WO2018221649A1 - Apcsの発現を抑制する核酸 - Google Patents
Apcsの発現を抑制する核酸 Download PDFInfo
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- WO2018221649A1 WO2018221649A1 PCT/JP2018/020941 JP2018020941W WO2018221649A1 WO 2018221649 A1 WO2018221649 A1 WO 2018221649A1 JP 2018020941 W JP2018020941 W JP 2018020941W WO 2018221649 A1 WO2018221649 A1 WO 2018221649A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
Definitions
- the present invention relates to a nucleic acid that suppresses the expression of APCS or a pharmaceutical composition containing the nucleic acid.
- APCS amyloid P component, serum
- SAP Serum amyloid P
- Pentraxin-2 has a pentameric structure of glycoprotein composed of 223 amino acids.
- APCS is a protein produced in the liver and is present in the blood at a relatively high concentration of 30-50 ⁇ g / mL.
- APCS has a biochemical property that binds to all types of amyloid fibrils in a calcium-dependent manner, and amyloid in patients with amyloid is known to contain as much as 20,000 mg of APCS.
- Non-Patent Document 1 Since APCS exists in amyloid in all amyloid-related disease patients, it is used as a diagnostic marker in amyloid-related disease patients (Non-patent Document 2).
- Amyloid-related diseases are diseases in which abnormal insoluble protein fibers known as amyloid fibrils are deposited in tissues and cause organ damage.
- APCS resistance to protease degradation and immune cell engulfment has been shown to stabilize APCS-bound amyloid, and by inhibiting the binding of APCS to amyloid, amyloid deposition in organs and It has been strongly suggested from studies using animal models and clinical studies that tissue damage associated therewith can be reduced (Non-Patent Documents 3 and 4). Although it can be expected that amyloid-related diseases can be prevented or treated by specifically suppressing the expression of APCS, no pharmaceuticals that specifically suppress the expression of APCS have been reported so far.
- RNA interference also referred to as RNA interference or RNAi
- RNAi RNA interference
- a method for suppressing gene expression itself Specifically, by introducing a double-stranded RNA having the same sequence as the target gene, it was found that the expression of the target gene was specifically suppressed, and short interfering RNA (siRNA) and (Patent Document 1).
- siRNA short interfering RNA
- Patent Document 2 an antisense method is known as a method for suppressing the expression of genes other than RNA interference.
- siRNA sequences targeting human APCS mRNA are disclosed, it is not known that the siRNA sequence suppresses human APCS expression (Patent Documents 3 and 4).
- An object of the present invention is to provide a nucleic acid capable of suppressing the expression of APCS.
- the present invention relates to the following (1) to (13).
- a double-stranded nucleic acid comprising a sense-strand nucleic acid and an antisense-strand nucleic acid and comprising a double-stranded region of at least 11 base pairs, wherein at least 17 nucleotides in the antisense-strand nucleic acid and many A double strand that suppresses the expression of the APCS gene, which is complementary to the target APCS mRNA sequence containing the base sequence represented by any of SEQ ID NOs: 1288 to 1930 in an oligonucleotide chain of 30 nucleotides in length Nucleic acid.
- the double-stranded region is 11 to 27 base pairs, and the second nucleotide from the 5 ′ end of the antisense strand nucleic acid is the second nucleotide from the 3 ′ end of the target APCS mRNA sequence.
- the double-stranded nucleic acid according to (1) which is complementary.
- the sense strand nucleic acid has a chain length of 21 nucleotides
- the antisense strand nucleic acid has a chain length of 21 nucleotides, according to any one of (1) to (3) Double-stranded nucleic acid.
- the double strand according to (4) comprising a double-stranded region of 19 base pairs, wherein 40 to 65% of the nucleotides in the double-stranded region are 2′-O-methyl modified nucleotides Nucleic acid.
- the sense strand nucleic acid includes a base sequence represented by any of SEQ ID NOs: 2 to 644 and 1931 to 1974, and correspondingly, the antisense strand nucleic acid includes SEQ ID NOs: 645 to 1287 and 1975 to The double-stranded nucleic acid according to any one of (1) to (7), comprising the base sequence represented by any of 2018.
- a pharmaceutical composition comprising the nucleic acid according to any one of (1) to (10).
- (12) A method for treating a disorder mediated by amyloid fibrils containing APCS, wherein the therapeutically effective amount of the nucleic acid according to any one of (1) to (10) or the nucleic acid according to (11) Administering a pharmaceutical composition of to a human in need of such treatment.
- the method according to (12), wherein the disorder is an amyloid-related disease.
- a nucleic acid capable of suppressing the expression of APCS can be provided.
- the APCS mRNA that is the target of the nucleic acid is homologous to SEQ ID NO: 1 registered as Genbank AccessionNo.NM_001639.3, and an APCS having a base sequence in which T in SEQ ID NO: 1 is replaced with U Full length mRNA.
- SEQ ID NO: 1 shows the base sequence of APCS 2nd strand cDNA.
- the base sequence of APCS mRNA and the base sequence of APCS 2nd strand cDNA are in a homologous relationship.
- nucleic acid of the present invention a nucleic acid containing a base sequence complementary to the APCS mRNA sequence is called an antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid Is referred to as a sense strand nucleic acid.
- the term “nucleic acid of the present invention” refers to a single-stranded nucleic acid of an antisense strand nucleic acid or a sense strand nucleic acid, and a double-stranded nucleic acid paired with an antisense strand nucleic acid and a sense strand nucleic acid. Used in the meaning of inclusion.
- 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, for example, RNA that is a polymer of ribonucleotides, or a polymer of deoxyribonucleotides.
- DNA a chimeric nucleic acid that is a polymer of ribonucleotides and deoxyribonucleotides, and a nucleotide polymer in which at least one nucleotide of these nucleic acids (RNA, DNA, and chimeric nucleic acid) is substituted with a molecule having a function equivalent to that nucleotide Is mentioned.
- Uracil (U) in RNA is uniquely read as thymine (T) in DNA.
- nucleotide derivatives obtained by modifying nucleotides.
- a nucleotide derivative although not particularly limited, for example, compared with RNA or DNA, it can improve or stabilize the nuclease resistance, can improve the affinity with the complementary strand nucleic acid, There is an advantage that cell permeability can be improved and / or can be visualized.
- nucleotide derivative examples include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and nucleotides in which two or more of the sugar moiety, phosphodiester bond, and base are simultaneously modified.
- the sugar moiety-modified nucleotide may be any nucleotide as long as it is modified or substituted with an arbitrary substituent for a part or all of the sugar sugar chemical structure, or substituted with an arbitrary atom. -Modified nucleotides are preferably used.
- 2′-modified nucleotides include, for example, the 2′-OH group of ribose is OR, R, R′OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl, Br, and I (R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, R ′ is alkylene, preferably alkylene having 1 to 6 carbon atoms, and two Rs in NR 2 are And 2'-modified nucleotides substituted with a substituent selected from (which may be the same or different).
- 2'-modified nucleotides in which the 2'-OH group of ribose is substituted with F or a methoxy group (also referred to as 2'-F modified nucleotide or 2'-O-methyl modified nucleotide, respectively) Is preferably used.
- the 2'-OH group of ribose is 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-cyanoethoxy group
- Examples also include 2'-modified nucleotides substituted with a substituent selected from the group consisting of:
- sugar-modified nucleotide examples include a crosslinked nucleic acid (BNA) having two cyclic structures by introducing a crosslinked structure into the sugar part, specifically, an oxygen at the 2 ′ position.
- BNA crosslinked nucleic acid
- LNA Locked Nucleic Acid
- EAA ethylene Ethylene bridged nucleic acid
- cEt Constrained Ethyl
- AmNA AmNA
- Sugar modified nucleotides include peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001). And peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].
- PNA peptide nucleic acids
- OPNA oxypeptide nucleic acids
- OPNA oxypeptide nucleic acids
- PRNA peptide ribonucleic acid
- any or all of the chemical structure of the nucleotide phosphodiester bond modified or substituted with any substituent or any atom may be used.
- Examples of phosphodiester bond-modified nucleotides include nucleotides in which the phosphodiester bond is replaced with phosphorothioate bonds, nucleotides in which the phosphodiester bond is replaced with phosphorodithioate bonds, and phosphodiester bonds are replaced with alkylphosphonate bonds. And nucleotides in which a phosphodiester bond is substituted with a phosphoramidate bond.
- the base-modified nucleotide may be any nucleotide as long as it is a part or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent or substituted with an arbitrary atom.
- Examples of the base-modified nucleotide include nucleotides in which the oxygen atom in the base is substituted with a sulfur atom, those in which the hydrogen atom in the base is substituted with an alkyl group having 1 to 6 carbon atoms, halogen, etc., the methyl group is hydrogen, hydroxy Those substituted with methyl, an alkyl group having 2 to 6 carbon atoms, or the like wherein the amino group is substituted with an alkyl group having 1 to 6 carbon atoms, an alkanoyl group with 1 to 6 carbon atoms, an oxo group, a hydroxy group, or the like Can be mentioned.
- nucleotide derivatives include nucleotides or the aforementioned nucleotide derivatives, lipids such as ligands, cholesterol, fatty acids, tocopherols, retinoids, saccharides such as N-acetylgalactosamine (GalNAc), galactose (Gal), mannose (Man), full antibodies Fragment antibody such as Fab, scFv, VHH, low density lipoprotein (LDL), protein such as human serum albumin, peptides such as RGD, NGR, R9, CPP, low such as phenazine, phenanthridine, anthraquinone, folic acid
- Other chemicals such as molecules, synthetic polymers such as synthetic polyamino acids, nucleic acid aptamers, pigments such as acridine, fluorescein, rhodamine, coumarin, Cy3 series, Alexa series (registered trademark), fluorophores such as black hole quenchers, Also added
- polyamine addition nucleotide derivatives examples include 6-FAM-added nucleotide derivatives and biotin-added nucleotide derivatives, and preferably include GalNAc-added nucleotide derivatives.
- nucleotide derivative may form a cross-linked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, and an ester structure, and a combination of at least two 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.
- “complementary” means a relationship that allows base pairing between two bases, for example, a slow hydrogen such as a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine.
- a slow hydrogen such as a relationship between adenine and thymine or uracil
- guanine and cytosine A double-stranded structure as a whole of the double-stranded region through a bond.
- each of the bases A, T (U), G, and C is a modified base.
- an antisense strand nucleic acid complementary to an APCS mRNA sequence contains a base sequence that is completely complementary to the partial base sequence of the mRNA, or a base sequence that is completely complementary Is meant to include a base sequence that may include substitution of one or more bases.
- the antisense strand nucleic acid is 1 to 8, preferably 1 to 6, more preferably 1 to 4, further preferably 1 to 3, particularly preferably 1 to 2, with respect to the target sequence of the target gene.
- the antisense strand nucleic acid may have one mismatch base.
- the antisense strand nucleic acid has a chain length of 21 nucleotides (21 base length)
- it is 1 to 6, preferably 1 to 5, more preferably with respect to the target nucleotide sequence of the target gene.
- nucleotide sequence is a sequence in which one or a plurality of bases are added and / or deleted in a nucleotide sequence that is completely complementary to the other nucleotide sequence.
- the antisense strand nucleic acid in the present invention may have one or two bulge bases in the target APCS mRNA sequence by adding a base in the base sequence that is completely complementary to the APCS mRNA sequence.
- the antisense strand nucleic acid or the sense strand nucleic acid may have one or two bulge bases.
- bulge base refers to a base that does not form a complementary base pair with the other nucleotide sequence.
- nucleic acid containing a base sequence that is completely complementary to the target APCS mRNA sequence is used.
- these nucleic acids 1 to 3 bases, preferably 1 to 2 bases, more preferably May be one in which one base is deleted, substituted or added.
- Nucleic acids that suppress APCS expression include single-stranded nucleic acids that are complementary to the target APCS mRNA sequence (antisense strand nucleic acids) or base sequences that are complementary to the target APCS mRNA sequence.
- a double-stranded nucleic acid consisting of a nucleic acid containing (antisense strand nucleic acid) and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid (sense strand nucleic acid) is preferably used.
- the expression of the APCS gene can be suppressed by the antisense strand nucleic acid.
- the double-stranded nucleic acid of the present invention is a double-stranded nucleic acid consisting of a sense-strand nucleic acid and an antisense-strand nucleic acid, and comprising a double-stranded region of at least 11 base pairs, and at least in the antisense-strand nucleic acid.
- an APCS gene that is complementary to a target APCS mRNA sequence comprising a base sequence represented by any of SEQ ID NOs: 1288-1930 in an oligonucleotide chain of 17 nucleotides and at most 30 nucleotides in length It is a double-stranded nucleic acid that suppresses
- the base sequences represented by any of SEQ ID NOs: 1288 to 1930 are listed in Table 1.
- the sense strand nucleic acid and the antisense strand nucleic acid may be composed of any nucleotide or nucleotide derivative.
- a nucleotide chain containing a base sequence complementary to the target APCS mRNA sequence is used as the antisense strand nucleic acid in the double-stranded nucleic acid of the present invention.
- the target APCS mRNA sequence is homologous to SEQ ID NO: 1, and includes the base sequence represented by any one of SEQ ID NOS: 1288 to 1930 among the APCS mRNA base sequences in which T in SEQ ID NO: 1 is read as U.
- the target APCS mRNA sequence is homologous to SEQ ID NO: 1, and the nucleotide sequence represented by any one of SEQ ID NOS: 1288 to 1930 among the APCS mRNA base sequences in which T in SEQ ID NO: 1 is replaced with U It is.
- an oligonucleotide chain having a chain length of at least 17 nucleotides and at most 30 nucleotides is complementary to the target APCS mRNA sequence.
- nucleotides constituting the antisense strand nucleic acid in the present invention preferably 11 to 27 nucleotides, more preferably 15 to 25 nucleotides, still more preferably 15 to 23 nucleotides, particularly preferably 17 to 21 nucleotides.
- oligonucleotides particularly more preferably 17-19 nucleotides, and even more preferably 19 nucleotides in length, are complementary to the target APCS mRNA sequence.
- the antisense strand nucleic acid and the sense strand nucleic acid comprise a double-stranded region of at least 11 base pairs, and the double-stranded region is usually 11-30 nucleotides, preferably 11-27 base pairs. More preferably, it is 15 to 25 base pairs, more preferably 15 to 23 base pairs, particularly preferably 17 to 21 base pairs, particularly preferably 17 to 19 base pairs, and particularly preferably 19 base pairs.
- the double-stranded nucleic acid containing a 2′-modified nucleotide is, for example, from a pair of sense strand / antisense strand sequences selected from the group consisting of the sense strand / antisense strand described in Table 3. A double-stranded nucleic acid.
- the suitable 2′-modified nucleotide content relative to the nucleotide of the sense strand nucleic acid may be 20 to 40%, 40 to 60%, or 60% to 100%. It may be.
- the suitable 2′-modified nucleotide content relative to the nucleotide of the antisense strand nucleic acid is preferably 0 to 40%, may be 10 to 20%, and may be 20 to 40%. It may be.
- the single-stranded antisense strand nucleic acid and the sense strand nucleic acid constituting the double-stranded nucleic acid usually comprise 17 to 30 bases, preferably 17 to 27 bases, and preferably 17 to 25 bases. More preferably, it consists of 17 to 25 bases, more preferably 19 to 25 bases.
- the base length of the antisense strand nucleic acid and the sense strand nucleic acid may be the same or different.
- the antisense strand nucleic acid preferably includes the base sequence represented by any of SEQ ID NOs: 645 to 1287 and 1975 to 2018, and the sense strand nucleic acids include those of SEQ ID NOs: 2 to 644 and 1931 to 1974. It is preferable to include a base sequence represented by either.
- the antisense strand nucleic acid is more preferably a nucleotide strand having the base sequence represented by any of SEQ ID NOs: 645 to 1287 and 1975 to 2018, and the sense strand nucleic acid is preferably SEQ ID NO: 2 to 644. And a nucleotide chain having a base sequence represented by any one of 1931 to 1974.
- the sense strand nucleic acid includes the base sequence represented by any of SEQ ID NOs: 2 to 644 and 1931 to 1974, and correspondingly, the antisense strand nucleic acid includes SEQ ID NO: 645 to It preferably includes a base sequence represented by any one of 1287 and 1975-2018.
- “correspondingly” means a combination of a sense strand nucleic acid and an antisense strand nucleic acid in a horizontal column described as a double-stranded nucleic acid number in Table 1 or Table 3.
- the sequence number corresponding to sequence number 2 is 645.
- the sense strand nucleic acid is a nucleotide strand having a base sequence represented by any of SEQ ID NOs: 2 to 644 and 1931 to 1974.
- the antisense strand nucleic acid is More preferred is a nucleotide chain having the base sequence represented by any of SEQ ID NOs: 645 to 1287 and 1975 to 2018.
- 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 ′ end side or 5 ′ end side following the double-stranded region, this duplex is not formed.
- Nucleotides or nucleotide derivatives are called overhangs. In the case of having an overhang, the nucleotide constituting the overhang may be ribonucleotide, deoxyribonucleotide, or a derivative thereof.
- the protruding portion usually comprising 1 to 6 bases, preferably 1 to 3 bases, at the 3 ′ end or 5 ′ end of at least one of the sense strand nucleic acid and the antisense strand nucleic acid Those having a protruding portion consisting of 2 bases are more preferably used, and examples thereof include those having a protruding portion consisting of dTdT (dT represents thymidine) or UU. Overhangs can be present only in antisense strand nucleic acids, only in sense strand nucleic acids, and in both antisense strand nucleic acids and sense strand nucleic acids.
- the 3 ′ end of the nucleic acid and It may have a protrusion at the 5 ′ end.
- the antisense strand nucleic acid used in the present invention is preferably an oligonucleotide strand consisting of at least 17 nucleotides and at most 30 nucleotides, comprising a double-stranded region of the sense strand nucleic acid and a subsequent overhang. It is sufficiently complementary to the target APCS mRNA sequence containing the base sequence represented by any of SEQ ID NOs: 1288 to 1930.
- 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 has no protruding portion at the 3 ′ end or 5 ′ end.
- a double-stranded nucleic acid that forms a blunt end, a double-stranded nucleic acid in which only a sense strand nucleic acid protrudes US2012 / 0040459
- “blunt end” means that there is no overhang at the end of a double-stranded nucleic acid.
- blunt ends for example, overhangs on both the 3 ′ end of the sense strand nucleic acid and the 5 ′ end of the antisense strand nucleic acid, and / or both the 5 ′ end of the sense strand nucleic acid and the 3 ′ end of the antisense strand nucleic acid
- blunt ends for example, overhangs on both the 3 ′ end of the sense strand nucleic acid and the 5 ′ end of the antisense strand nucleic acid, and / or both the 5 ′ end of the sense strand nucleic acid and the 3 ′ end of the antisense strand nucleic acid.
- the 3 ′ end of the sense strand nucleic acid and the 5 ′ end of the antisense strand nucleic acid and / or the 5 ′ end of the sense strand nucleic acid and the 3 ′ end of the antisense strand nucleic acid form a blunt end.
- the 3 'end of the sense strand nucleic acid and the 5' end of the antisense strand nucleic acid, or the 5 'end of the sense strand nucleic acid and the 3' end of the antisense strand nucleic acid form a blunt end.
- the sense strand nucleic acid Preferably has a chain length of 21 nucleotides and the antisense strand nucleic acid has a chain length of 23 nucleotides.
- a double-stranded nucleic acid having a protruding portion on both the antisense strand nucleic acid and the sense strand nucleic acid is also preferable. It is preferable that the 3 'end of the sense strand nucleic acid and the 3' end of the antisense strand nucleic acid, or the 5 'end of the sense strand nucleic acid and the 5' end of the antisense strand nucleic acid have an overhang.
- the sense strand nucleic acid has a length of 21 nucleotides
- the antisense strand nucleic acid has a length of 21 nucleotides. Is preferred.
- both the 3 'end and the 5' end of the sense strand nucleic acid or both the 3 'end and the 5' end of the antisense strand nucleic acid have protrusions.
- the second nucleotide from the 5 ′ end of the antisense strand nucleic acid of the present invention is preferably completely complementary to the second ribonucleotide from the 3 ′ end of the target APCS mRNA sequence, and the 5 ′ end of the antisense strand nucleic acid. It is more preferable that the 2nd to 7th ribonucleotides from 2 to 7 are completely complementary to the 2nd to 7th ribonucleotides from the 3 ′ end of the target APCS mRNA sequence, respectively. More preferably, the second ribonucleotide is completely complementary to the 2nd to 11th ribonucleotides from the 3 ′ end of the target APCS mRNA sequence, respectively.
- the 11th nucleotide from the 5 ′ end of the antisense strand nucleic acid in the present invention is preferably completely complementary to the 11th ribonucleotide from the 3 ′ end of the target APCS mRNA sequence, and the 5 ′ end of the antisense strand nucleic acid
- the ninth to thirteenth ribonucleotides are completely complementary to the ninth to thirteenth ribonucleotides from the 3 ′ end of the target APCS mRNA sequence, respectively, and 7 to 15 from the 5 ′ end of the antisense strand nucleic acid.
- the second ribonucleotide is completely complementary to the 7th to 15th ribonucleotides from the 3 ′ end of the target APCS mRNA sequence, respectively.
- 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.
- Known methods using chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, and CEM method [Nucleic Acid Research, 35, 3287 (2007)].
- ABI3900 high-throughput nucleic acid synthesizer The nucleic acid of the present invention can be synthesized by (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.
- the sense strand nucleic acid synthesized and purified and the antisense strand nucleic acid in an appropriate ratio for example, 0.1 to 10 equivalents of the sense strand nucleic acid, preferably 0.5 to 1 equivalent to 1 equivalent of the antisense strand nucleic acid.
- the mixture is usually annealed, but the mixture may be used directly without the annealing step.
- Annealing may be performed under any conditions that can form a double-stranded nucleic acid, but usually, an antisense strand nucleic acid and a sense strand nucleic acid are mixed in an approximately equimolar amount, and then at about 94 ° C. for about 5 minutes. After heating, it is performed by gradually cooling to room temperature.
- the enzymatic transcription method include 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.
- 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, microinjection method or the like.
- the nucleic acid of the present invention may contain a ligand.
- the nucleic acid of the present invention may be modified with one or more ligands or fluorophores at the 5 ′ end and / or 3 ′ end and / or within the sequence. Also called conjugate nucleic acid.
- the 5'-end and / or 3'-end and / or the inside of the sequence can be modified by reacting a modifying agent capable of reacting on the solid phase.
- a conjugated nucleic acid by previously synthesizing and purifying a nucleic acid into which a functional group such as an amino group, mercapto group, azide group, or triple bond has been introduced and then allowing a modifier to act on the functional group. it can.
- a functional group such as an amino group, mercapto group, azide group, or triple bond
- the ligand may be any molecule that has an affinity for biomolecules.
- lipids such as cholesterol, fatty acid, tocopherol, and retinoid, N-acetylgalactosamine (GalNAc), galactose (Gal), and mannose (Man) Saccharides such as full antibodies, fragment antibodies such as scFV, Fab and VHH, proteins such as low density lipoprotein (LDL) and human serum albumin, peptides such as RGD, NGR, R9 and CPP, low such as folic acid Examples include molecules, synthetic polymers such as synthetic polyamino acids, and nucleic acid aptamers. These can also be used in combination.
- fluorophore examples include Cy3 series, Alexa (registered trademark) series, and black hole quencher (registered trademark).
- 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.
- nucleic acid of the present invention a vector that is introduced into a cell and expressed therein may be used.
- 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), And pENTR / U6 (manufactured by Invitrogen). Since the nucleic acid of the present invention is expressed in cells, use of the vector is included in the technical scope of the nucleic acid of the present invention.
- 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 can also be used.
- virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like.
- the antisense strand nucleic acid and the sense strand nucleic acid in the present invention are, for example, the bases of the APCS 2nd strand cDNA of the full length mRNA of human APCS registered as Genbank Accession No. NM_001639.3. It can be designed based on the sequence (SEQ ID NO: 1).
- suppression of APCS mRNA expression in non-human species by comparing the cDNA sequence of human APCS full-length mRNA with the nucleotide sequence of non-human species APCS full-length mRNA It is also possible to design antisense strand nucleic acids and sense strand nucleic acids.
- the nucleic acid having APCS expression suppressing activity includes the antisense strand nucleic acid of the present invention containing a base sequence complementary to the APCS mRNA sequence, and the base sequence complementary to the base sequence of the antisense strand nucleic acid. And a double-stranded nucleic acid comprising the sense strand nucleic acid of the present invention and having an APCS expression-suppressing activity.
- the APCS expression can be suppressed by introducing the double-stranded nucleic acid of the present invention into a cell.
- the double-stranded nucleic acid of the present invention can suppress the expression of APCS 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.
- Evaluation of the APCS mRNA expression inhibitory activity of the double-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid etc. into a human cell line using a cationic liposome etc. This can be done by quantifying the expression level of APCS mRNA.
- Evaluation of the APCS ⁇ mRNA expression inhibitory activity of the double-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid etc. into a human cell line using a cationic liposome etc. This can be done by quantifying the expression level of mRNA.
- the nucleic acid having an APCS expression-suppressing activity is a nucleic acid having a base sequence complementary to a part of the base sequence of APCS mRNA, and suppresses the expression of APCS.
- a single-stranded nucleic acid is mentioned.
- the single-stranded nucleic acid usually consists of 8 to 30 bases, preferably 12 to 30 bases, more preferably 12 to 20 bases.
- examples of the single-stranded nucleic acid include a single-stranded nucleic acid that is an antisense strand nucleic acid in the double-stranded nucleic acid of the present invention.
- the expression of APCS can be suppressed.
- the single-stranded nucleic acid of the present invention can suppress the expression of APCS mRNA after being introduced into cells at a concentration of several pM to several ⁇ M and then cultured for 24 hours or more, for example 48 hours.
- Evaluation of the APCS mRNA expression inhibitory activity of the antisense strand nucleic acid of the present invention was carried out by transfecting the nucleic acid etc. into a human cell line etc. using a cationic liposome etc. This can be done by quantifying the expression level of APCS mRNA.
- composition of the present invention also relates to a pharmaceutical composition containing a nucleic acid such as a double-stranded nucleic acid and a single-stranded nucleic acid of the present invention as an active ingredient.
- the pharmaceutical composition of the present invention can be used as a therapeutic or prophylactic agent for amyloid-related diseases.
- the present invention also provides a method for treating a disorder mediated by amyloid fibrils containing APCS. In the method of treatment, a disorder mediated by amyloid fibrils containing APCS can be treated by administering the nucleic acid of the present invention or the pharmaceutical composition of the present invention to a human in need of such treatment. it can.
- Examples of the disorder mediated by amyloid fibrils containing APCS include amyloid-related diseases.
- the double-stranded nucleic acid of the present invention can be used alone in a therapeutic method, but as shown below, by administering it to a human as an active ingredient in the pharmaceutical composition of the present invention, a medicinal effect can be exerted.
- the pharmaceutical composition of the present invention can further contain a carrier effective for transferring the nucleic acid of the present invention into cells.
- the carrier effective for transferring the nucleic acid into the cell include a cationic carrier.
- the cationic carrier include cationic liposomes and cationic polymers.
- a carrier utilizing a viral envelope may be used as an effective carrier for transferring nucleic acids into cells.
- the cationic polymer include JetSI (Qbiogene) and Jet-PEI (polyethyleneimine; Qbiogene).
- Examples of the carrier using the virus envelope include GenomeOne (HVJ-E liposome; Ishihara Sangyo Co., Ltd.).
- the pharmaceutical composition containing the nucleic acid of the present invention and the carrier can be prepared by methods known to those skilled in the art.
- the pharmaceutical composition of the present invention can be prepared by mixing an appropriate concentration carrier dispersion and a nucleic acid solution.
- a cationic carrier since the nucleic acid is usually negatively charged in an aqueous solution, the pharmaceutical composition of the present invention can be easily prepared by mixing in an aqueous solution by a conventional method.
- the aqueous solvent used for preparing the pharmaceutical 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.
- the pharmaceutical composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersion device, a high-pressure emulsification device or the like.
- the optimum method and conditions for the preparation of the pharmaceutical composition comprising the nucleic acid of the present invention and the carrier 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. it can.
- compositions comprising a composite particle comprising a nucleic acid and a lead particle and a composite membrane and a lipid membrane that coats the composite particle as required is suitably used.
- the lead particles include lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, and fine particle preparations.
- liposomes are used, and cationic liposomes are more 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 preparations, etc., and lipid aggregates, liposomes, emulsion particles
- a complex formed by combining a polymer, a metal colloid, and / or a fine particle formulation and the like with another compound eg, sugar, lipid, inorganic compound, etc.
- the lipid membrane covering the composite particles include non-cationic lipids, lipids that prevent aggregation of particles, and cationic lipids as constituent components.
- the pharmaceutical composition of the present invention can be prepared, for example, according to the method described in International Publication No. 2006/080118.
- the mixing ratio between the nucleic acid and the carrier contained in the pharmaceutical composition of the present invention is usually 1 to 200 parts by weight of the carrier per 1 part by weight of the nucleic acid.
- the mixing ratio is preferably 2.5 to 100 parts by mass of the carrier and more preferably 7 to 25 parts by mass of the carrier with respect to 1 part by mass of the nucleic acid.
- the average particle diameter of the particles constituting the pharmaceutical composition of the present invention is preferably about 10 nm to 300 nm, more preferably about 30 nm to 200 nm, and further 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 innocuous compounds (including those that are compositions) that affect the biological activity of the pharmaceutical composition of the present invention. It is something that is not given.
- Examples of pharmaceutically acceptable carriers or diluents include, but are not limited to, salt solutions, sugar solutions, glycerol solutions, and ethanol.
- the pharmaceutical composition of the present invention is provided in a form that contains an effective amount of nucleic acid 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, and external preparations such as ointments and lotions.
- the concentration range of the nucleic acid of the present invention which is an 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 to 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, and / or a pH adjuster. Any pharmaceutically acceptable additive can be added in an appropriate step before or after dispersion of the complex.
- the pH of the solution is usually adjusted to about 5.0 to about 8.5, preferably about 6.0 to about 8.0.
- the solution is preferably subjected to sterilization such as filtration sterilization using a membrane filter or the like.
- 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.
- a solution include an electrolyte solution such as water for injection and physiological saline, a glucose solution, and other general infusion solutions.
- the amount of the solution used varies depending on the application and the like and is not particularly limited, but is preferably 0.5 to 2 times the amount of the solution 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, and suppositories.
- the dose of the pharmaceutical composition of the present invention is preferably determined in consideration of the patient's condition such as drug, dosage form, age, and body weight, administration route, and the nature and degree of the disease.
- the mass is usually 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.
- Example 1 Synthesis of Double-Stranded Nucleic Acid
- Example 2 Measurement of Knockdown Activity of APCS mRNA Double-stranded nucleic acid synthesized in Example 1 and RNAiMax transfection reagent (Thermo Fisher Scientific, catalog number 13778150) were added to a 384-well culture plate Opti-MEM I Reduced. 20 ⁇ L of siRNA / RNAiMax mixture diluted with Serum Medium medium (Thermo Fisher Scientific, catalog number 31985070) was added. RMG-I cells (JCRB cell bank, JCRB0172), a cell line derived from human ovarian cancer, are seeded in 384-well culture plates at 10,000 cells / 40 ⁇ L / well, respectively, at 37 ° C and 5% CO 2 conditions. The cells were cultured for 24 hours.
- Ham's F-12 Nutrient Mix medium (Thermo Fisher Scientific, catalog number 11765-047) containing 10% fetal calf serum (Thermo Fisher Scientific, catalog number 10091-148) was used. The final concentration of double-stranded nucleic acid was 1 nM. The cells were then washed with DPBS, no calcium, no magnesium (Thermo Fisher Scientific, catalog number 14190-144), and TaqMan® Fast Cells-to-CT TM kit (Thermo Fisher) CDNA was synthesized by the following method using the catalog number 4939003 manufactured by Scientific.
- RNA extract 10 ⁇ L of a solution prepared by mixing Lysis solution (kit contents) and DNase I (kit contents) in a ratio of 100: 1 was added and mixed for 5 minutes. 1 ⁇ L of Stop Solution (kit contents) was added and mixed for 2 minutes to obtain an RNA extract. Mix 5 ⁇ L of 2 ⁇ RT Buffer (kit contents), 0.5 ⁇ L of 20 ⁇ RT Enzyme Mix (kit contents), 2.5 ⁇ L of Nuclease-free Water, and 2 ⁇ L of RNA extract. Reaction at 95 ° C. for 5 minutes to synthesize cDNA.
- ACTB is a constitutively expressed gene, measured as an internal control, and corrected for APCS gene expression.
- the APCS mRNA relative expression value when each siRNA was introduced was calculated with 1.0 as the APCS mRNA amount when RMG-I cells were treated with only the transfection reagent without adding siRNA. This experiment was performed twice, and the minimum value of APCS mRNA relative expression level is shown in Table 1. Table 1 is divided and shown as follows for convenience.
- Table 2 shows the full-length base sequence of APCS-2nd strand cDNA.
- the final concentration of the synthesized double-stranded nucleic acid was 1 nM or 0.1 nM, and the same operation as in Example 2 was performed except that the double-stranded nucleic acid was introduced into RMG-1 cells and cultured for 24 hours.
- the APCS mRNA relative expression level was calculated. This experiment was performed 4 times, and the median APCS mRNA relative expression value is shown in Table 3.
- N (M) represents 2′-O-methyl-RNA
- N (F) represents 2′-F-RNA.
- N represents A, C, G, or U. Table 3 is divided and shown as follows for convenience.
- nucleic acid having APCS expression inhibitory activity a nucleic acid having APCS 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 APCS expression and can be used as a therapeutic or prophylactic agent for amyloid-related diseases.
- SEQ ID NO: 1 Shows the full-length base sequence of APCS 2nd strand cDNA.
- SEQ ID NOs: 2 to 644 This shows the base sequence of the sense strand nucleic acid.
- SEQ ID NOs: 645 to 1287 This shows the base sequence of the antisense strand nucleic acid.
- SEQ ID NOs: 1288 to 1930 This shows the base sequence of the target APCS mRNA sequence.
- SEQ ID NOs: 1931 to 1974 This shows the base sequence of the sense strand nucleic acid.
- SEQ ID NOs: 1975-2018 This shows the base sequence of antisense strand nucleic acid.
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Abstract
Description
また、APCSはカルシウム依存的にすべての種類のアミロイド線維に結合する生化学的特性を持ち、アミロイドを有する患者のアミロイド中にも20,000 mgもの多量のAPCSが含まれていることが知られている(非特許文献1)。
APCSはすべてのアミロイド関連疾患患者におけるアミロイド中に存在することから、アミロイド関連疾患患者における診断マーカーとして用いられている(非特許文献2)。
プロテアーゼによる分解や免疫細胞による貪食に対するAPCSの耐性により、APCSが結合したアミロイドを安定化し得ることが明らかとなっており、また、APCSのアミロイドへの結合を阻害することで臓器へのアミロイド沈着及びそれに伴う組織障害を軽減できることが動物モデルを用いた研究ならびに臨床研究より強く示唆されている(非特許文献3,4)。APCSの発現を特異的に抑制することにより、アミロイド関連疾患を予防又は治療できると期待できるが、APCSの発現を特異的に抑制する医薬品はこれまでに報告されていない。
(1) センス鎖核酸及びアンチセンス鎖核酸からなり、少なくとも11個の塩基対の二重鎖領域を含む二本鎖核酸であって、前記アンチセンス鎖核酸中の、少なくとも17個のヌクレオチドかつ多くとも30個のヌクレオチドの鎖長のオリゴヌクレオチド鎖において、配列番号1288~1930のいずれかで表される塩基配列を含む標的APCS mRNA配列と相補的である、APCS遺伝子の発現を抑制する二本鎖核酸。
(2) 前記二重鎖領域が11~27個の塩基対であり、前記アンチセンス鎖核酸の5’末端から2番目のヌクレオチドが、前記標的APCS mRNA配列の3’末端から2番目のヌクレオチドと相補する、(1)に記載の二本鎖核酸。
(3) 前記センス鎖核酸の3’末端及び前記アンチセンス鎖核酸の5’末端、並びに/又は、前記センス鎖核酸の5’末端及び前記アンチセンス鎖核酸の3’末端が、平滑末端を形成する、(1)又は(2)に記載の二本鎖核酸。
(4) 前記センス鎖核酸は、21個のヌクレオチドの鎖長を有し、かつ前記アンチセンス鎖核酸は、21個のヌクレオチドの鎖長を有する、(1)~(3)のいずれかに記載の二本鎖核酸。
(5) 19個の塩基対の二重鎖領域を含み、該二重鎖領域内の40~65%のヌクレオチドが2’-O-メチル修飾ヌクレオチドである、(4)に記載の二本鎖核酸。
(6) 前記アンチセンス鎖核酸は、配列番号645~1287及び1975~2018のいずれかで表される塩基配列を含む、(1)~(5)のいずれかに記載の二本鎖核酸。
(7) 前記センス鎖核酸は、配列番号2~644及び1931~1974のいずれかで表される塩基配列を含む、(1)~(6)のいずれかに記載の二本鎖核酸。
(8) 前記センス鎖核酸は、配列番号2~644及び1931~1974のいずれかで表される塩基配列を含み、それに対応して、前記アンチセンス鎖核酸は、配列番号645~1287及び1975~2018のいずれかで表される塩基配列を含む、(1)~(7)のいずれかに記載の二本鎖核酸。
(9) リガンドを含む、(1)~(8)のいずれか一項に記載の二本鎖核酸。
(10) (1)~(9)のいずれか一項に記載の二本鎖核酸の、アンチセンス鎖核酸のみからなる一本鎖核酸。
(11) (1)~(10)のいずれか一項に記載の核酸を含む、医薬組成物。
(12) APCSを含んだアミロイド線維により媒介される障害を治療する方法であって、治療上有効量の、(1)~(10)のいずれか一項に記載の核酸又は(11)に記載の医薬組成物を、そのような治療を必要とするヒトに投与するステップを含む方法。
(13) 前記障害が、アミロイド関連疾患である、(12)に記載の方法。
本発明において、APCS mRNA配列に対して相補的な塩基配列を含む核酸をアンチセンス鎖核酸と称し、アンチセンス鎖核酸の塩基配列に対して相補的な塩基配列を含む核酸をセンス鎖核酸と称する。本明細書において「本発明の核酸」という場合、特に断らない限り、アンチセンス鎖核酸又はセンス鎖核酸の一本鎖核酸、並びにアンチセンス鎖核酸及びセンス鎖核酸が対形成した二本鎖核酸を包含する意味で用いられる。
ヌクレオチド誘導体を用いることで、特に限定されるものではないが、例えばRNA若しくはDNAと比較して、ヌクレアーゼ耐性を向上若しくは安定化させることができる、相補鎖核酸とのアフィニティーを向上させることができる、細胞透過性を向上させることができる、及び/又は可視化することができるといった利点がある。
2’-修飾ヌクレオチドとしては、リボースの2’-OH基がF又はメトキシ基で置換された2’-修飾ヌクレオチド(それぞれ、2’-F修飾ヌクレオチド又は2’-O-メチル修飾ヌクレオチドとも言う)が好ましく用いられる。
2’-修飾ヌクレオチドとしては、リボースの2’-OH基が2-(methoxy)ethoxy基、3-aminopropoxy基、2-[(N,N-dimethylamino)oxy]ethoxy基、3-(N,N-dimethylamino)propoxy基、2-[2-(N,N-dimethylamino)ethoxy]ethoxy基、2-(methylamino)-2-oxoethoxy基、2-(N-methylcarbamoyl)ethoxy 基、及び2-cyanoethoxy 基からなる群から選択される置換基で置換された2’-修飾ヌクレオチド等も挙げられる。
糖部修飾ヌクレオチドとしては、ペプチド核酸(PNA)[Acc. Chem. Res., 32, 624 (1999)]、オキシペプチド核酸(OPNA)[J. Am. Chem. Soc., 123, 4653 (2001)]、及びペプチドリボ核酸(PRNA)[J. Am. Chem. Soc., 122, 6900 (2000)]等も挙げられる。
リン酸ジエステル結合修飾ヌクレオチドとしては、例えばリン酸ジエステル結合がホスホロチオエート結合に置換されたヌクレオチド、リン酸ジエステル結合がホスホロジチオエート結合に置換されたヌクレオチド、リン酸ジエステル結合がアルキルホスホネート結合に置換されたヌクレオチド、及びリン酸ジエステル結合がホスホロアミデート結合に置換されたヌクレオチド等が挙げられる。
塩基修飾ヌクレオチドとしては、例えば塩基内の酸素原子が硫黄原子で置換されたヌクレオチド、塩基内の水素原子が炭素数1~6のアルキル基、ハロゲン等で置換されたもの、メチル基が水素、ヒドロキシメチル、炭素数2~6のアルキル基等で置換されたもの、アミノ基が炭素数1~6のアルキル基、炭素数1~6のアルカノイル基、オキソ基、ヒドロキシ基等に置換されたものが挙げられる。なお、シトシン(C)の代わりに5-メチルシトシン(5-mC)を塩基修飾ヌクレオチドとして用いることも、本発明の好ましい形態の一つである。
ヌクレオチド誘導体は、核酸内の他のヌクレオチド又はヌクレオチド誘導体とアルキレン構造、ペプチド構造、ヌクレオチド構造、エーテル構造、及びエステル構造、並びにこれらの少なくとも2つを組み合わせた構造等の架橋構造を形成してもよい。
具体的には、アンチセンス鎖核酸が21個のヌクレオチドの鎖長(21塩基長)の場合には、標的遺伝子の標的ヌクレオチド配列に対して1~6個、好ましくは1~5個、より好ましくは1~4個、さらに好ましくは1~3個、特に好ましくは1~2個、特により好ましくは1個のミスマッチ塩基を有してもよく、そのミスマッチの位置は、それぞれのアンチセンス鎖におけるヌクレオチド配列の5’末端又は3’末端であってもよい。
本明細書において「相補的」とは、一方のヌクレオチド配列が、他方のヌクレオチド配列と完全に相補する塩基配列において、1つ若しくは複数の塩基が付加及び/又は欠失した配列である場合を包含する。例えば、本発明におけるアンチセンス鎖核酸は、APCS mRNA配列に対して完全に相補する塩基配列における塩基の付加により、標的APCS mRNA配列に1個又は2個のバルジ塩基を有してもよい。
本発明においては、アンチセンス鎖核酸若しくはセンス鎖核酸において、1個又は2個のバルジ塩基を有してもよい。
本発明においては、アンチセンス鎖核酸により、APCS遺伝子の発現を抑制することができる。
配列番号1288~1930のいずれかで表される塩基配列は、表1に記載される。
本発明の二本鎖核酸において、センス鎖核酸及びアンチセンス鎖核酸は、いずれのヌクレオチド又はヌクレオチド誘導体から構成されてよい。
標的APCS mRNA配列は、配列番号1に相同であり、配列番号1におけるTがUと読み替えられたAPCS mRNAの塩基配列のうち、配列番号1288~1930のいずれかで表される塩基配列を含む。好ましくは、標的APCS mRNA配列は、配列番号1に相同であり、配列番号1におけるTがUと読み替えられたAPCS mRNAの塩基配列のうち、配列番号1288~1930のいずれかで表される塩基配列である。
本発明の二本鎖核酸におけるアンチセンス鎖核酸を構成するヌクレオチドのうち、少なくとも17個のヌクレオチドかつ多くとも30個のヌクレオチドの鎖長のオリゴヌクレオチド鎖が、標的APCS mRNA配列と相補的である。
本発明においてアンチセンス鎖核酸を構成するヌクレオチドのうち、好ましくは11~27個のヌクレオチド、より好ましくは15~25個のヌクレオチド、さらに好ましくは15~23個のヌクレオチド、特に好ましくは17~21個のヌクレオチド、特により好ましくは17~19個のヌクレオチド、特にさらに好ましくは19個のヌクレオチドの鎖長のオリゴヌクレオチド鎖において、標的APCS mRNA配列と相補的である。
2’-修飾ヌクレオチドを含む二本鎖核酸として具体的には、例えば、表3に記載のセンス鎖/アンチセンス鎖からなる成る群から選択される1対のセンス鎖/アンチセンス鎖の配列からなる二本鎖核酸が挙げられる。
本発明において、センス鎖核酸のヌクレオチドに対して、好適な2’-修飾ヌクレオチドの含有量として、20~40%であってもよく、40~60%であってもよく、60%~100%であってもよい。
本発明において、アンチセンス鎖核酸のヌクレオチドに対して、好適な2’-修飾ヌクレオチドの含有量として、0~40%であることが好ましく、10~20%であってもよく、20~40%であってもよい。
本発明においては、アンチセンス鎖核酸は、配列番号645~1287及び1975~2018のいずれかで表される塩基配列を有するヌクレオチド鎖であることがより好ましく、センス鎖核酸は、配列番号2~644及び1931~1974のいずれかで表される塩基配列を有するヌクレオチド鎖であることがより好ましい。
本発明の二本鎖核酸は、センス鎖核酸は、配列番号2~644及び1931~1974のいずれかで表される塩基配列を含み、それに対応して、アンチセンス鎖核酸は、配列番号645~1287及び1975~2018のいずれかで表される塩基配列を含むことが好ましい。
本明細書において、それに対応してとは、表1又は表3において二本鎖核酸番号として記載される横一列のカラムにおけるセンス鎖核酸とアンチセンス鎖核酸の組み合わせであることを意味し、配列番号n(n=2~644)に対応する配列番号は、n+643となる。具体的に例示して説明すると、配列番号2に対応する配列番号は645である。
本発明の二本鎖核酸は、センス鎖核酸は、配列番号2~644及び1931~1974のいずれかで表される塩基配列を有するヌクレオチド鎖であり、それに対応して、アンチセンス鎖核酸は、配列番号645~1287及び1975~2018のいずれかで表される塩基配列を有するヌクレオチド鎖であることがより好ましい。
本発明の二本鎖核酸としては、例えばDicer等のリボヌクレアーゼの作用により上記の二本鎖核酸を生成する核酸分子(WO2005/089287)や、3’末端や5’末端の突出部を有さず平滑末端を形成する二本鎖核酸、センス鎖核酸のみが突出した二本鎖核酸(US2012/0040459)等を用いることもできる。本発明において、「平滑末端」とは、2本鎖核酸の末端に突出部を有さないことを指す。平滑末端として、例えば、センス鎖核酸の3’末端及びアンチセンス鎖核酸の5’末端の両方、及び/又は、センス鎖核酸の5’末端及びアンチセンス鎖核酸の3’末端の両方に突出部を有しない2本鎖核酸がある。
本発明におけるアンチセンス鎖核酸の5’末端から11番目のヌクレオチドが、標的APCS mRNA配列の3’末端から11番目のリボヌクレオチドと完全に相補であることが好ましく、アンチセンス鎖核酸の5’末端から9~13番目のリボヌクレオチドが、それぞれ標的APCS mRNA配列の3’末端から9~13番目のリボヌクレオチドと完全に相補であることがより好ましく、アンチセンス鎖核酸の5’末端から7~15番目のリボヌクレオチドが、それぞれ標的APCS mRNA配列の3’末端から7~15番目のリボヌクレオチドと完全に相補であることがさらに好ましい。
公知の化学合成を用いる方法として、ホスホロアミダイト法、ホスホロチオエート法、ホスホトリエステル法、及びCEM法[Nucleic Acid Research, 35, 3287 (2007)]等が挙げられ、例えば、ABI3900ハイスループット核酸合成機(アプライドバイオシステムズ社製)により本発明の核酸を合成することができる。合成が終了した後は、固相からの脱離、保護基の脱保護、及び目的物の精製等を行う。精製により、純度90%以上、好ましくは95%以上の核酸を得るのが望ましい。
二本鎖核酸の場合には、合成及び精製したセンス鎖核酸及びアンチセンス鎖核酸を適当な比率、例えば、アンチセンス鎖核酸1当量に対して、センス鎖核酸0.1~10当量、好ましくは0.5~2当量、より好ましくは0.9~1.1当量、さらに好ましくは等モル量で混合した後、通常混合したものをアニーリングするが、アニーリングする工程を省いて混合したものを直接用いてもよい。
アニーリングは、二本鎖核酸を形成できる条件であればいかなる条件で行ってもよいが、通常、アンチセンス鎖核酸及びセンス鎖核酸をほぼ等モル量で混合した後、94℃程度で5分程度加熱したのち、室温まで徐冷することにより行われる。
酵素的転写法としては、目的の塩基配列を有したプラスミド又はDNAを鋳型としてファージRNAポリメラーゼ、例えば、T7、T3、又はSP6RNAポリメラーゼを用いた転写による方法が挙げられる。
細胞内において、本発明の核酸が発現することから、当該ベクターを用いることは、本発明の核酸の技術的範囲に含まれる。
本発明におけるアンチセンス鎖核酸及びセンス鎖核酸は、例えば、Genbank Accession No.NM_001639.3として登録されているヒトAPCSの完全長mRNAのAPCS 2nd strand cDNAの塩基配列(配列番号1)に基づいて設計することができる。なお、ヒトのAPCSの完全長mRNAのcDNAの塩基配列とヒト以外の生物種のAPCSの完全長mRNAのcDNAの塩基配列とを比較することにより、ヒト以外の生物種でAPCS mRNAの発現を抑制するアンチセンス鎖核酸及びセンス鎖核酸を設計することもできる。
また、該一本鎖核酸としては、例えば、本発明の二本鎖核酸におけるアンチセンス鎖核酸である一本鎖核酸が挙げられる。
本発明は、本発明の二本鎖核酸及び一本鎖核酸等の核酸を有効成分として含有する医薬組成物にも関する。本発明の医薬組成物は、アミロイド関連疾患の治療剤又は予防剤として用いることができる。
また、本発明においては、APCSを含んだアミロイド線維により媒介される障害を治療する方法も提供する。当該治療方法においては、本発明の核酸又は本発明の医薬組成物を、そのような治療を必要とするヒトに投与することにより、APCSを含んだアミロイド線維により媒介される障害を治療することができる。
APCSを含んだアミロイド線維により媒介される障害としては、例えば、アミロイド関連疾患があげられる。本発明の二本鎖核酸は、単独で治療方法に用いることもできるが、以下に示すとおり、本発明の医薬組成物における有効成分としてヒトに投与することにより、薬効を発揮させることができる。
該複合粒子を被覆する脂質膜としては、例えば非カチオン性脂質、粒子の凝集を阻止する脂質、及びカチオン性脂質等を構成成分とするものが挙げられる。
本発明の医薬組成物は、例えば国際公開第2006/080118号等に記載の方法に従って調製することができる。
液剤のpHは、通常、約5.0~約8.5、好ましくは約6.0~約8.0に調整される。
液剤は、好ましくは、メンブレンフィルター等を用いた濾過滅菌等の滅菌処理を行う。
シグマアルドリッチ社に依頼して、配列番号2~644に示すリボヌクレオチドから成るセンス鎖核酸、配列番号645~1287に示すリボヌクレオチドから成るアンチセンス鎖核酸、及びそれらをアニーリングさせた二本鎖核酸(配列番号n(n=2~644)に示すセンス鎖核酸と配列番号[n+643]に示すアンチセンス鎖核酸とが対をなす)を合成した。
384ウェルの培養プレートに、実施例1で合成した二本鎖核酸とRNAiMaxトランスフェクション試薬(Thermo Fisher Scientific社製、カタログ番号13778150)をOpti-MEM I Reduced Serum Medium培地(Thermo Fisher Scientific社製、カタログ番号31985070)で希釈したsiRNA/RNAiMax混合液20 μLを添加した。ヒト卵巣癌由来の細胞株であるRMG-I細胞(JCRB細胞バンク、JCRB0172)を、10,000細胞/40 μL/ウェルとなるよう各々384ウェルの培養プレートに播種し、37℃、5% CO2条件下で24時間培養した。培地は、10%ウシ胎仔血清(Thermo Fisher Scientific社製、カタログ番号10091-148)を含むHam’s F-12 Nutrient Mix培地(Thermo Fisher Scientific社製、カタログ番号11765-047)を用いた。二本鎖核酸の終濃度は1 nMとなるようにした。その後、細胞をDPBS, no calcium, no magnesium(Thermo Fisher Scientific社製、カタログ番号14190-144)で洗浄し、各々のプレートからTaqMan(登録商標) Fast Cells-to-CT(商標)キット(Thermo Fisher Scientific社製、カタログ番号4399003)を用いて以下の方法でcDNAを合成した。Lysis solution(キット内容物)とDNase I(キット内容物)を100:1で混合した溶液を10 μLずつ添加し、5分間混合した。Stop Solution(キット内容物)を1 μLずつ添加、2分間混合し、RNA抽出液とした。5 μLの2×RT Buffer(キット内容物)、0.5 μLの20×RT Enzyme Mix(キット内容物)、2.5 μLのNuclease-free Water、2 μLのRNA抽出液を混合し、これを37℃60分、95℃5分で反応させ、cDNAを合成した。このcDNA 2 μLをMicroAmp(登録商標) EnduraPlate(商標) Optical 384-Well Clear Reaction Plates with Barcode(Thermo Fisher Scientific社製、カタログ番号4483285)に加え、更に5 μLのTaqMan(登録商標) Fast Universal PCR Master Mix(Thermo Fisher Scientific社製、カタログ番号4352042)、2.5 μLのDISTILLED WATER (ULTRAPURE)(Thermo Fisher Scientific社製、カタログ番号10977015)、0.25 μLのhuman APCSプローブ、0.25 μLのhuman ACTBプローブを添加した。QuantStudio(商標) 7 Flexを用いて、human APCS遺伝子及びhuman ACTB(actin beta)遺伝子のリアルタイムPCRをおこなった。ACTBは構成的発現遺伝子であり内部対照として測定し、APCS遺伝子発現量を補正した。siRNAを添加せずにトランスフェクション試薬だけでRMG-I細胞を処理した時のAPCS mRNA量を1.0として、各siRNAを導入した時のAPCS mRNA相対発現量値を算出した。本実験を2回おこない、APCS mRNA相対発現量値の最小値を表1に示した。表1は、便宜上、以下のとおり分割して示す。
ジーンデザイン社に依頼して、配列番号1931~1974に示すリボヌクレオチドから成るセンス鎖核酸、配列番号1975~2018に示すリボヌクレオチドから成るアンチセンス鎖核酸、及びそれらをアニーリングさせた二本鎖核酸(配列番号n(n=1931~1974)に示すセンス鎖と配列番号[n+44]に示すアンチセンス鎖とが対をなす)を合成した。
合成した二本鎖核酸の終濃度は1 nM、又は0.1 nMとなるようにし、当該二本鎖核酸をRMG-1細胞に導入し、24時間培養した以外は、実施例2と同様の操作を行って、APCS mRNA相対発現量を算出した。本実験を4回おこない、APCS mRNA相対発現量値の中央値を表3に示した。表3において、N(M)は2’-O-methyl-RNAを、N(F)は2’-F-RNAを示す。ここで、NはA、C、G又はUを示す。表3は、便宜上、以下のとおり分割して示す。
配列番号2~644 センス鎖核酸の塩基配列を示す。
配列番号645~1287 アンチセンス鎖核酸の塩基配列を示す。
配列番号1288~1930 標的APCS mRNA配列の塩基配列を示す。
配列番号1931~1974 センス鎖核酸の塩基配列を示す。
配列番号1975~2018 アンチセンス鎖核酸の塩基配列を示す。
Claims (13)
- センス鎖核酸及びアンチセンス鎖核酸からなり、少なくとも11個の塩基対の二重鎖領域を含む二本鎖核酸であって、前記アンチセンス鎖核酸中の、少なくとも17個のヌクレオチドかつ多くとも30個のヌクレオチドの鎖長のオリゴヌクレオチド鎖において、配列番号1288~1930のいずれかで表される塩基配列を含む標的APCS mRNA配列と相補的である、APCS遺伝子の発現を抑制させる二本鎖核酸。
- 前記二重鎖領域が11~27個の塩基対であり、前記アンチセンス鎖核酸の5’末端から2番目のヌクレオチドが、前記標的APCS mRNA配列の3’末端から2番目のデオキシリボヌクレオチドと相補する、請求項1に記載の二本鎖核酸。
- 前記センス鎖核酸の3’末端及び前記アンチセンス鎖核酸の5’末端、並びに/又は、前記センス鎖核酸の5’末端及び前記アンチセンス鎖核酸の3’末端が、平滑末端を形成する、請求項1又は2に記載の二本鎖核酸。
- 前記センス鎖核酸は、21個のヌクレオチドの鎖長を有し、かつ前記アンチセンス鎖核酸は、21個のヌクレオチドの鎖長を有する、請求項1~3のいずれか1項に記載の二本鎖核酸。
- 19個の塩基対の二重鎖領域を含み、該二重鎖領域内の40~65%のヌクレオチドが2’-O-メチル修飾ヌクレオチドである、請求項4に記載の二本鎖核酸。
- 前記アンチセンス鎖核酸は、配列番号645~1287及び1975~2018のいずれかで表される塩基配列を含む、請求項1~5のいずれか1項に記載の二本鎖核酸。
- 前記センス鎖核酸は、配列番号2~644及び1931~1974のいずれかで表される塩基配列を含む、請求項1~6のいずれか1項に記載の二本鎖核酸。
- 前記センス鎖核酸は、配列番号2~644及び1931~1974のいずれかで表される塩基配列を含み、それに対応して、前記アンチセンス鎖核酸は、配列番号645~1287及び1975~2018のいずれかで表される塩基配列を含む、請求項1~7のいずれか1項に記載の二本鎖核酸。
- リガンドを含む、請求項1~8のいずれか一項に記載の二本鎖核酸。
- 請求項1~9のいずれか一項に記載の二本鎖核酸の、アンチセンス鎖核酸のみからなる一本鎖核酸。
- 請求項1~10のいずれか一項に記載の核酸を含む、医薬組成物。
- APCSを含んだアミロイド線維により媒介される障害を治療する方法であって、治療上有効量の、請求項1~10のいずれか一項に記載の核酸又は請求項11に記載の医薬組成物を、そのような治療を必要とするヒトに投与するステップを含む方法。
- 前記障害が、アミロイド関連疾患である、請求項12に記載の方法。
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Cited By (3)
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WO2019100039A1 (en) * | 2017-11-20 | 2019-05-23 | Alnylam Pharmaceuticals, Inc. | Serum amyloid p component (apcs) irna compositions and methods of use thereof |
US11229663B2 (en) * | 2017-11-20 | 2022-01-25 | Alnylam Pharmaceuticals, Inc. | Serum amyloid P component (APCS) iRNA compositions and methods of use thereof |
WO2020111280A1 (ja) * | 2018-11-30 | 2020-06-04 | 協和キリン株式会社 | 核酸複合体 |
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EP3633037A1 (en) | 2020-04-08 |
US20200190514A1 (en) | 2020-06-18 |
JPWO2018221649A1 (ja) | 2020-04-02 |
CN110832078A (zh) | 2020-02-21 |
KR20200014320A (ko) | 2020-02-10 |
EP3633037A4 (en) | 2021-03-24 |
AU2018277476A1 (en) | 2020-01-02 |
TW201903149A (zh) | 2019-01-16 |
CA3065149A1 (en) | 2018-12-06 |
US11466272B2 (en) | 2022-10-11 |
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