base sequence of the antisense oligonucleotide of the present invention include a partial base sequence of CFB mRNA (cDNA) represented by SEQ ID NOs: 53 to 103 described in Table 1. Therefore, the antisense oligonucleotide of the present invention is an oligonucleotide capable of hybridizing under stringent conditions to the target base sequence represented by any of SEQ ID NOs: 53 to 103. As long as the antisense oligonucleotide of the present invention can hybridize to any one of the above target base sequences under stringent conditions, not only the sequence completely complementary to the target base sequence but also 1 to the target base sequence. Also included are those containing sequences having several, preferably 1 to 3, more preferably 1 or 2, particularly preferably 1 mismatch. Therefore, the antisense oligonucleotide of the present invention includes a base sequence complementary to the target base sequence represented by any of SEQ ID NOs: 53 to 103 and a sequence completely complementary to the target base sequence. , 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base deleted, substituted, inserted or added may be used.

Specific preferred specific base sequences are described herein, but at least 8 selected from the antisense base sequences represented by any of SEQ ID NOs: 2 to 52 described in Table 1 Antisense oligonucleotides having a length of 8 to 80 bases containing a series of bases are also preferred antisense oligonucleotides. More preferably 9 or more, still more preferably 10 or more, still more preferably 11 or more, particularly preferably 12 or more, most preferably selected from each antisense base sequence described in Table 1 Antisense oligonucleotides containing 13 consecutive bases and having a length of 8 to 80 bases, preferably 8 to 30 bases, are also preferred antisense oligonucleotides.

Typical preferred antisense oligonucleotides include at least 8, more preferably 9 or more, even more preferably 10 or more, and even more preferably 11 from the 5 ′ end of each antisense base sequence listed in Table 1. Oligonucleotides comprising one or more, particularly preferably 12 or more consecutive nucleobases are included. Similarly, preferable antisense oligonucleotides include at least 8 or more, more preferably 9 or more, still more preferably 10 or more, and still more preferably 11 from the 3 ′ end of each antisense base sequence described in Table 1. Oligonucleotides comprising one or more, particularly preferably 12 or more consecutive nucleobases are included. Most preferably, it is an oligonucleotide containing each antisense base sequence described in Table 1.

The antisense oligonucleotide of the present invention includes each antisense base sequence described in Table 1, and lacks 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base in the antisense base sequence. A deletion, substitution, insertion or addition may be used.

As the antisense oligonucleotide of the present invention, an antisense oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 104 to 154 and 155 described in Table 2 is preferably used. Among these, it is preferable to use an antisense oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 118, 125, 150 and 155.

Antisense oligonucleotides of the present invention include antisense oligonucleotides described in Table 2 and Table 3, each having an antisense oligonucleotide sequence having a CFB relative expression level of 0.5 or less. It is more preferable to use nucleotides, more preferably antisense oligonucleotides having a CFB relative expression level of 0.3 or less, and particularly preferably antisense oligonucleotides having a CFB relative expression level of 0.1 or less.

When the antisense oligonucleotide of the present invention is introduced into a cell, it binds to complementary mRNA and mRNA precursor, and sterically inhibits translation of the mRNA and mRNA precursor into a protein, thereby inhibiting the CFB gene. Can be suppressed.

In addition, the antisense oligonucleotide of the present invention introduced into a cell may bind to complementary mRNA and mRNA precursor in the cell and cleave the mRNA and mRNA precursor. As such an example, an action through RNaseH, which is an endonuclease that cleaves RNA strands of RNA and DNA double strands, is known. When the antisense oligonucleotide of the present invention forms a duplex with mRNA and mRNA precursor in the cell, it is recognized by endogenous RNase H and can enzymatically degrade the complementary mRNA strand.

In order to induce cleavage of mRNA and mRNA precursor by RNaseH, an antisense oligonucleotide having 4 to 80 continuous DNA regions is preferable. In this case, the antisense oligonucleotide preferably has 0 to 80% sugar-modified nucleotides, more preferably 10 to 60%, and even more preferably 20 to 50%. In addition, the number of continuous DNA regions having a sugar-modified nucleotide is more preferably 4 to 20, more preferably 4 to 15, and most preferably 5 to 10. Furthermore, the position of the sugar-modified nucleotide in the antisense oligonucleotide is preferably arranged in the vicinity of the 5 ′ end and / or in the vicinity of the 3 ′ end, and the position within 30% of the total length from the 5 ′ end and / or More preferably, it is arranged at a position within 30% of the total length from the 3 ′ end, and the sugar-modified nucleotide is located at a position within 25% of the total length from the 5 ′ end of the antisense oligonucleotide and / or More preferably, it is arranged at a position within 25% of the total length from the 3 ′ end.

When introduced into a cell, the antisense oligonucleotide of the present invention also binds to genomic DNA encoding a complementary CFB, inhibits transcription of the DNA to an mRNA precursor, and inhibits expression of the CFB gene. Can be suppressed.

In the antisense oligonucleotide of the present invention, it is particularly preferable that the vicinity of the 5 'end and / or the vicinity of the 3' end is a sugar-modified nucleotide. In the present specification, the vicinity of the 5 ′ end is a sugar moiety-modified nucleotide means that 1 to 4, preferably 2 to 4 consecutive nucleotides from the 5 ′ end are sugar part-modified nucleotides, The vicinity of the 3 ′ end is a sugar part modified nucleotide means that 1 to 4, preferably 2 to 4 consecutive nucleotides from the 3 ′ end are sugar part modified nucleotides. That is, the antisense oligonucleotide of the present invention is preferably an antisense oligonucleotide in which 1 to 4 nucleotides from the 5 ′ end are sugar-modified nucleotides, more preferably 2 to 4 from the 5 ′ end. In which the nucleotide is a sugar-modified nucleotide. The antisense oligonucleotide of the present invention is preferably an antisense oligonucleotide in which 1 to 4 nucleotides from the 3 ′ end are sugar-modified nucleotides, more preferably 2 to 4 nucleotides from the 3 ′ end. Can be used in which is a sugar-modified nucleotide.

The method for producing the antisense oligonucleotide of the present invention is not particularly limited, and includes a method using a known chemical synthesis, an enzymatic transcription method, or the like. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method [Nucleic® Acid® Research, 35, 3287] (2007)]. For example, ABI3900 high-throughput nucleic acid synthesis Can be synthesized by a machine (Applied Biosystems). After the synthesis is completed, elimination from the solid phase, deprotection of the protecting group, purification of the target product, and the like are performed. It is desirable to obtain an antisense oligonucleotide having a purity of 90% or more, preferably 95% or more by purification. As an enzymatic transcription method for producing the antisense oligonucleotide of the present invention, a transcription method using a phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template. Can be mentioned.

The antisense oligonucleotide of the present invention can be introduced into cells using a transfection carrier, preferably a cationic carrier such as a cationic liposome. It can also be directly introduced into cells by the calcium phosphate method, electroporation method or microinjection method.

The antisense oligonucleotide of the present invention can also induce suppression of target gene expression by forming a double strand with a complementary oligonucleic acid and introducing it into a cell as a double-stranded nucleic acid (International Publication No. 2005). / 113571). In this case, the position at which the double-stranded nucleic acid is modified with a ligand is preferably the 5 'end or 3' end of a complementary oligonucleic acid.

2. Inhibition of CFB Expression by the Antisense Oligonucleotide of the Present Invention The antisense oligonucleotide of the present invention has a cDNA base sequence (SEQ ID NO: 1) corresponding to the full-length mRNA of human CFB or a genomic sequence corresponding to the mRNA precursor. Can be designed based on. The cDNA of human CFB full-length mRNA is registered, for example, as Genbank Accession No. NM_001710, and the genomic sequence containing the human CFB mRNA precursor is, for example, Genbank Accession No. It is registered.

Among the nucleic acid consisting of a base sequence complementary to a part of the target base sequence of the CFB gene, the antisense oligonucleotide having CFB expression suppression activity is selected from the group described in Table 1 or Table 2, for example. And antisense oligonucleotides composed of antisense base sequences. The length of the antisense oligonucleotide is 8 to 80 bases, preferably 8 to 30 bases. For example, it can be 8-20 bases, 10-20 bases, 13-20 bases, 13-16 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases.

CFB expression can be suppressed by introducing these antisense oligonucleotides into cells. For example, the antisense oligonucleotide of the present invention can suppress the expression of CFB mRNA after being introduced into cells at a concentration of several nM to several μM and then cultured for 24 hours or more, for example 48 hours.

The evaluation of the CFB mRNA expression inhibitory activity of the antisense oligonucleotide of the present invention can be carried out using the antisense oligonucleotide as a cationic liposome or the antisense oligonucleotide as it is, or the antisense oligonucleotide. Can be bound to some ligand, introduced into a human cell line, etc., cultured for a certain period of time, and then quantified for the expression level of CFB mRNA in the human cell line.

The antisense oligonucleotide of the present invention may contain a ligand. The ligand may be one that directly modifies the 5 ′ end, 3 ′ end and / or the sequence interior of the antisense oligonucleotide of the present invention.

The ligand contained in the antisense oligonucleotide of the present invention may be any molecule that has affinity for biomolecules.For example, lipids such as cholesterol, fatty acids, tocopherols, retinoids, N-acetylgalactosamine (GalNAc), Sugars such as galactose (Gal) and mannose (Man), full antibodies, fragment antibodies such as Fab, scFV, VHH, proteins such as low density lipoprotein (LDL), human serum albumin, RGD, NGR, R9, CPP, etc. Peptides, low molecular weight compounds such as folic acid, synthetic polymers such as synthetic polyamino acids, nucleic acid aptamers, and the like can be mentioned, but the invention is not limited thereto, and these can be used in appropriate combinations.

As a method for adding a ligand to the antisense oligonucleotide of the present invention, for example, at the time of extension reaction on a solid phase, by reacting a modifying agent capable of reacting on the solid phase, the 5 ′ end, 3 ′ end and However, the present invention is not limited to this. Alternatively, a conjugated nucleic acid can be obtained by previously synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifier to act on them.

3. Pharmaceutical composition of the present invention The present invention relates to a pharmaceutical composition containing the antisense oligonucleotide of the present invention as an active ingredient (hereinafter also referred to as the pharmaceutical composition of the present invention).

The pharmaceutical composition can further contain a carrier effective for transferring the antisense oligonucleotide into the cell. The pharmaceutical composition of the present invention comprises atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), C3 Nephritis, membranous nephropathy, rapid progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis) ) And the like.

Examples of the carrier effective for transferring the antisense oligonucleotide into the cell include a cationic carrier. Examples of the cationic carrier include cationic liposomes and cationic polymers. Further, a carrier utilizing a viral envelope may be used as a carrier effective for transferring the antisense oligonucleotide into the cell. Examples of cationic liposomes include liposomes containing 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol (hereinafter also referred to as liposome A), oligofectamine (Invitrogen), lipofectin ( Invitrogen), Lipofectamine (Invitrogen), Lipofectamine 2000 (Invitrogen), DMRIE-C (Invitrogen), GeneSilencer (Gene Therapy Systems), TransMessenger (QIAGEN), TransIT TKO (Mirus), etc. Is preferably used. As the cationic polymer, JetSI (Qbiogene), Jet-PEI (polyethyleneimine; Qbiogene) and the like are preferably used. As a carrier using a virus envelope, GenomeOne (HVJ-E liposome; Ishihara Sangyo Co., Ltd.) is preferably used.

The pharmaceutical composition of the present invention comprising the above-described carrier in the antisense oligonucleotide of the present invention can be prepared by methods known to those skilled in the art. For example, it can be prepared by mixing a carrier dispersion having an appropriate concentration and an antisense oligonucleotide solution. When a cationic carrier is used, the antisense oligonucleotide is negatively charged in an aqueous solution, and therefore can be easily prepared by mixing in an aqueous solution by a conventional method. Examples of the aqueous solvent used for preparing the composition include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution. In addition, conditions such as pH and temperature when preparing the composition can be appropriately selected by those skilled in the art. For example, in the case of liposome A, prepared by gradually adding an antisense oligonucleotide in 10% maltose aqueous solution to 16 mg / ml liposome dispersion in 10% maltose aqueous solution with stirring at pH 7.4 and 25 ° C. can do.

The composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersion device or a high-pressure emulsification device if necessary. The optimum method and conditions for the preparation of the composition comprising the carrier and the antisense oligonucleotide depend on the carrier to be used, and those skilled in the art can select the optimum method for the carrier to be used without being bound by the above method. it can.

The pharmaceutical composition of the present invention comprises a composite particle comprising an antisense oligonucleotide and a lead particle as constituents and a lipid membrane covering the composite particle, wherein the constituent of the lipid membrane is soluble A liposome containing a liquid containing the polar organic solvent at a concentration capable of dispersing the constituent components of the lipid membrane and the composite particles can also be suitably used. Examples of the lead particles include lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, and fine particle preparations, and liposomes are preferably used. The lead particles in the present invention may be composed of a complex obtained by combining two or more lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle formulations, etc., and lipid aggregates, liposomes, emulsion particles, A complex formed by combining a polymer, a metal colloid, a fine particle preparation, and the like with another compound (eg, sugar, lipid, inorganic compound, etc.) may be used as a constituent component.

Examples of the lipid membrane that coats the composite particles include neutral lipids and polyethylene glycol-phosphatidylethanolamine as constituent components.

The liposome can be prepared, for example, according to the method described in WO2006 / 080118.

The mixing ratio of the antisense oligonucleotide and the carrier contained in the pharmaceutical composition of the present invention is suitably 1 to 200 parts by weight of the carrier with respect to 1 part by weight of the antisense oligonucleotide. Preferably, the amount is 2.5 to 100 parts by weight, more preferably 10 to 20 parts by weight, based on 1 part by weight of the antisense oligonucleotide.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent in addition to the above carrier. A pharmaceutically acceptable carrier or diluent or the like is an essentially chemically inert and harmless composition that does not affect the biological activity of the pharmaceutical composition of the present invention at all. Examples of such carriers or diluents include but are not limited to salt solutions, sugar solutions, glycerol solutions, ethanol and the like.

The pharmaceutical composition of the present invention can be suitably used for the treatment or prevention of disorders mediated by abnormalities in the alternative pathway of complement. In the present specification, disorders mediated by abnormalities in the alternative complement pathway include atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), Membranoproliferative glomerulonephritis (MPGN), C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE)) Psoriasis, optic neuritis, myasthenia gravis, etc.). Therefore, the pharmaceutical composition of the present invention comprises atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN) , C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis It can be used as a therapeutic or prophylactic agent such as

The pharmaceutical composition of the present invention can be provided in a form that contains an amount of the complex effective for treating or preventing a disease and can be appropriately administered to a patient. The preparation form of the pharmaceutical composition of the present invention may be, for example, injections, eye drops, liquids for inhalation, etc., for example, external preparations such as ointments, lotions and the like.

In the case of a liquid, the concentration range of the pharmaceutical composition of the present invention is usually 0.001 to 25% (w / v), preferably 0.01 to 5% (w / v), more preferably 0.1 to 2% ( w / v). The pharmaceutical composition of the present invention may contain an appropriate amount of any pharmaceutically acceptable additive, for example, an emulsification aid, a stabilizer, an isotonic agent, a pH adjuster and the like. Any pharmaceutically acceptable additive can be added in an appropriate step before or after dispersion of the complex.

The pharmaceutical composition of the present invention can also be provided as a lyophilized preparation. The lyophilized preparation can be prepared by dispersing the antisense oligonucleotide and the carrier and then lyophilizing. The lyophilization treatment can be performed by a conventional method. For example, a predetermined amount of the complex solution after the above dispersion treatment is aseptically dispensed into a vial, preliminarily dried for about 2 hours under a condition of about -40 to -20 ° C, and about 0 to 10 ° C. Primary drying under reduced pressure, followed by secondary drying under reduced pressure at about 15-25 ° C. and lyophilization. Then, for example, by replacing the inside of the vial with nitrogen gas and stoppering, a freeze-dried preparation of the pharmaceutical composition of the present invention can be obtained.

When the pharmaceutical composition of the present invention is provided as a lyophilized preparation, the pharmaceutical composition of the present invention can be redissolved and used by adding any appropriate solution. Examples of such a solution include electrolytes such as water for injection and physiological saline, glucose solution, and other general infusion solutions. The amount of this solution varies depending on the application and is not particularly limited, but it is preferably 0.5 to 2 times the amount before lyophilization, or 500 ml or less.

The pharmaceutical composition of the present invention can be administered to animals including humans, for example, intravenous administration, intraarterial administration, oral administration, tissue administration, transdermal administration, transmucosal administration, or rectal administration. It is preferable to administer by an appropriate method according to the symptoms. In particular, intravenous administration, transdermal administration, and transmucosal administration are preferably used. Moreover, local administration, such as local administration in cancer, can also be performed. Examples of the dosage form suitable for these administration methods include various injections, oral preparations, drops, absorbents, eye drops, ointments, lotions, suppositories and the like.

The dosage of the pharmaceutical composition of the present invention is preferably determined in consideration of the drug, dosage form, patient condition such as age and weight, administration route, nature and degree of disease, etc. The mass of nucleotide is 0.1 mg to 10 g / day, preferably 1 mg to 500 mg / day per day for an adult. In some cases, this may be sufficient, or vice versa. It can also be administered once to several times a day, and can be administered at intervals of 1 to several days.

4. Therapeutic methods The present invention is further a method of treating a disorder mediated by an alternative pathway alternative comprising a therapeutically effective amount of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention. , A method comprising the step of administering to a human in need of such treatment (the treatment method of the invention).

The treatment method of the present invention is preferably an atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapidly progressive It is a method of treating diseases such as glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis) Thus, it is characterized in that a therapeutically effective amount of the antisense oligonucleotide of the present invention or the pharmaceutical composition of the present invention is administered to a human in need of the treatment. Other processes and conditions are not limited at all.

For the treatment method of the present invention, for example, the administration method, dose, preparation method and the like of the pharmaceutical composition of the present invention can be used.

Hereinafter, the present invention will be described with reference to examples. However, the present invention is not limited to these examples.

Measurement of knockdown activity of CFB mRNA in human cells Huh7 cells (obtained from JCRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition) on a 96-well culture plate, a cell line derived from human liver cancer Cells / 80 μL / well were seeded. As the medium, a DMEM medium (Life Technologies, catalog number 11095-098) containing 10% fetal bovine serum (FBS) was used. Antisense oligonucleotides described in Table 2 were synthesized by gene design and used. In the antisense oligonucleotides in Table 2, lower case letters indicate DNA, upper case letters indicate LNA, and C (M) indicates 5-methylcytosine LNA. In any antisense oligonucleotide, each nucleotide has a phosphodiester bond substituted with a phosphorothioate bond. The antisense oligonucleotide and Lipofectamine LTX & Plus reagent (Life Technology, catalog number 15338) are diluted with Opti-MEM medium (Life Technology, catalog number 11058-021) to terminate the antisense oligonucleotide. 20 μL of antisense oligonucleotide / Lipofectamine LTX mixed solution was added to each 96-well culture plate so that the concentration was 30 nM, and cultured at 37 ° C. under 5% CO ₂ condition for 24 hours. After that, the cells were washed with PBS (Phosphate buffered saline) and described in the instructions attached to the product from each plate using the Cells-to-Ct kit (Applied Biosystems, catalog number: AM1728). CDNA was synthesized according to the method. Add 5 μL of this cDNA to a MicroAmpOptical 96-well plate (Applied Biosystems, catalog number 4326659), then add 10 μL TaqMan Gene Expression Master Mix (Applied Biosystems, catalog number 4369016), 3 μL of UltraPure Distilled Water (Life Technologies) (Cat. No. 10977-015), 1 μL human CFB probe, and 1 μL human β-actin probe were added. Real-time PCR of human CFB gene and human β-actin was performed using ABI7900 HT real-time PCR system. β-actin is a constitutively expressed gene, measured as an internal control, and corrected for CFB expression. The relative expression level of CFB mRNA when each antisense oligonucleotide was introduced was calculated with 1.0 as the amount of CFB mRNA when Huh7 cells were treated with only the transfection reagent without adding the antisense oligonucleotide. This experiment was performed a plurality of times, and the average value of the relative expression level of CFB mRNA is shown in Table 2.

In Vivo Knockdown Activity of Antisense Oligonucleotide in Mice Among the antisense oligonucleotides obtained in Example 1, the in vivo knockdown activity of SEQ ID NOS: 118, 125, and 150 was measured by the following method. Each antisense oligonucleotide was diluted with phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque).
Mice (BALB / cA, obtained from Clea Japan) were bred and conditioned, and then each antisense oligonucleotide was administered subcutaneously at a dose of 3 mg / kg. As a control group, only DPBS was subcutaneously administered to mice. Each antisense oligonucleotide or control group was administered to 3 mice each.
Three days after administration, animals were euthanized, livers were collected and stored frozen in liquid nitrogen. A sample of frozen liver is attached to the product using Trizol® NA Isolation Regents (manufactured by Life Technologies, catalog number 15596026) and Magna Pure 96 Cellular NA NA Volume Volume Kit (manufactured by Roche, catalog number 05467535001). Total RNA was collected according to the method described in the written instructions. Furthermore, using the transcripter first strand CDNA synthesis kit (Roche, catalog number 04897030001), according to the method described in the instructions attached to the product, cDNA by reverse transcription reaction using the obtained total RNA as a template Was made. Using the obtained cDNA as a template, a Tackman (registered trademark) Gene Expression Assays probe (Applied Biosystems) as a probe, a Quant Studio 12 Kaflex Real-Time PC system (ABI) was used and attached The CFB gene and constitutively expressed gene glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase, hereinafter referred to as Gapdh) gene by PCR reaction according to the method described in the instruction manual The amount of mRNA amplification was measured by PCR reaction, and the semi-quantitative value of CFB mRNA was calculated using the amount of mRNA amplification of Gapdh as an internal control. Similarly, the CFB mRNA expression rate was determined from the CFB mRNA quasi-quantitative value, with the CFB mRNA quasi-quantitative value in the control group measured similarly. The relative expression rate of the obtained CFB mRNA is shown in FIG.

In vitro and in vivo knockdown activity of antisense oligonucleotides (SEQ ID NO: 155) with different modified nucleic acids C (M) C (M) AtgttgtgC modified with the antisense base sequence (SEQ ID NO: 23) obtained in Example 1 (M) AA (SEQ ID NO: 155) was newly synthesized by Gene Design, and in vitro and in vivo knockdown activity was measured. Here, lowercase letters indicate DNA, uppercase letters indicate LNA, and C (M) indicates 5-methylcytosine LNA. In any antisense oligonucleotide, each nucleotide has a phosphodiester bond substituted with a phosphorothioate bond.
The in vitro knockdown activity was adjusted 3 times in the same manner as in Example 1 by adjusting the final concentration of the antisense oligonucleotide (SEQ ID NO: 155) to 30 nM, 10 nM, 3 nM, 1 nM, and 0.3 nM, respectively. The average value of the results is shown in Table 3. In vivo knockdown activity is shown in FIG. 2, in which antisense oligonucleotide (SEQ ID NO: 155) was subcutaneously administered to mice at 10 mg / kg and measured in the same manner as in Example 2.

This application is based on Japanese Patent Application No. 2016-21128 (filing date: February 5, 2016) filed in Japan, the contents of which are hereby incorporated by reference.

The present invention provides an antisense oligonucleotide having CFB expression-inhibiting activity, a pharmaceutical composition containing the antisense oligonucleotide as an active ingredient, and the like. The antisense oligonucleotide and the pharmaceutical composition of the present invention suppress the expression of CFB, atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 Nephritis, membranous nephropathy, rapid progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis) It is useful in the treatment and prevention of disorders mediated by abnormalities in the alternative pathway of the complement such as).

Claims (13)

1. Antisense oligonucleotide that suppresses the expression of complement factor B, which can hybridize under stringent conditions to a nucleic acid comprising the target base sequence represented by any of SEQ ID NOs: 53 to 103, 8 to 80 bases Long antisense oligonucleotide.
2. An antisense oligonucleotide that suppresses the expression of complement factor B, comprising 8 to 80 bases in length, comprising at least 8 consecutive bases of the antisense base sequence represented by any of SEQ ID NOs: 2 to 52 Oligonucleotide.
3. An antisense oligonucleotide having a length of 8 to 80 bases comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 53 to 103.
4. The antisense oligonucleotide according to claim 3, comprising the base sequence represented by any of SEQ ID NOs: 2 to 52.
5. The antisense oligonucleotide according to claim 4, comprising a sequence in which one to several bases are deleted, substituted, inserted or added in the base sequence represented by any of SEQ ID NOs: 2 to 52.
6. An antisense oligonucleotide consisting of the base sequence represented by any of SEQ ID NOs: 2 to 52.
7. The antisense oligonucleotide according to any one of claims 1 to 6, wherein the vicinity of the 5 'end and / or the vicinity of the 3' end is composed of sugar-modified nucleotides.
8. An antisense oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 104 to 154 and 155.
9. An antisense oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 118, 125, 150 and 155.
10. The antisense oligonucleotide according to any one of claims 1 to 9, comprising a ligand.
11. A pharmaceutical composition comprising the antisense oligonucleotide according to any one of claims 1 to 10.
12. A method of treating a disorder mediated by an alternative pathway alternative comprising a therapeutically effective amount of an antisense oligonucleotide according to any one of claims 1 to 10 or claim 11. Administering a pharmaceutical composition to a human in need of such treatment.
13. The disorders include atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapid progressive glomerulonephritis (RPGN) The method according to claim 12, which is acute kidney injury (AKI), asthma, or autoimmune disease.

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