WO2018117253A1 - Nucleic acid inhibiting expression of complement factor b - Google Patents

Abstract

Provided are: a nucleic acid having an activity of inhibiting the expression of complement factor B; a medicinal composition comprising the nucleic acid; and a prophylactic or therapeutic agent, said agent comprising the nucleic acid, for 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), ANCA associated vasculitis, lupus nephritis, asthma or an autoimmune disease.

Description

Nucleic acid that suppresses expression of complement factor B

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

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), Known to be associated with the onset of diseases such as ANCA-related vasculitis, lupus nephritis, asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic myelitis, myasthenia gravis) Patent Documents 2 and 3). 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 4), 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 the antisense sequence targeting the human complement factor B gene has been disclosed (Patent Document 3). Moreover, although a part of siRNA sequence targeting the gene is disclosed (Patent Document 4), the present invention is a sequence different from these sequences.

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

Proc Natl Acad Sci USA, 94, 8720-8725 (1997) Immunological Reviews 223, 300-316 (2008) J Am Soc Nephrol. 26, 2917-2929 (2015) Invitation to complement, Medical View, p. 18 (2011)

An object of the present invention is to provide a nucleic acid 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 (15).
(1) a double-stranded nucleic acid comprising a sense strand and an antisense strand, and comprising a double-stranded region of at least 11 base pairs, wherein at least 17 nucleotides and at most 30 in the antisense strand A double-stranded nucleic acid that reduces the expression of the CFB gene that is complementary to a target CFB mRNA sequence selected from the group described in Tables 1-1 to 1-3 .
(2) The antisense strand, wherein the double-stranded region is 11 to 27 base pairs and is complementary to a target CFB mRNA sequence selected from the group described in Table 1-1 to Table 1-3 The double-stranded nucleic acid according to (1), wherein the second nucleotide from the 5 ′ end of the target is complementary to the second ribonucleotide from the 3 ′ end of the target CFB mRNA sequence.
(3) The double-stranded nucleic acid according to (1), wherein the 3 ′ end of the sense strand and the 5 ′ end of the antisense strand form a blunt end.
(4) The double-stranded nucleic acid according to (1), wherein the sense strand is 21 nucleotides in length and the antisense strand is 21-23 nucleotides in length.
(5) The antisense strand comprises a sequence selected from the group described in the “antisense strand” of Table 1-1 to Table 1-3 and Table 2-1 to Table 2-2. A double-stranded nucleic acid according to 1.
(6) The sense strand comprises a sequence selected from the group described in the “sense strand” of Table 1-1 to Table 1-3 and Table 2-1 to Table 2-2. Double-stranded nucleic acid.
(7) Two pairs according to (1), comprising a pair of sense strands / antisense strands selected from the group consisting of the sense strand / antisense strand described in Table 1-1 to Table 1-3 Strand nucleic acid.
(8) The double-stranded nucleic acid according to any one of (1) to (7), comprising a 2′-modified nucleotide.
(9) The double-stranded nucleic acid according to (8), wherein 50 to 100% of the nucleotides in the double-stranded region are 2′-modified nucleotides.
(10) A pair of sense strands / antisense selected from the group consisting of the sense strand / antisense strand described in Table 2-1 to Table 2-2, Table 3-1 to Table 3-2, and Table 4 The double-stranded nucleic acid according to (1), comprising a sense strand sequence.
(11) The double-stranded nucleic acid according to any one of (1) to (10), comprising a ligand.
(12) A single-stranded nucleic acid comprising only an antisense strand of the double-stranded nucleic acid according to any one of (1) to (11).
(13) A pharmaceutical composition comprising the nucleic acid according to any one of (1) to (12).
(14) A method for treating a disorder mediated by an abnormality in the alternative pathway of the complement, comprising an effective amount of the nucleic acid according to any one of (1) to (12) or the pharmaceutical according to (13) Administering the composition to a human in need of such treatment.
(15) 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), ANCA-related vasculitis, lupus nephritis, asthma or autoimmune disease.

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

The CFB gene (gene encoding CFB) targeted by the nucleic acid of the present invention has a cDNA base sequence (SEQ ID NO: 1) corresponding to the full-length mRNA of human CFB registered as Genbank Accession No. NM_001710 It is said that naturally occurring variants thereof (see, for example, Hum. Mutat. 31: E1445-E1460 (2010), refsnp No. rs12614, rs641153, etc.) can also serve as target genes for the nucleic acids of the present invention. Not too long.
In addition, mRNA of CFB gene of biological species other than human can also be a target gene of the nucleic acid of the present invention. For example, Genbank Accession No. NM_008198 or NM_001142706 is the cDNA base sequence corresponding to the full-length mRNA of the mouse CFB gene, Genbank Accession No. NM_212466 is the cDNA base sequence corresponding to the full-length mRNA of the rat CFB gene, and the cynomolgus CFB gene is Examples of the cDNA base sequence corresponding to the full-length mRNA include Genbank Accession No. XM_005553440.2, and examples of the cDNA base sequence corresponding to the full-length mRNA of the rhesus monkey CFB gene include Genbank Accession No. XM_015136029.1.

1. Nucleic acid of the present invention In the present invention, a nucleic acid containing a base sequence complementary to CFB mRNA is called an antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of an antisense strand nucleic acid is Also referred to as sense strand nucleic acid. In the present specification, unless otherwise specified, the term “nucleic acid of the present invention” is used to include an antisense strand nucleic acid, a sense strand nucleic acid, and a double-stranded nucleic acid paired with a sense strand and an antisense strand nucleic acid. .

The nucleic acid 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 the nucleotides, such as RNA or deoxyribonucleotide polymers that are ribonucleotide polymers. DNA, chimeric nucleic acids composed of RNA and DNA, 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, derivatives containing at least one molecule having a function equivalent to nucleotide in these nucleic acids are also included in the nucleic acid of the present invention. Uracil (U) can be uniquely read as thymine (T).

Examples of molecules having a function 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 RNA or DNA, In order to increase affinity, to increase cell permeability, or to visualize, a molecule in which ribonucleotides or deoxyribonucleotides are modified is 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 substituted with any atom. '-Modified nucleotides are preferably used.

2′-modified nucleotides include, for example, the 2′-OH group of ribose is H, OR, R, R′OR, SH, SR, NH ₂ , NHR, NR ₂ , N ₃ , CN, F, Cl, Br and Substituted with a substituent selected from the group consisting of I (R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, and R ′ is alkylene, preferably alkylene having 1 to 6 carbon atoms) Examples include 2'-modified nucleotides, preferably 2'-OH groups substituted with H, F or methoxy groups, more preferably 2'-OH groups substituted with F or methoxy groups. . In addition, 2'-OH group is 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- A substituent selected from the group consisting of [2- (N, N-dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) ethoxy group and 2-cyanoethoxy group Examples thereof include substituted nucleotides.
The 2′-modified nucleotide is preferably contained in an amount of 50 to 100%, more preferably 70 to 100%, and still more preferably 90 to 100% with respect to the nucleotide in the double-stranded nucleic acid region. . As a double-stranded nucleic acid to which 2′-modified nucleotides are applied, for example, a pair of senses selected from the group consisting of the sense strand / antisense strand described in Table 3-1, Table 3-2 and Table 4 There is a double-stranded nucleic acid consisting of a strand / antisense strand sequence. In Table 3-1, Table 3-2 and Table 4, N (M) represents 2'-O-methyl-RNA, N (F) represents 2'-F-RNA, and p represents phosphorylation. . Here, N represents A, C, G, or U.

In addition, examples of the sugar-modified nucleotide include a crosslinked structure-type artificial nucleic acid (BNA) having two circular structures by introducing a crosslinked structure into the sugar moiety, specifically, the 2′-position. Locked Nucleic Acid (LNA) [Tetrahedron Letters, 38, 8735, (1997) and Tetrahedron, 54, 3607, (1998)] Ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)], Constrained Ethyl (cEt) [The Journal of Organic 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)] can give.
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 ( PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like are also exemplified as sugar-modified nucleotides.

The phosphodiester bond-modified nucleotide is any nucleotide that has been modified or substituted with an arbitrary substituent for a part or all of the chemical structure of the phosphodiester bond of the nucleotide, or with any atom. 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 thereof include nucleotides in which a diester bond is substituted with a phosphoramidate bond.

As the base-modified nucleotide, any or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent or substituted with an arbitrary atom may be used. In which oxygen atoms are substituted with sulfur atoms, hydrogen atoms are substituted with alkyl groups having 1 to 6 carbon atoms, halogens, etc., methyl groups are hydrogen, hydroxymethyl, alkyl groups with 2 to 6 carbon atoms, etc. And those in which the 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 such as phenazine, phenanthridine, anthraquinone, folic acid, synthetic polymers such as synthetic polyamino acids, nucleic acid aptamers, acridine, fluorescein, rhodamine, coumarin dyes, Cy3 series, Alexa series (registered trademark), Black hole quencher And other chemical substances such as a fluorophore added 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 combination of at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.

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

In the present specification, “complementary” means a relationship that allows base pairing between two bases, for example, a moderate hydrogen such as a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine. It means a double-stranded structure as a whole double-stranded region through a bond.

As used herein, “complementary” includes not only a case where two nucleotide sequences are completely complementary, but also a 0-30%, 0-20% or 0-10% mismatch base between the nucleotide sequences. For example, an antisense strand complementary to CFB mRNA means that one or more base substitutions may be included in the base sequence that is completely complementary to the partial base sequence of the mRNA. Specifically, the antisense strand contains 1 to 8, preferably 1 to 6, 1 to 4, 1 to 3, particularly 2 or 1 mismatch bases to the target sequence of the target gene. You may have. For example, if the antisense strand is 21 bases long, it may have 6, 5, 4, 3, 2 or 1 mismatch bases with respect to the target sequence of the target gene. The position of the mismatch may be the 5 ′ end or 3 ′ end of each sequence.
The term “complementary” includes a case where one nucleotide sequence is a sequence in which one or more bases are added and / or deleted in a base sequence that is completely complementary to the other nucleotide sequence. For example, CFB mRNA and the antisense strand nucleic acid of the present invention have one or two bulge bases in the antisense strand and / or target CFB mRNA region due to addition and / or deletion of a base in the antisense strand. May be.

As long as the nucleic acid of the present invention is a nucleic acid containing a base sequence complementary to a partial base sequence of CFBCFmRNA and / or a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid, any nucleic acid can be used. It may be composed of nucleotides or derivatives thereof. In the double-stranded nucleic acid of the present invention, a nucleic acid containing a base sequence complementary to the target CFB mRNA sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid form a double strand. However, the length of the sequence capable of forming a duplex is usually 11 to 35 bases, preferably 15 to 30 bases, more preferably 17 to 25 bases, and 17 to 23 bases. Bases are more preferred, with 19 to 23 bases being particularly preferred. It is also preferred that it consists of 21 to 23 bases.

As the antisense strand nucleic acid of the present invention, a nucleic acid containing a base sequence complementary to the target CFB mRNA sequence is used. Among these nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 A base deleted, substituted or added may be used.

The nucleic acid that suppresses CFB expression is a nucleic acid containing a base sequence complementary to the target CFB mRNA sequence, and is complementary to the single-stranded nucleic acid that suppresses CFB expression or the target CFB mRNA sequence. A double-stranded nucleic acid that consists of a nucleic acid containing a basic sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid and suppresses the expression of CFB is preferably used.

In the present invention, a double-stranded nucleic acid refers to a nucleic acid having a double-stranded region in which two nucleotide chains are paired. A double-stranded region refers to a portion where nucleotides constituting a double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand. The double-stranded region is usually 11 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, still more preferably 17 to 21 base pairs, and particularly preferably 17 to 19 base pairs.

The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 11 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and 15 to 25 bases. More preferably, it consists of 17-23 bases, most preferably 19-23 bases. It is also preferred that it consists of 21 to 23 bases.

When the double-stranded nucleic acid of the present invention has an additional nucleotide or nucleotide derivative that does not form a duplex on the 3 ′ side or 5 ′ side following the double-stranded region, this is referred to as an overhang. Call. In the case of having an overhang, the nucleotide constituting the overhang may be ribonucleotide, deoxyribonucleotide or a derivative thereof.

As the double-stranded nucleic acid having a protruding portion, one having a protruding portion consisting of 1 to 6 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used. Those having a protruding portion are preferably used, and examples thereof include those having a protruding portion made of dTdT or UU. Overhangs can be on the antisense strand only, the sense strand only, and both the antisense and sense strands. The antisense strand is an oligonucleotide strand consisting of at least 17 nucleotides and at most 30 nucleotides including a double-stranded region and a subsequent overhang, and is described in Table 1-1 to Table 1-3. Is sufficiently complementary to a target CFB mRNA sequence selected from the selected group. Furthermore, the double-stranded nucleic acid of the present invention includes, for example, a nucleic acid molecule (WO2005 / 089287) that generates the above-mentioned double-stranded nucleic acid by the action of a ribonuclease such as Dicer, and a 3′-end or 5′-end overhang. Alternatively, a double-stranded nucleic acid that forms a blunt end, a double-stranded nucleic acid in which only the sense strand protrudes (US2012 / 0040459), and the like can also be used.

As the double-stranded nucleic acid of the present invention, a nucleic acid having the same sequence as the base sequence of the target gene or its complementary strand may be used, but the 5 ′ end or 3 ′ of at least one strand of the nucleic acid may be used. A double-stranded nucleic acid comprising a nucleic acid from which one to four bases have been deleted and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid may be used.

The double-stranded nucleic acid of the present invention includes a double-stranded RNA (dsRNA) in which RNAs form a double strand, a double-stranded DNA (dsDNA) in which DNAs form a double strand, or a double strand of RNA and DNA. It may be a hybrid nucleic acid that forms a strand. Alternatively, one or both of the double strands may be a chimeric nucleic acid of DNA and RNA. Double-stranded RNA (dsRNA) is preferred.

The second nucleotide from the 5 ′ end of the antisense strand of the present invention is preferably complementary to the second ribonucleotide from the 3 ′ end of the target CFB mRNA sequence, and 2-7 from the 5 ′ end of the antisense strand. More preferably, the second nucleotide is completely complementary to the 2-7 th ribonucleotide from the 3 ′ end of the target CFB mRNA sequence, and the 2-11 th nucleotide from the 5 ′ end of the antisense strand is the target CFB More preferably, it is completely complementary to ribonucleotides 2 to 11 from the 3 ′ end of the mRNA sequence. The 11th nucleotide from the 5 ′ end of the antisense strand in the nucleic acid of the present invention is preferably complementary to the 11th ribonucleotide from the 3 ′ end of the target CFBCF mRNA sequence, and the 5 ′ end of the antisense strand. It is more preferable that the 9th to 13th nucleotides are completely complementary to the 9th to 13th ribonucleotides from the 3 ′ end of the target CFB mRNA sequence, and the 7th to 15th nucleotides from the 5 ′ end of the antisense strand Is more preferably completely complementary to the 7th to 15th ribonucleotides from the 3 ′ end of the target CFB mRNA sequence.

The method for producing the nucleic acid of the present invention is not particularly limited, and examples thereof include a method using known chemical synthesis or an enzymatic transcription method. Examples of methods using known chemical synthesis 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 a nucleic acid having a purity of 90% or more, preferably 95% or more by purification. In the case of a double-stranded nucleic acid, the synthesized and purified sense strand and antisense strand are in an appropriate ratio, for example, 0.1 to 10 equivalents, preferably 0.5 to 2 equivalents of sense strand to 1 equivalent of antisense strand. Preferably, 0.9 to 1.1 equivalents, more preferably equimolar amounts are mixed and then annealed, or the mixed product may be used directly without the step of annealing. Annealing may be performed under any conditions that can form a double-stranded nucleic acid, but usually, the sense strand and the antisense strand are mixed in approximately equimolar amounts and then heated at about 94 ° C. for about 5 minutes. Then, it is performed by slowly cooling to room temperature. Examples of the enzymatic transcription method for producing the nucleic acid of the present invention include a transcription method using a phage RNA polymerase such as T7, T3, or SP6 RNA polymerase using a plasmid or DNA having the target base sequence as a template.

The nucleic acid 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.

In place of the nucleic acid of the present invention, a vector that can be introduced into cells and expressed can be used. Specifically, the nucleic acid or the like can be expressed by inserting the sequence encoding the nucleic acid of the present invention downstream of the promoter in the expression vector, constructing the expression vector, and introducing it into a cell. Expression vectors include pCDNA6.2-GW / miR (Invitrogen), pSilencer® 4.1-CMV (Ambion), pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), pENTR / U6 (Invitrogen) etc. can be mentioned.

It is also possible to use a recombinant viral vector produced by inserting a sequence encoding the nucleic acid of the present invention downstream of a promoter in a viral vector and introducing the vector into a packaging cell. Examples of virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like.

2. Nucleic acid having CFB expression inhibitory activity The antisense strand and the sense strand of the present invention are, for example, the nucleotide sequence (sequence) of cDNA (sense strand) of full-length mRNA of human CFB registered as Genbank Accession No. NM_001710. It can be designed based on the number 1). In addition, by comparing the cDNA sequence of full-length mRNA of human CFB with the cDNA sequence of full-length mRNA of CFB from other species, an antisense strand that suppresses CFB mRNA expression in multiple species. The sense strand can also be designed.

The nucleic acid having CFB expression suppressing activity includes the antisense strand nucleic acid of the present invention containing a base sequence complementary to CFB に 対 し て mRNA and the nucleic acid of the present invention containing a base sequence complementary to the base sequence of the nucleic acid. Examples thereof include double-stranded nucleic acids consisting of a sense strand nucleic acid and having CFB expression suppression activity. The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 11 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and more preferably 15 to 25 bases. More preferably, it consists of 17 to 23 bases, most preferably 19 to 23 bases. It is also preferred that it consists of 21 to 23 bases. The double-stranded nucleic acid usually has a double-stranded region consisting of 15 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, and even more preferably 15 to 21 base pairs.

CFB expression can be suppressed by introducing these double-stranded nucleic acids into cells. For example, the double-stranded nucleic acid of the present invention can suppress the expression of CFB mRNA after being introduced into cells at a concentration of several pM to several nM and then cultured for 24 hours or more, for example 48 hours.

In addition, the evaluation of the CFB 発 現 mRNA expression inhibitory activity of the double-stranded nucleic acid of the present invention is carried out by transfecting the nucleic acid or the like into a human cell line using a cationic liposome or the like, culturing for a certain time, Can be carried out by quantifying the expression level of CFB mRNA.

In addition to the above-mentioned double-stranded nucleic acid, the nucleic acid having a CFB expression suppressing activity is a nucleic acid having a base sequence complementary to a part of the base sequence of CFB の mRNA, and suppresses the expression of CFB. Single-stranded nucleic acid is exemplified. The single-stranded nucleic acid constituting the nucleic acid usually consists of 8 to 30 bases, preferably 12 to 30 bases, more preferably 12 to 20 bases. It is also preferred that it consists of 21 to 23 bases.

CFB expression can be suppressed by introducing these single-stranded nucleic acids into cells. For example, the single-stranded nucleic acid of the present invention can suppress the expression of CFB mRNA after being introduced into cells at a concentration of several pM to several nM and then cultured for 24 hours or more, for example 48 hours.

The evaluation of the CFB mRNA expression inhibitory activity of the single-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid or the like into a human cell line using a cationic liposome, etc. This can be done by quantifying the expression level of CFB mRNA in the strain.

The nucleic acid of the present invention may contain a ligand. The ligand may directly modify the 5 ′ end, 3 ′ end and / or the inside of the sequence of the nucleic acid of the present invention.

The ligand contained in the nucleic acid of the present invention may be a molecule having affinity for a biomolecule.For example, lipids such as cholesterol, fatty acid, tocopherol, retinoid, N-acetylgalactosamine (GalNAc), galactose (Gal ), Sugars such as mannose (Man), full antibodies, fragment antibodies such as Fab, scFV, VHH, proteins such as low density lipoprotein (LDL), human serum albumin, peptides such as RGD, NGR, R9, CPP, Examples include low molecular weight compounds such as folic acid, synthetic polymers such as synthetic polyamino acids, or nucleic acid aptamers, but the invention is not limited to these, and these may be used in appropriate combinations.

As a method for adding a ligand to the nucleic acid of the present invention, for example, at the time of extension reaction on a solid phase, a modifying agent capable of reacting on the solid phase is allowed to react, thereby 5 ′ end, 3 ′ end and / or sequence. Modifications can be made to the inside, but are not limited thereto. 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.

3. Pharmaceutical composition of the present invention The present invention also relates to a pharmaceutical composition containing a nucleic acid such as the above double-stranded nucleic acid or single-stranded nucleic acid as an active ingredient.

The pharmaceutical composition of the present invention can further contain a carrier effective for transferring nucleic acid into cells. 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), ANCA-related vasculitis, lupus nephritis, asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic nerve Such as myelitis and myasthenia gravis).

Examples of carriers that are effective for transferring nucleic acids into cells include cationic carriers. Examples of the cationic carrier include cationic liposomes and cationic polymers. Further, a carrier utilizing a viral envelope may be used as an effective carrier for transferring nucleic acids into cells. As the cationic polymer, JetSI (Qbiogene), Jet-PEI (polyethyleneimine; Qbiogene) and the like are preferably used. As a carrier using a viral envelope, GenomeOne (registered trademark) (HVJ-E liposome; Ishihara Sangyo Co., Ltd.) is preferably used.

The composition containing the nucleic acid of the present invention and the above carrier 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 a nucleic acid solution. When a cationic carrier is used, since the nucleic acid is usually negatively charged in an aqueous solution, it 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.

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 containing the carrier and the nucleic acid depend on the carrier to be used. Therefore, those skilled in the art can select the optimum method for the carrier to be used without being bound by the above method.

In addition, the pharmaceutical composition of the present invention includes, for example, a composite organic particle composed of nucleic acid and lead particles and a lipid membrane that coats the composite particle, and a polar organic solvent in which the component of the lipid film is soluble. Liposomes in which a liquid containing the polar organic solvent is present in a concentration that can disperse the components of the lipid membrane and the composite particles can also be dispersed in the liquid that is contained are preferably used, but are not limited thereto. 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 non-cationic lipids, lipids that prevent particle aggregation, and cationic lipids as constituent components.

The composition can be prepared according to the method described in, for example, International Publication No. 2006/080118 Pamphlet.

The mixing ratio of the nucleic acid 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 nucleic acid. The amount is preferably 2.5 to 100 parts by weight, more preferably 7 to 25 parts by weight, based on 1 part by weight of the nucleic acid.

The average size of the pharmaceutical composition of the present invention is preferably about 10 nm to 300 nm, more preferably about 30 nm to 200 nm, and even more preferably about 50 nm to 150 nm.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent in addition to the above carrier. Pharmaceutically acceptable carriers or diluents and the like are essentially chemically inert and harmless compositions that do 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), ANCA-related vasculitis, lupus nephritis, asthma, autoimmune disease ( For example, 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), ANCA-related vasculitis, lupus nephritis, asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis , Neuromyelitis optica, myasthenia gravis, etc.) or the like.

The pharmaceutical composition of the present invention contains an amount of the complex effective for treating or preventing a disease and is provided in a form that 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 active ingredient in the pharmaceutical composition of the present invention is usually 0.001 to 25% (w / v), preferably 0.1 to 10% (w / v), more preferably 0.5 ~ 5% (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 prepared as a lyophilized preparation. A freeze-dried preparation can be prepared by subjecting a nucleic acid and a carrier to dispersion treatment and then freeze-drying treatment. 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.

The freeze-dried preparation can be used after re-dissolving 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 dosage forms suitable for these administration methods include various injections, oral preparations, drops, absorbents, eye drops, ointments, lotions, suppositories and the like.

The dose of the pharmaceutical composition of the present invention is preferably determined in consideration of nucleic acid, dosage form, patient condition such as age and weight, administration route, nature and degree of disease, etc. As an adult, it is 0.1 mg to 10 g / day, preferably 1 mg to 500 mg / day per day. 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 disorders mediated by abnormalities of the alternative complement pathway, wherein a therapeutically effective amount of a nucleic acid of the invention or a pharmaceutical composition of the invention is treated as such. A method (the treatment method of the present invention) comprising the step of administering to a human in need of proper treatment.

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 Glomerulonephritis (RPGN), acute kidney injury (AKI), ANCA-related vasculitis, lupus nephritis, asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis) A method for treating the above diseases, wherein a therapeutically effective amount of the nucleic acid 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 Cells / 80 μL / well were seeded. The medium used was DMEM medium (Life Technologies, catalog number 08458-16) containing 10% fetal bovine serum (FBS). As double-stranded nucleic acids, those shown in Table 1-1 to Table 1-3 were synthesized and used by Gene Design. Specifically, it is composed of a double-stranded nucleic acid obtained by annealing a sense strand consisting of ribonucleotides shown in SEQ ID NOs: 2 to 124 and an antisense strand consisting of ribonucleotides shown in SEQ ID NOs: 125 to 247 (SEQ ID NO: n (n = 2 to 124) and the antisense strand shown in SEQ ID NO: [n + 123] make a pair). The double-stranded nucleic acid and RNAiMax transfection reagent described in Table 1-1 to Table 1-3 (manufactured by Life Technology Co., Ltd., catalog number: 1401251) are mixed with Opti-MEM medium (manufactured by Life Technology Co., Ltd., catalog number 11058-021) And add 20 μL of siRNA / RNAiMax mixture to each 96-well culture plate so that the final concentration of double-stranded nucleic acid is 1 nM or 0.1 nM, and 37 ° C and 5% CO ₂ conditions. Cultured for 24 hours. Thereafter, the cells were washed with PBS (Phosphate buffered saline), and each plate was attached to the product using the SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR kit (manufactured by Toyobo, catalog number: SCQ-101). CDNA was synthesized according to the method described in the instructions. Add 5 μL of this cDNA to a MicroAmpOptical 96-well plate (Applied Biosystems, Catalog No. 4326659), then add 10 μL of TaqMan (registered trademark) Gene Expression Master Mix (Applied Biosystems, Catalog No. 4369016), 3 μL of UltraPure Distilled Water (manufactured by Life Technologies, catalog number: 10977-015), 1 μL of human CFB probe, and 1 μL of human β-actin probe were added. Using the QuantStudio (registered trademark) 12K Flex real-time PCR system, real-time PCR of human CFB gene and human β-actin was performed. 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 double-stranded nucleic acid was introduced was calculated with 1.0 as the amount of CFB mRNA when Huh7 cells were treated with transfection reagent alone without adding siRNA. This experiment was performed a plurality of times, and the average value of the relative expression level of CFB mRNA was shown in Table 1-1 to Table 1-3 (Table 1-1 to Table 1-3 may be collectively referred to as Table 1. Table 2, Table 1). The same applies to 3). Table 2 shows the sense strand sequence and antisense strand sequence of a double-stranded nucleic acid different from Table 1 that targets the same sequence as the target CFB mRNA sequence listed in Table 1 (in Tables 1 and 2). , The same last four digits of the double-stranded nucleic acid number target the same CFB mRNA sequence). Furthermore, sequences obtained by 2'-modifying the nucleotide sugar part of the sequences shown in Table 2 are shown in Table 3 (in Tables 2 and 3, the last four digits of the double-stranded nucleic acid numbers are the same, but the base sequences are the same) (This corresponds to a sequence that is different but not modified).

Measurement of CFB mRNA knockdown activity in human cells of modified siRNA Huh7 cells, a cell line derived from human liver cancer, on a 96-well culture plate (obtained from JCRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition) Was seeded at 10,000 cells / 80 μL / well. The medium used was DMEM medium (Life Technologies, catalog number 08458-16) containing 10% fetal bovine serum (FBS). As double-stranded nucleic acids, those listed in Table 4 were synthesized and used by Gene Design. The double-stranded nucleic acid described in Table 4 is obtained by 2′-modifying the sugar part of the nucleotide with respect to some of the double-stranded nucleic acids described in Table 1 or Table 2. The double-stranded nucleic acid and RNAiMax transfection reagent listed in Table 4 (life technology, catalog number: 1401251) are diluted with Opti-MEM medium (life technology, catalog number 11058-021). 20 μL of siRNA / RNAiMax mixture was added to each 96-well culture plate so that the final concentration of the chain nucleic acid was 0.1 nM or 0.03 nM, and cultured for 24 hours at 37 ° C. under 5% CO ₂ . Thereafter, the cells were washed with PBS (Phosphate buffered saline), and each plate was attached to the product using the SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR kit (manufactured by Toyobo, catalog number: SCQ-101). CDNA was synthesized according to the method described in the instructions. Add 5 μL of this cDNA to a MicroAmpOptical 96-well plate (Applied Biosystems, Catalog No. 4326659), then add 10 μL of TaqMan (registered trademark) Gene Expression Master Mix (Applied Biosystems, Catalog No. 4369016), 3 μL of UltraPure Distilled Water (manufactured by Life Technologies, catalog number: 10977-015), 1 μL of human CFB probe, and 1 μL of human β-actin probe were added. Using the QuantStudio (registered trademark) 12K Flex real-time PCR system, real-time PCR of human CFB gene and human β-actin was performed. 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 double-stranded nucleic acid was introduced was calculated with 1.0 as the amount of CFB mRNA when Huh7 cells were treated with transfection reagent alone without adding siRNA. This experiment was performed 3 times, and the average value of the relative expression level of CFB mRNA is shown in Table 4.

This application is based on Japanese Patent Application No. 2016-250264 filed in Japan (filing date: December 23, 2016), the contents of which are hereby incorporated by reference.

The present invention provides a nucleic acid having CFB expression inhibitory activity, a pharmaceutical composition containing the nucleic acid as an active ingredient, and the like. The nucleic acid and 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, membrane Nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), ANCA-related vasculitis, lupus nephritis, asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, It is useful in the treatment and prevention of disorders mediated by abnormalities in the alternative pathway of the complement such as myasthenia gravis).

Claims (15)

A double-stranded nucleic acid consisting of a sense strand and an antisense strand, comprising a double-stranded region of at least 11 base pairs, comprising at least 17 nucleotides and at most 30 nucleotides in said antisense strand A double-stranded nucleic acid that reduces the expression of a CFB gene that is complementary to a target CFB mRNA sequence selected from the group described in Table 1-1 to Table 1-3 in an oligonucleotide chain of a chain length. The double-stranded region is 11 to 27 base pairs, and 5 'of the antisense strand is complementary to a target CFBCF mRNA sequence selected from the group described in Table 1-1 to Table 1-3. The double-stranded nucleic acid according to claim 1, wherein the second nucleotide from the end complements the second ribonucleotide from the 3 'end of the target CFB mRNA sequence. The double-stranded nucleic acid according to claim 1, wherein the 3 'end of the sense strand and the 5' end of the antisense strand form a blunt end. The double-stranded nucleic acid according to claim 1, wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 to 23 nucleotides in length. The antisense strand according to claim 1, wherein the antisense strand comprises a sequence selected from the group described in "antisense strand" in Table 1-1 to Table 1-3 and Table 2-1 to Table 2-2. Double-stranded nucleic acid. The two strands according to claim 1, wherein the sense strand includes a sequence selected from the group described in "Sense strand" in Table 1-1 to Table 1-3 and Table 2-1 to Table 2-2. Strand nucleic acid. The double-stranded nucleic acid according to claim 1, comprising a pair of sense strand / antisense strand sequences selected from the group consisting of the sense strand / antisense strand described in Table 1-1 to Table 1-3. The double-stranded nucleic acid according to any one of claims 1 to 7, comprising a 2'-modified nucleotide. The double-stranded nucleic acid according to claim 8, wherein 50 to 100% of the nucleotides in the double-stranded region are 2'-modified nucleotides. A pair of sense strand / antisense strand selected from the group consisting of the sense strand / antisense strand described in Table 2-1 to Table 2-2, Table 3-1 to Table 3-2, and Table 4 The double-stranded nucleic acid according to claim 1, comprising a sequence. The double-stranded nucleic acid according to any one of claims 1 to 10, comprising a ligand. A single-stranded nucleic acid comprising only the antisense strand of the double-stranded nucleic acid according to any one of claims 1 to 11. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 12. A method of treating a disorder mediated by an alternative pathway alternative comprising a therapeutically effective amount of a nucleic acid according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13. Administering to a human in need of such treatment. 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 14, which is acute kidney injury (AKI), ANCA-related vasculitis, lupus nephritis, asthma or autoimmune disease.