WO2020246560A1 - 化学修飾核酸を導入した安定型標的編集ガイドrna - Google Patents
化学修飾核酸を導入した安定型標的編集ガイドrna Download PDFInfo
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- WO2020246560A1 WO2020246560A1 PCT/JP2020/022200 JP2020022200W WO2020246560A1 WO 2020246560 A1 WO2020246560 A1 WO 2020246560A1 JP 2020022200 W JP2020022200 W JP 2020022200W WO 2020246560 A1 WO2020246560 A1 WO 2020246560A1
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- oligonucleotide
- residue
- target
- pharmaceutically acceptable
- acceptable salt
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Images
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Definitions
- the present invention relates to a stable target editing guide RNA into which a chemically modified nucleic acid has been introduced.
- RNA is a nucleic acid molecule to which DNA information is copied, and unlike DNA, it is a transient genetic information molecule in which synthesis and degradation are repeated. Therefore, modification of RNA information can give the target organism a temporary non-permanent genetic information modification effect. That is, although RNA modification technology is a gene modification technology like DNA modification, its properties are significantly different.
- Patent Document 1 describes a target-directed portion containing an antisense sequence complementary to a part of a target RNA and an RNA editing entity existing in a cell and capable of editing a nucleotide. Oligonucleotide constructs for site-specific editing of nucleotides in a target RNA sequence are described, including recruitment moieties that can bind and recruit. Further, for example, Patent Document 2 describes a site-specific RNA mutation introduction method in which double-stranded specific adenosine deaminase (ADAR) is allowed to act on a complex of a target RNA and a target editing guide RNA. In addition, Non-Patent Document 1 describes that a modified nucleic acid is introduced into an antisense oligonucleotide that recruits ADAR.
- ADAR double-stranded specific adenosine deaminase
- the oligonucleotide construct described in Patent Document 1 has a stem-loop structure having a specific repetitive sequence as a recruitment portion in addition to a target directional site, and requires 16 or more oligonucleotides in the recruitment portion. .. Further, the target editing guide RNA described in Patent Document 2 requires an ADAR binding region having a stem-loop structure consisting of a specific sequence of 40 or 49 residues in addition to the antisense region.
- the oligonucleotides applied to the RNA modification technique described in Patent Documents 1 or 2 are all intramolecularly having a certain length for binding to ADAR in addition to the site forming a duplex with the target RNA.
- the main chain region is indispensable, and the total length as an oligonucleotide tends to be inevitably long.
- One aspect of the present invention is to provide a target editing guide RNA that has a small number of nucleotides added to a target recognition site, has excellent stability in vivo, and can induce site-specific editing in cells. ..
- (1-1) A first oligonucleotide that identifies a target RNA, a second oligonucleotide that is linked to the 3'side of the first oligonucleotide, and a third oligonucleotide that can form a complementary pair with the second oligonucleotide.
- the first oligonucleotide includes a first linking portion that links the second oligonucleotide and the third oligonucleotide, and the first oligonucleotide includes a target-corresponding nucleotide residue corresponding to the adenosine residue in the target RNA and the target-corresponding nucleotide residue.
- the target is linked to the 5'side of the target-corresponding nucleotide residue and is linked to the 3'side of the target-corresponding nucleotide residue with a 10 to 30-residue oligonucleotide having a base sequence complementary to the target RNA.
- oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof.
- the first link contains at least one selected from the group consisting of 4 or 5 residue oligonucleotides and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units (1-).
- the first oligonucleotide is the oligonucleotide according to any one of (1-1) to (1-3) containing a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.
- the first oligonucleotide consists of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- the oligonucleotide according to any one of (1-1) to (1-4) containing at least one modified nucleotide residue selected from the group, or a pharmaceutically acceptable salt thereof.
- At least one of the 3'side and 5'side 1 residues of the target-corresponding nucleotide residue is a 2'-O-alkylribonucleotide residue and a 2'-deoxy-2.
- At least one of the second oligonucleotide and the third oligonucleotide is a 2'-O-alkylribonucleotide residue, a 2'-deoxy-2'-fluororibonucleotide residue, and a 2'-deoxyribonucleotide.
- a hereditary disease is a disease that can be treated by converting an adenosine residue in a target RNA into an inosine residue.
- oligonucleotide according to any one of (1-1) to (1-10) for use in the prevention or treatment of a disease, or a pharmaceutically acceptable salt thereof.
- the first oligonucleotide includes a first link portion that links the second oligonucleotide and the third oligonucleotide, and the first oligonucleotide includes a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA and the target-corresponding nucleotide residue.
- the target is linked to the 5'side of the target-corresponding nucleotide residue and is linked to the 3'side of the target-corresponding nucleotide residue with a 10 to 30 residue oligonucleotide having a base sequence complementary to the target RNA. It consists of 3 to 6 residue oligonucleotides having a base sequence complementary to RNA, the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues. At least one residue selected from the target-corresponding nucleotide residue and the counter region consisting of one residue on each of the 3'side and 5'side thereof is a nucleotide residue other than the natural ribonucleotide residue. Yes, an oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof.
- the first link contains at least one selected from the group consisting of 4 or 5 residue oligonucleotides and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units (2-2).
- the first oligonucleotide is the oligonucleotide according to any one of (2-1) to (2-8) containing a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.
- the first oligonucleotide consists of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- the oligonucleotide according to any one of (2-1) to (2-9) containing at least one modified nucleotide selected from the group, or a pharmaceutically acceptable salt thereof.
- the first oligonucleotide consists of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- the oligonucleotide according to any one of (2-1) to (2-9) containing at least one modified nucleotide selected from the group, or a pharmaceutically acceptable salt thereof.
- At least one of the 1 residues on the 3'side and 1'side of the target-corresponding nucleotide residue is a 2'-O-alkylribonucleotide residue and 2'-deoxy-2.
- At least one of the second oligonucleotide and the third oligonucleotide is a 2'-O-alkylribonucleotide residue, a 2'-deoxy-2'-fluororibonucleotide residue, and a 2'-deoxyribonucleotide.
- the first oligonucleotide includes a first link that links the second oligonucleotide and the third oligonucleotide, and the first oligonucleotide has a target-corresponding nucleotide residue corresponding to the adenosine residue in the target RNA and the target-correspondence.
- oligonucleotide 10 to 30 residues having a base sequence complementary to the target RNA and is linked to the target RNA. It consists of 3 to 6 residue oligonucleotides having a complementary base sequence, the number of residues of the second oligonucleotide is 5 to 8, and the number of residues of the third oligonucleotide is 5 to 8.
- the second oligonucleotide and the third oligonucleotide are all composed of 2'-O-alkylribonucleotides, and a counter region consisting of the target-corresponding nucleotide residue and one residue each on the 3'side and 5'side thereof.
- the first link contains at least one selected from the group consisting of 4 or 5 residue oligonucleotides and polyalkyleneoxy groups consisting of 2 to 8 alkyleneoxy units (2-19).
- the oligonucleotide, or a pharmaceutically acceptable salt thereof is selected from the group consisting of 4 or 5 residue oligonucleotides and polyalkyleneoxy groups consisting of 2 to 8 alkyleneoxy units (2-19).
- oligonucleotide according to any one of (2-18) to (2-25), wherein the target-corresponding nucleotide residue is a cytidine residue, a uridine residue, an adenosine residue, or a derivative thereof. , Or its pharmaceutically acceptable salt.
- (2-27) The oligonucleotide according to (2-26), wherein the target-corresponding nucleotide residue is a cytidine residue or a derivative thereof, or a pharmaceutically acceptable salt thereof.
- the first oligonucleotide consists of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- the first oligonucleotide consists of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- At least one of the 1 residues on the 3'side and the 5'side of the target-corresponding nucleotide residue is a 2'-O-alkylribonucleotide residue and 2'-deoxy-2.
- (2-31) The oligonucleotide according to any one of (2-18) to (2-30), wherein the second oligonucleotide has a nucleotide sequence of GGGUGG and the third oligonucleotide has a nucleotide sequence of CCACCU. , Or its pharmaceutically acceptable salt.
- the first oligonucleotide includes a first linking portion that links the second oligonucleotide and the third oligonucleotide, and the first oligonucleotide includes a target-corresponding nucleotide residue corresponding to the adenosine residue in the target RNA and the target-corresponding nucleotide residue.
- the target is linked to the 5'side of the target-corresponding nucleotide residue and is linked to the 3'side of the target-corresponding nucleotide residue with a 10 to 30-residue oligonucleotide having a base sequence complementary to the target RNA. It consists of 3 to 6 residue oligonucleotides having a base sequence complementary to RNA, the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues. At least one residue selected from the target-corresponding nucleotide residue and the counter region consisting of one residue on each of the 3'side and 5'side thereof is a nucleotide residue other than the natural ribonucleotide residue. Yes, an oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof.
- the first link contains at least one selected from the group consisting of 4 or 5 residue oligonucleotides and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units (3-2).
- the first oligonucleotide is the oligonucleotide according to any one of (3-1) to (3-3) containing a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.
- the first oligonucleotide consists of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- the oligonucleotide according to any one of (3-1) to (3-4) containing at least one modified nucleotide residue selected from the group, or a pharmaceutically acceptable salt thereof.
- (3-7) The oligonucleotide according to any one of (3-1) to (3-6), wherein the target-corresponding nucleotide residue contains a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.
- At least one of the 1 residues on the 3'side and the 5'side of the target-corresponding nucleotide residue is a 2'-O-alkylribonucleotide residue and 2'-deoxy-2.
- At least one of the second oligonucleotide and the third oligonucleotide is a 2'-O-alkylribonucleotide residue, a 2'-deoxy-2'-fluororibonucleotide residue, and a 2'-deoxyribonucleotide.
- Oligonucleotides linked to the 5'side of the target-corresponding nucleotide residue are alternately linked with 2'-deoxy-2'-fluoronucleotide residues and 2'-O-alkylribonucleotide residues.
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide residue is 2'-deoxy-2'-.
- the oligonucleotide linked to the 5'side of the target-corresponding nucleotide residue has a base sequence in which crosslinked nucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked (3).
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide residue is a crosslinked nucleotide residue (3-13).
- the oligonucleotide, or a pharmaceutically acceptable salt thereof is a crosslinked nucleotide residue (3-13).
- the oligonucleotide linked to the 5'side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and crosslinked nucleotide residues are alternately linked.
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide is 2'-deoxy-2'-fluoronucleotide.
- the oligonucleotide according to (3-14) which is a residue, or a pharmaceutically acceptable salt thereof.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue has a base sequence to which the 2'-O-alkylribonucleotide residue is linked (3-1). ) To (3-15), or a pharmaceutically acceptable salt thereof.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue is alternately linked with a 2'-O-alkylribonucleotide residue and a crosslinked nucleotide residue.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue is any one of (3-1) to (3-17) consisting of 4 to 6 residues.
- Each of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide has any of (3-1) to (3-19) in which nucleotide residues are linked by a phosphorothioate bond.
- (3-22) A drug containing the oligonucleotide according to any one of (3-1) to (3-21) or a pharmaceutically acceptable salt thereof.
- (3-23) A therapeutic agent for a hereditary disease containing the oligonucleotide according to any one of (3-1) to (3-21) or a pharmaceutically acceptable salt thereof.
- the hereditary disease is a disease that can be treated by converting an adenosine residue in a target RNA into an inosine residue.
- the hereditary disease is a hereditary disease caused by a mutation of a guanosine residue to an adenosine residue in a gene.
- the first oligonucleotide includes a first linking portion that links the second oligonucleotide and the third oligonucleotide, and the first oligonucleotide includes a target-corresponding nucleotide residue corresponding to the adenosine residue in the target RNA and the target-corresponding nucleotide residue.
- the target is linked to the 5'side of the target-corresponding nucleotide residue and is linked to the 3'side of the target-corresponding nucleotide residue with a 10 to 30 residue oligonucleotide having a base sequence complementary to the target RNA. It consists of 3 to 6 residue oligonucleotides having a base sequence complementary to RNA, the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues. At least one residue selected from the target-corresponding nucleotide residue and the counter region consisting of one residue on each of the 3'side and 5'side thereof is a nucleotide residue other than the natural ribonucleotide residue. Yes, an oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof.
- the first link contains at least one selected from the group consisting of 4 or 5 residue oligonucleotides and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units (4-2).
- oligonucleotide according to any one of (4-1) to (4-5), which comprises a second link containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide. Or its pharmaceutically acceptable salt.
- the first oligonucleotide is the oligonucleotide according to any one of (4-1) to (4-8) containing a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.
- the first oligonucleotide consists of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- the first oligonucleotide consists of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- At least one of the 1 residues on the 3'side and 1'side of the target-corresponding nucleotide residue is a 2'-O-alkylribonucleotide residue and 2'-deoxy-2.
- At least one of the second oligonucleotide and the third oligonucleotide is a 2'-O-alkylribonucleotide residue, a 2'-deoxy-2'-fluororibonucleotide residue, and a 2'-deoxyribonucleotide.
- the first oligonucleotide includes a first link that links the second oligonucleotide and the third oligonucleotide, and the first oligonucleotide has a target-corresponding nucleotide residue corresponding to the adenosine residue in the target RNA and the target-correspondence.
- oligonucleotide 10 to 30 residues having a base sequence complementary to the target RNA and is linked to the target RNA. It consists of 3 to 6 residue oligonucleotides having a complementary base sequence, the number of residues of the second oligonucleotide is 5 to 8, and the number of residues of the third oligonucleotide is 5 to 8.
- the second oligonucleotide and the third oligonucleotide are all composed of 2'-O-alkylribonucleotides, and a counter region consisting of the target-corresponding nucleotide residue and one residue each on the 3'side and 5'side thereof.
- At least one residue selected from is an oligonucleotide residue other than the native ribonucleotide residue, and all phosphate diester bond modifications are phosphorothioate bonds, and oligos that induce site-specific editing on the target RNA.
- a nucleotide, or a pharmaceutically acceptable salt thereof is an oligonucleotide residue other than the native ribonucleotide residue, and all phosphate diester bond modifications are phosphorothioate bonds, and oligos that induce site-specific editing on the target RNA.
- the first link contains at least one selected from the group consisting of 4 or 5 residue oligonucleotides and polyalkyleneoxy groups consisting of 2 to 8 alkyleneoxy units (4-19).
- the oligonucleotide, or a pharmaceutically acceptable salt thereof is selected from the group consisting of 4 or 5 residue oligonucleotides and polyalkyleneoxy groups consisting of 2 to 8 alkyleneoxy units (4-19).
- oligonucleotide according to any one of (4-18) to (4-22), which comprises a second link containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide. Or its pharmaceutically acceptable salt.
- oligonucleotide according to any one of (4-18) to (4-25), wherein the target-corresponding nucleotide residue is a cytidine residue, a uridine residue, an adenosine residue, or a derivative thereof. , Or its pharmaceutically acceptable salt.
- the first oligonucleotide consists of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- the first oligonucleotide consists of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, crosslinked nucleotide residues, and 2'-deoxyribonucleotides.
- At least one of the 1 residues on the 3'side and 1'side of the target-corresponding nucleotide residue is a 2'-O-alkylribonucleotide residue and 2'-deoxy-2.
- the oligonucleotide linked to the 5'side of the target-corresponding nucleotide residue is alternately linked with a 2'-deoxy-2'-fluoronucleotide residue and a 2'-O-alkylribonucleotide residue.
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide residue is 2'-deoxy-2'-.
- the oligonucleotide linked to the 5'side of the target-corresponding nucleotide residue has a base sequence in which crosslinked nucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked (4).
- -1 The oligonucleotide according to any one of (4-31), or a pharmaceutically acceptable salt thereof.
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide residue is a crosslinked nucleotide residue (4-35).
- the oligonucleotide linked to the 5'side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and crosslinked nucleotide residues are alternately linked.
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide residue is 2'-deoxy-2'-.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue has a base sequence to which the 2'-O-alkylribonucleotide residue is linked (4-1). ) To (4-37), or a pharmaceutically acceptable salt thereof.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue is alternately linked with a 2'-O-alkylribonucleotide residue and a crosslinked nucleotide residue.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue is any one of (4-1) to (4-39) consisting of 4 to 6 residues.
- (4-41) Described in any of (4-1) to (4-40), wherein the second oligonucleotide and the third oligonucleotide have a base sequence in which a 2'-O-alkylribonucleotide residue is linked. Oligonucleotide, or a pharmaceutically acceptable salt thereof.
- Each of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide has any of (4-1) to (4-41) in which nucleotide residues are linked by a phosphorothioate bond.
- Method for prevention or treatment of. (4-51) The method according to (4-50), wherein the disease is a hereditary disease.
- the hereditary disease is a hereditary disease caused by a mutation in a gene from a guanosine residue to an adenosine residue.
- a target editing guide RNA that has a small number of nucleotides added to a target recognition site, is excellent in in vivo stability, and can induce site-specific editing.
- the term "process” is included in this term not only as an independent process but also as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes. ..
- the content of each component in the composition means the total amount of the plurality of substances present in the composition when a plurality of substances corresponding to each component are present in the composition, unless otherwise specified.
- embodiments of the present invention will be described in detail. However, the embodiments shown below exemplify oligonucleotides for embodying the technical idea of the present invention, and the present invention is not limited to the oligonucleotides shown below.
- the oligonucleotides that induce site-specific editing for the target RNA are the first oligonucleotide that identifies the target RNA and the second oligonucleotide that is linked to the 3'side of the first oligonucleotide. And a third oligonucleotide that can form a complementary pair with the second oligonucleotide, and a first link that connects the second oligonucleotide and the third oligonucleotide.
- the first oligonucleotide is a 10 to 30 residue that is linked to the target-corresponding nucleotide residue corresponding to the adenosine residue in the target RNA and the 5'side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA. It consists of a base oligonucleotide and an oligonucleotide of 3 to 6 residues linked to the 3'side of the target-corresponding nucleotide residue and having a base sequence complementary to the target RNA.
- the number of residues of the second oligonucleotide is 5 to 8
- the number of residues of the third oligonucleotide is 5 to 8.
- At least one residue selected from the target-corresponding nucleotide residue and the counter region consisting of one residue on each of the 3'and 5'sides is a nucleotide residue other than the native ribonucleotide residue.
- the target editing guide RNA has a second oligonucleotide, a first link and a third oligonucleotide on the 3'side of the first oligonucleotide that identifies the target RNA, with a counter region other than the native ribonucleotide residue.
- a nucleotide residue By having a nucleotide residue, it is possible to induce excellent site-specific editing with excellent intracellular stability. This is, for example, in a state where ADAR, which catalyzes target editing, recognizes a double-stranded region consisting of a target RNA and a first oligonucleotide, does not form a double strand with the target RNA, and is free from the target RNA.
- the second oligonucleotide, first link and third oligonucleotide that may be present enhances its editing activity. That is, in the target editing guide RNA, the first oligonucleotide functions as a complementary region (antisense region; ASR) to the target RNA, and at least a part of the second oligonucleotide, the first link portion and the third oligonucleotide is edited. It is considered to function as an enhancement region, an ADAR coupling region (ADAR recruiting region; ARR), and the like.
- ASR antisense region
- the target editing guide RNA induces site-specific editing for the target RNA, for example, by recruiting ADAR, which catalyzes the target editing, to the target RNA.
- ADAR is an enzyme that converts adenosine residues in double-stranded RNA into inosine residues by a hydrolytic deamination reaction, and is widely present in mammalian cells. Since the inosine residue is similar in structure to the guanosine residue, it is translated as a guanosine residue when translating the RNA information, and as a result, the RNA information is edited. When such RNA editing occurs in the portion encoding an amino acid, amino acid substitution or the like occurs even though there is no DNA mutation on the genome. As ADAR in mammals, ADAR1, ADAR2 and ADAR3 having different genes are known.
- the target editing guide RNA enhances the target editing activity of at least ADAR1 or ADAR2.
- the ADAR existing in the cell can be recruited to the target RNA to induce site-specific editing on the target RNA.
- the nucleotide constituting the target editing guide RNA may be composed of any of a natural ribonucleotide (RNA), a natural deoxyribonucleotide (DNA), an RNA / DNA chimera, and a modified nucleotide which is a modified product thereof.
- the nucleotides that make up the target editing guide RNA can be linked to each other by phosphodiester bonds that bind to the hydroxyl groups of the sugar moiety of the nucleoside.
- the phosphoric acid diester bond can be formed by utilizing the 2'-hydroxyl group, the 3'-hydroxyl group, or the 5'-hydroxyl group of the sugar moiety.
- the nucleotides that make up the target editing guide RNA can form a natural 3'-5'phosphodiester bond.
- At least one of the nucleotides that make up the target editing guide RNA may be a modified nucleotide.
- Examples of the modified nucleotide include those with a modified sugar moiety, those with a modified phosphate diester bond, those with a modified base, and combinations thereof.
- modified sugar moieties include 2'-O-alkylated ribonucleotides of D-ribofuranose (eg, 2'-O-methylated, 2'-O-aminoethylated, 2'-O. -Propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, 2'-O-propargylation, etc.); Cross-linked ribonucleotides cross-linked between the 2'and 4'positions of D-ribofuranose (eg, 2'-O, 4'-C-ethyleneation, 2'-O, 4'-C-methyleneation, 2'-O, 4'-C-propyleneation, 2'-O, 4'-C-tetramethyleneation, 2'-O, 4'-C-pentamethyleneation, 2'-S, 4'-C -Methyleneation, 2'-deoxy-2'-
- Modified phosphate diester bonds include phosphorothioate bonds (including optically active substances derived from asymmetric phosphorus atoms), methylphosphonate bonds, methylthiophosphonate bonds, phosphologithioate bonds, phosphoromidate bonds, etc. Can be mentioned.
- Base-modified bases include halogenated; methylated, ethylened, propylated, isopropylized, cyclopropylated, butylated, isobutylated, s-butylated, t-butylated, cyclobutylated, etc. Alkylation of 1 to 6 or 1 to 4; hydroxylation; amination; deaminolation; demethylation and the like. Specifically, 5-methylation, 5-fluorolation, 5-bromolation, 5-iodolation, N4-methylation, etc. of cytosine; 5-demethylation (uracil), 5-fluorolation, 5-fluorolysis of thymine, etc. Bromomination, 5-iodolation, etc .; N6-methylation, 8-bromolation, etc. of adenine; N2-methylation, 8-bromoization, etc. of guanine;
- the first oligonucleotide contained in the target editing guide RNA identifies the target RNA.
- the target RNA is not particularly limited as long as it contains an adenosine residue to be edited, and may be either cellular RNA or viral RNA, and is usually an mRNA precursor or mRNA encoding a protein. Editing sites in the target RNA may be in untranslated regions, splice regions, exons, introns, or regions that affect the stability, structure, or function of RNA.
- the target RNA may also contain mutations to be modified or altered. Alternatively, the target RNA may have its sequence mutated to encode a phenotype different from the native form.
- the target RNA is preferably an RNA encoding a protein, and specifically, the encoded protein is used for signal transduction of serotonin receptor, glutamate receptor, membrane potential-dependent potassium channel, STAT3, NFkBIA, MAPK14 and the like. Examples include phosphorylated proteins involved.
- Target editing guide RNA can be applied, for example, to the treatment of hereditary diseases.
- the hereditary disease may be, for example, a disease that can be treated by converting an adenosine residue in the target RNA to an inosine residue.
- the hereditary disease may be, for example, a hereditary disease caused by a mutation in a gene from a guanosine residue to an adenosine residue.
- Hereditary diseases include, for example, cystic fibrosis, leukoderma, ⁇ -1 antitrypsin deficiency, Alzheimer's disease, muscular dystrophy lateral sclerosis, asthma, ⁇ -salasemia, CADASIL syndrome, Charcoe Marie Tooth.
- hereditary diseases include type I citrullinemia (ASS1), type II citrullinemia (SLC25A13), hemophilia (Factor V Leiden), primary hypercystinuria (AGXT), and distal type.
- Myopathy Cystic fibrosis: cyclic fibrosis (CFCFTR), Homocystinuria (CBS), Huller syndrome: Mucopolysaccharidosis (IDUA (SLC26A1)), Alexander's disease (GFAP), Retinal pigment degeneration (EYS), Phenylketonuria (PAH), hemochromatosis (HFE), Gilbert syndrome (UGT1A1) and the like can be mentioned.
- the names in parentheses are the target gene names.
- the first oligonucleotide is linked to the target-corresponding nucleotide residue corresponding to the adenosine residue to be edited in the target RNA and the 5'side of the target-corresponding nucleotide residue with respect to the corresponding base sequence of the target RNA. It is linked to the 5'side oligonucleotide of 10 to 30 residues having a complementary base sequence and the 3'side of the target corresponding nucleotide residue, and has a base sequence complementary to the corresponding base sequence of the target RNA. It consists of 3 to 6 residues of 3'side oligonucleotide.
- the oligonucleotides linked to the 5'side and the 3'side of the target-corresponding nucleotide residue form a double strand with the target RNA, thereby identifying the target RNA and the editing target site in the target RNA.
- the region consisting of the target-corresponding nucleotide residue, one residue on the 3'side, and one residue on the 5'side will be referred to as a counter region for convenience, and the region other than the counter region of the first oligonucleotide will be referred to.
- the target-corresponding nucleotide residue is a nucleotide residue corresponding to the adenosine residue to be edited, and is, for example, a cytidine residue, a uridine residue, an adenosine residue, or a derivative thereof.
- the target-corresponding nucleotide residue is preferably a base that does not form a base pair with the adenosine residue to be edited, more preferably a cytidine residue or a derivative thereof, and further preferably a cytidine residue.
- the base sequence of the oligonucleotide linked to the 5'side or 3'side of the target-corresponding nucleotide residue is a base sequence complementary to the corresponding base sequence of the target RNA.
- the number of oligonucleotide residues linked to the 5'side of the target-corresponding nucleotide residue is, for example, 10 or more, 12 or more, 14 or more, or 15 or more, and 26 or less, 20 or less, or from the viewpoint of specificity for the target RNA. It is 16 or less.
- the number of oligonucleotide residues linked to the 5'side of the target-corresponding nucleotide residue may be 10 or more and 20 or less, and 13 or more and 20 or less, or 13 or more and 15 or less, from the viewpoint of specificity for the target RNA. You can. Further, the number of oligonucleotide residues linked to the 3'side of the target-corresponding nucleotide residue may be 3 or 3 or 4 from the viewpoint of editing activity. The number of oligonucleotide residues linked to the 3'side of the target-corresponding nucleotide residue may be 4 to 6, 4 or 5, or 5 or 6 from the viewpoint of ADAR specificity.
- At least one, at least two, or all three residues selected from the counter region may be nucleotide residues other than native ribonucleotide (RNA) residues, preferably. , At least 1 residue, at least 2 residues, or all 3 residues are modified nucleotide residues.
- the modified nucleotide residue in the counter region may have at least one of the sugar moiety and the phosphodiester bond modified, at least the sugar moiety may be modified, at least the phosphate diester bond may be modified, or the sugar. Partial and phosphodiester bonds may be modified.
- the targeting nucleotide residue may be a cytidine residue having a phosphorothioate bond.
- each 1 residue on the 5'side or 3'side of the target-corresponding nucleotide residue may be modified with at least one of a sugar moiety and a phosphate diester bond, and at least the sugar moiety may be modified.
- At least the phosphodiester bond may be modified, or the sugar moiety and the phosphodiester bond may be modified.
- Modification of the sugar moiety in the counter region may be, for example, 2'-O-alkylation, 2'-deoxy-2'-fluorolation, or the like.
- oligonucleotide residues are modified with at least one of the sugar moiety and the phosphodiester bond. It may be, at least the sugar moiety may be modified, at least the phosphate diester bond may be modified, or the sugar moiety and the phosphodiester bond may be modified. Modifications of the sugar moiety in the non-counter region include, for example, 2'-O-alkylation, 2'-deoxy-2'-fluorocation, cross-linking between the 2'and 4'positions, 2'-deoxylation (DNA). ) Etc.
- the plurality of modified nucleotides may be arranged consecutively or separatedly.
- the first oligonucleotide may be composed of all nucleotide residues linked by a phosphorothioate bond.
- the oligonucleotides linked to the 5'side of the target-corresponding nucleotide residue of the first oligonucleotide are 2'-deoxy-2'-fluoronucleotide residue, 2'-O-alkylribonucleotide residue and crosslinked nucleotide residue. It may have a base sequence in which two kinds of modified nucleotide residues selected from the group consisting of are alternately linked. That is, the oligonucleotide linked to the 5'side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked.
- It may have, and may have a base sequence in which crosslinked nucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked, and may have a 2'-deoxy-2'-fluoronucleotide residue. It may have a base sequence in which crosslinked nucleotide residues are alternately linked.
- the two modified nucleotide residues used here have a base sequence in which they are alternately linked, the oligonucleotide of interest is linked (here, 5'side of the target-corresponding nucleotide residue of the first oligonucleotide).
- the two modified nucleotide residues have a base sequence in which the two modified nucleotide residues are partially alternately linked, or the entire oligonucleotide of interest has two modified nucleotide residues alternating with each other. It means both having a base sequence linked to, and is used in the same meaning in the following description of the present specification.
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide residue is 2'-deoxy-2'-. It may be a modified nucleotide residue selected from the group consisting of fluoronucleotide residues, 2'-O-alkylribonucleotide residues and crosslinked nucleotide residues.
- the third nucleotide residue counting in the 5'direction from the target-corresponding nucleotide residue is the 2'-deoxy-2'-fluoronucleotide residue.
- the third nucleotide residue counted in the 5'direction from the target-corresponding nucleotide residue is a nucleotide residue that does not include the target-corresponding nucleotide residue itself and is adjacent to the target-corresponding oligonucleotide residue in the 5'direction.
- the first is the third nucleotide residue counting in the 5'direction.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue may have a base sequence to which the 2'-O-alkylribonucleotide residue is linked.
- the oligonucleotide linked to the 3'side of the target-corresponding nucleotide residue is a 2'-deoxy-2'-fluoronucleotide residue, a 2'-O-alkylribonucleotide residue and cross-linking.
- Two types selected from the group consisting of type nucleotide residues may have a base sequence in which two types are alternately linked, and 2'-deoxy-2'-fluoronucleotide residues and cross-linked nucleotide residues are alternately linked. It may have a base sequence to be linked, and may have a base sequence in which 2'-O-alkylribonucleotide residues and crosslinked nucleotide residues are alternately linked.
- the first oligonucleotide has a 2'-deoxy-2'-fluoronucleotide residue, 2'-, which is the second nucleotide residue counted in the 3'direction from the target-corresponding nucleotide residue. It may be one selected from the group consisting of O-alkylribonucleotide residues and cross-linked nucleotide residues, and may be cross-linked nucleotide residues.
- the second nucleotide residue counted in the 3'direction from the target-corresponding nucleotide residue does not include the target-corresponding nucleotide residue itself, and 1 nucleotide residue is adjacent to the target-corresponding oligonucleotide residue in the 3'direction.
- the second nucleotide residue is shown as the second, counting in the 3'direction.
- the second oligonucleotide and the third oligonucleotide consist of 5 to 8 residues, 5 to 7 residues, or 6 residues, respectively, and have a base sequence capable of forming a complementary pair with each other.
- the number of residues of the second oligonucleotide and the third oligonucleotide may be the same or different.
- the base sequence of the second oligonucleotide includes GGGUGG, GGGUG, GGUGG, GGGU, GGUG, GUGG, GGG, GGU, GUG, UGG, GG, GC, GA, GU, UC, UG, UA, UU, CG, CA, Nucleotide sequences including CU, CC, AG, AA, AC, AU and the like can be mentioned.
- the second oligonucleotide also consists of a sequence consisting of two or three consecutive guanines (GG or GGG), a sequence consisting of successive uracils and guanines (UG) and a sequence consisting of consecutive guanines, uracils and guanines (GUG).
- the second oligonucleotide may also have a sequence of contiguous guanine, uracil and guanine (GUG) or a sequence of contiguous uracil, uracil and guanine (UGG) that binds to the first link. ..
- the base sequence of the third oligonucleotide may be selected so as to form a complementary pair with the base sequence of the second oligonucleotide.
- the fact that the second oligonucleotide and the third oligonucleotide can form a complementary pair means that all the residues of the second oligonucleotide are the Watson-click type base pair with all the residues of the corresponding third oligonucleotide. It means that the second oligonucleotide and the third oligonucleotide contain 1 or 2 mismatched base pairs and 1 or 2 fluctuating base pairs.
- the mismatched base pair means a thermodynamically unstable base pair
- the fluctuating base pair means a thermodynamically stable non-Watson-click type base pair such as GU base pair.
- Wobble base pairs may be formed, for example, at the 3'end of the third oligonucleotide.
- the second and third oligonucleotides may have at least one residue, at least three residues, or all residues, respectively, modified at least one of the sugar moiety and the phosphodiester bond, and at least the sugar moiety is modified. At least the phosphate diester bond may be modified, or the sugar moiety and the phosphodiester bond may be modified. Modification of the sugar moiety in the second and third oligonucleotides may be, for example, 2'-O-alkylation, 2'-deoxy-2'-fluorolation, 2'-deoxylation (DNA conversion) and the like. When the second or third oligonucleotide has a plurality of modified nucleotides, the plurality of modified nucleotides may be arranged consecutively or separatedly.
- the second and third oligonucleotides may have a base sequence in which 2'-O-alkylribonucleotide residues are linked.
- the nucleotide residues of the second and third oligonucleotides may all be linked by a phosphorothioate bond.
- the first linking portion may contain at least one selected from the group consisting of 4 or 5 residue oligonucleotides and polyalkyleneoxy groups consisting of 1 to 8 alkyleneoxy units.
- the first linking portion has a phosphate diester bond or a modified phosphate diester bond on the 3'side of the second oligonucleotide and the 5'side of the third oligonucleotide, respectively. That is, the first linking portion is a phosphate diester bond or a modified phosphate diester bond with the hydroxyl group of the sugar portion at the 3'end of the second oligonucleotide and the hydroxyl group of the sugar portion at the 5'end of the third oligonucleotide, respectively. May be combined.
- the second and third oligonucleotides may be configured to form complementary pairs to form a stem structure, and the first connecting portion to form a loop structure.
- the base sequence thereof is specifically, for example, GCUAA; UNCG fold type such as UCCG, UACG, UGCG, UCCG; GAAA, GUAA, GCAA, GGAA.
- GNRA fold type such as GAGA, GUGA, GCGA, GGGA; GGUG fold type; CUUG fold type; AGNN fold type such as AGUU, AGUC, AGUG, AGUA, AGCU, AGCC, AGCG, AGCA and the like.
- the base sequence of the first linking portion may have a GCUAA, UNCG fold type base sequence, or a GNRA fold type base sequence, and may have a UCCG base sequence.
- the first link portion contains 4 or 5 residues of oligonucleotide
- at least 1 residue or at least 3 residues of the nucleotide residues constituting the first link portion may be modified nucleotide residues.
- the modified nucleotide in the first link may have at least one of the sugar moiety and the phosphodiester bond modified, at least the sugar moiety may be modified, at least the phosphate diester bond may be modified, or the sugar. Partial and phosphodiester bonds may be modified.
- the carbon number of the alkyleneoxy unit may be, for example, 2 to 4, 2 to 3, or 2. That is, the alkyleneoxy unit may be an ethyleneoxy unit, a propyleneoxy unit, or a butyleneoxy unit.
- the number of alkyleneoxy units constituting the polyalkyleneoxy group may be, for example, 1 to 8, 5 to 7, or 6.
- Each alkyleneoxy unit constituting the polyalkyleneoxy group may be the same or different.
- the first connecting portion may contain a polyethyleneoxy group having 1 to 8 units, and may contain a hexaethyleneoxy group.
- the polyalkyleneoxy group is connected to the hydroxyl group of the sugar moiety at the 3'end of the second oligonucleotide via a phosphodiester bond or a modified phosphodiester bond. It may be linked to the hydroxyl group of the sugar moiety at the 5'end of the third oligonucleotide.
- the first linking portion may be composed of only an oligonucleotide, may be composed of a nucleotide and an alkyleneoxy unit, or may be composed of only an alkyleneoxy unit.
- the target editing guide RNA can exhibit excellent target editing activity even when the first link has a loop structure containing an alkyleneoxy unit.
- the target editing guide RNA may have a second link between the first oligonucleotide and the second oligonucleotide. That is, the first oligonucleotide and the second oligonucleotide may be linked at the second linking portion.
- the second connecting portion includes, for example, an alkyleneoxy unit.
- the alkylene portion of the alkyleneoxy unit may have 2 to 8 or 2 to 6 carbon atoms.
- the second connecting portion may contain 1 to 8 units or 1 to 6 units of alkyleneoxy units having 2 or 3 carbon atoms, and 1 to 8 units or 1 to 6 units of alkyleneoxy units having 2 or 3 carbon atoms.
- the second connecting portion may contain an alkyleneoxy group having 3 to 6 carbon atoms.
- the polyalkyleneoxy group constituting the second linking portion is the hydroxyl group of the sugar portion at the 3'end of the first oligonucleotide and 5 of the second oligonucleotide via a phosphate diester bond or a modified phosphate diester bond. 'It may be connected to the hydroxyl group of the sugar portion at the end.
- the target editing guide RNA can be referred to a known document (see, for example, Nucleic Acids Research, 12, 4539 (1984)) using a commercially available synthesizer (for example, model 392 by the phosphoramidite method manufactured by PerkinElmer). It can be synthesized according to the described method.
- a commercially available synthesizer for example, model 392 by the phosphoramidite method manufactured by PerkinElmer. It can be synthesized according to the described method.
- a commercially available reagent may be used, or a reagent appropriately synthesized according to a method described in a known document may be used.
- a phosphorothioate bond can be introduced by reacting a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride.
- a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride.
- Oligonucleotides may be used in the form of pharmaceutically acceptable salts.
- a “pharmaceutically acceptable salt” is a salt of an oligonucleotide. Such salts include alkali metal salts such as sodium salts, potassium salts and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts and nickel salts.
- Metal salts such as cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholinic salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine Salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N, N'-dibenzylethylenediamine salts, chloroprocine salts, prokine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylammonium salts, tris (hydroxy) Amine salts such as organic salts such as methyl) aminomethane salts; hydrohydrochlorides, hydrochlorides, hydrobromates, hydrohalogenates such as hydroiodide, nitrates,
- oligonucleotide and the pharmaceutically acceptable salt thereof may also exist as a solvate (for example, a hydrate), and such a solvate may be used.
- Oligonucleotides and pharmaceutically acceptable salts thereof may contain optically active substances derived from asymmetric phosphorus atoms, and the oligonucleotides of the present invention also include such optically active substances.
- optically active substances can be synthesized by known methods (eg, Org. Lett., 114,967 (2009), Bioorganic & Medical Chemistry Letters, 8,2359 (1998), etc.).
- the tablet is mixed with itself or an appropriate pharmaceutically acceptable excipient, diluent, etc. , Capsules, granules, powders, syrups, etc., or parenterally, such as injections, suppositories, patches, or external preparations.
- formulations include excipients (eg, sugar derivatives such as lactose, sucrose, grape sugar, mannitol, sorbitol; starch derivatives such as corn starch, bailesho starch, ⁇ -sulfate, dextrin; cellulose derivatives such as crystalline cellulose.
- excipients eg, sugar derivatives such as lactose, sucrose, grape sugar, mannitol, sorbitol
- starch derivatives such as corn starch, bailesho starch, ⁇ -sulfate, dextrin
- cellulose derivatives such as crystalline cellulose.
- Arabic gum dextran; organic excipients such as pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; calcium carbonate Carbonates such as; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, stearic acid; metal stearate such as calcium stearate, magnesium stearate; talc; colloidal silica Waxes such as bead wax, gay wax; boric acid; adipic acid; sulfate such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; sodium lauryl sulfate, lauryl sulfate such as magnesium lauryl sulfate : Silicas such as silicate silicate, silicate hydrate; the above-mentioned starch derivatives, etc.
- emulsifiers eg, colloidal clays such as bentonite, beagum; metal hydroxides such as magnesium hydroxide, aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate, calcium stearate; Cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters, etc., stabilizers (such as methylparaben, propylparaben) Paraoxybenzoic acid esters; alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; timeroser It is produced by a well-known method using additives such as le; dehydroacetic acid; sorbic acid, etc.), flavoring agents (for example, commonly used sweeteners, acidulants), sodium lauryl s
- the therapeutic agent containing the oligonucleotide, a pharmaceutically acceptable salt or solvate thereof preferably contains 0.1 to 250 ⁇ moles / ml of oligonucleotide, preferably 1 to 50 ⁇ moles / ml. Also, a predetermined amount of oligonucleotide, a pharmaceutically acceptable salt or solvate thereof, 0.02 to 10% w / v carbohydrate or polyhydric alcohol, and 0.01 to 0.4% w / v.
- the therapeutic agent may be composed of a pharmaceutically acceptable surfactant.
- carbohydrate at least one of a monosaccharide and a disaccharide is particularly preferable.
- these carbohydrates and polyhydric alcohols include glucose, galactose, mannose, lactose, maltose, mannitol and sorbitol. These may be used alone or in combination.
- surfactant examples include polyoxyethylene sorbitan mono to tri-ester, alkylphenyl polyoxyethylene, sodium taurocholate, sodium collate, and polyhydric alcohol ester. Of these, particularly preferred are polyoxyethylene sorbitan mono to triesters, and here, particularly preferred esters are oleate, laurate, stearate and palmitate. These may be used alone or in combination.
- Therapeutic agents containing oligonucleotides, pharmaceutically acceptable salts or solvates thereof are more preferably 0.03M to 0.09M pharmaceutically acceptable neutral salts such as sodium chloride, potassium chloride and the like. / Or may contain calcium chloride.
- Therapeutic agents containing oligonucleotides, pharmaceutically acceptable salts or solvates thereof can more preferably contain 0.002 to 0.05 M pharmaceutically acceptable buffering agents.
- preferred buffers include sodium citrate, sodium glycinate, sodium phosphate, tris (hydroxymethyl) aminomethane. These buffers may be used alone or in combination.
- the above-mentioned therapeutic agent may be supplied in a solution state.
- a lysate injection
- the therapeutic agent of the present invention also includes those in a lyophilized state for use after reconstitution with a solution so that each component has a predetermined concentration range.
- Amino acids such as albumin and glycine may be further contained for the purpose of promoting the solubility of the freeze-dried product.
- a pharmaceutically acceptable salt or solvate thereof is administered to a human, for example, about 0.01 mg / kg to 100 mg / kg (body weight), preferably 0.1 mg / kg / day for an adult.
- Subcutaneous injection, intravenous drip infusion, or intravenous injection may be performed at a dose of kg to 20 mg / kg (body weight) in one or several divided doses, but the dose and frequency of administration are determined by the type and symptoms of the disease. It can be changed as appropriate depending on the age and administration method.
- treatment method of a disease includes a step of administering the above-mentioned therapeutic agent to a subject.
- treatment may be any treatment given to the disease, such as treatment, improvement, suppression of progression (prevention of exacerbation), prevention, etc. (preferably treatment or prevention) of the disease. ).
- the subject of treatment may be a warm-blooded animal including humans, or a non-human warm-blooded animal.
- target RNA and target editing guide RNA which is an oligonucleotide that induces site-specific editing for target RNA, are combined in the presence of adenosine deaminase. Including the step of contacting. By partially forming a double strand of the target editing guide RNA with the target RNA and recruiting adenosine deaminase, the adenosine residue contained in the target RNA can be site-specifically converted into an inosine residue.
- the site-specific editing method of the target RNA may further include the step of preparing the target editing guide RNA.
- the site-specific editing method of the target RNA can be performed, for example, by introducing the above-mentioned target editing guide RNA into a eukaryotic cell having the target RNA.
- the target editing guide RNA can be appropriately selected and applied from various methods used in nucleic acid medicines as a method for introducing RNA into eukaryotic cells.
- the site-specific editing method for the target RNA may be performed in vitro or in vivo.
- the invention uses a target editing guide RNA in the manufacture of a pharmaceutical composition used in the treatment of a disease (eg, a hereditary disease), a target editing guide RNA in the treatment of a disease (eg, a hereditary disease). Also includes targeted editing guide RNAs used in the use of, the treatment of diseases (eg, hereditary diseases).
- the underlined part corresponds to the first oligonucleotide (ASR), and the target RNA is Rluc_sRNA, which will be described later. It is considered that the oligonucleotide of Reference Example 1 can have the following stem-loop structure, for example.
- Examples 1 to 50 The oligonucleotide compounds of Examples 1 to 46 were obtained by introducing modified nucleotides as shown below based on the oligonucleotide sequence of Reference Example 1. Further, the oligonucleotide compounds of Examples 47 to 50 have the first oligonucleotide (ASR) having the sequence shown below, and are obtained by introducing a modified nucleotide as shown below. These oligonucleotide compounds were synthesized by the phosphoramidite method in the same manner as in Reference Example 1. In addition, when synthesizing the part "18" in the sequence, DMT Hexaethylene Glycol phosphoramidite (ChemGene, catalog number: CLP-9765) was used.
- ASR first oligonucleotide
- the "DNA” portion was synthesized using the phosphoramidite form described in Nucleic Acids Research 11, 4538 (1984).
- the portion of "2'-O, 4'-C-methylene nucleoside” was synthesized using the phosphoramidite compound described in International Publication No. 99/14226.
- the part of "2'-deoxy-2'-fluoronucleoside” is J.I. Med. Chem. , 36, 831 (1993), was synthesized using the phosphoramidite compound.
- Molecular weight in the table indicates the measured value by negative ion ESI mass spectrometry.
- sequence in the "sequence" in the table, uppercase letters are RNA, lowercase letters are DNA, N (M) is 2'-O-methylation of D-ribofuranose, and N (F) is 2'-deoxy-2'of D-ribofuranose.
- N (L) is 2'-O, 4'-C-methyleneation of D-ribofuranose, N (E) is 2'-O, 4'-C-ethylene of D-ribofuranose Shows the change.
- Rluc_WT RNA after the editing reaction was purified by phenol / chloroform extraction and ethanol precipitation.
- cDNA was synthesized with Rluc_WT_BamR01 primer (SEQ ID NO: 8) using Primescript Reverse Transcriptase II (TaKaRa).
- PCR (30 cycles, denaturation: 98 ° C., 10 seconds, annealing: 55 ° C., 15 seconds, elongation) using Rluc_WT_EcoF01 primer (SEQ ID NO: 9), Rluc_WT_BamR01 primer (SEQ ID NO: 8), and PrimeStar GXL DNA Polymerase (TaKaRa).
- the obtained cDNA was cloned into the pUC19 plasmid as an insert DNA as follows.
- the insert DNA and pUC19 plasmid were digested with restriction enzymes at 37 ° C. for 1 hour using EcoRI (TaKaRa) and BamHI (TaKaRa), and then each DNA was purified by phenol / chloroform extraction and ethanol precipitation.
- the pUC19 plasmid after digestion with restriction enzymes and the insert DNA were mixed so as to have a molar ratio of 1: 3, and a ligation reaction was carried out using DNA Ligation Kit ⁇ Mighty Mix> (TaKaRa).
- the obtained ligation sample was transformed into DH5 ⁇ , cultured overnight at 37 ° C. on an LB agar plate, and then a plasmid was extracted using QIAprep Spin Miniprep Kit (QIAGEN).
- the nucleotide sequence of the obtained plasmid DNA was analyzed to obtain a sequence in which A122 of Rluc_WT was mutated to G (Rluc_K41R) and a plasmid DNA in which Rluc_K41R was cloned (pUC19-Rluc_K41R).
- the 5'side fragment is the Rluc_NheF01 primer (SEQ ID NO: 10) and the RL_W104X_RV primer (SEQ ID NO: 11), and the 3'side fragment is the Rluc_XhoR01 primer (SEQ ID NO: 12) and the RL_W104X_FW primer (SEQ ID NO: 13).
- PrimeStar GXL DNA Primerase TaKaRa
- each PCR product was diluted 100-fold, and 2nd PCR (30 cycles, denaturation: 98 ° C., 10 seconds, annealing: 55 ° C., 15 seconds) was performed by PrimeStar GXL DNA Polymerase (TaKaRa) using Rluc_NheF01 primer and Rluc_XhoR01 primer. , Extension: 68 ° C., 60 seconds) and purified by phenol / chloroform extraction and ethanol precipitation to obtain DNA (Rluc_K41R_W104X) (SEQ ID NO: 16) having a sequence in which G311 of Rluc_K41R was mutated to A.
- the obtained DNA (Rluc_K41R_W104X) and psiCHECK TM- 2 Vector (promega) were subjected to restriction enzyme digestion at 37 ° C. for 1 hour using NheI (TaKaRa) and XhoI (TaKaRa), followed by phenol / chloroform extraction and ethanol. Each DNA was purified by precipitation.
- Rluc_K41R_W104X and psiCHECK TM- 2 Vector after digestion with restriction enzymes were mixed so as to have a molar ratio of 3: 1 and a ligation reaction was carried out using DNA Ligation Kit ⁇ Mighty Mix> (TaKaRa).
- the obtained ligation sample was transformed into DH5 ⁇ and cultured overnight at 37 ° C. using an LB agar plate.
- the selected colonies were cultured overnight at 37 ° C. in LB liquid medium, and plasmids were extracted using QIAprep Spin Miniprep Kit (QIAGEN). Then, the sequence of the plasmid obtained by the nucleotide sequence analysis was confirmed.
- PCR 30 cycles (denaturation: 98 ° C., 10 seconds, annealing: 98 ° C., 10 seconds, annealing) using T7_Rluc_sRNA_F01 primer (SEQ ID NO: 14), Rluc_sRNA_R01 primer (SEQ ID NO: 15), and PrimeStar GXL DNA Polymerase (TaKaRa):
- T7_Rluc_sRNA_F01 primer SEQ ID NO: 14
- Rluc_sRNA_R01 primer SEQ ID NO: 15
- PrimeStar GXL DNA Polymerase TaKaRa
- Rluc_sRNA (SEQ ID NO: 17) was obtained by in vitro transcription using AmpliScribe T7 Kit (Epicenter Biotechnology). The synthesized Rluc_sRNA was cut out and purified using a 5% polyacrylamide gel containing 8M urea, and used for the subsequent tests.
- Rluc_WT_RNA The sequences of Rluc_WT_RNA, Rluc_K41R_W104X and Rluc_sRNA, and the sequences of the primers used above are shown below.
- the underline in the table indicates the adenosine residue of the editing target.
- the sequence of the obtained cDNA was subjected to a sequencing reaction using Rluc_sRNA_F01 primer (SEQ ID NO: 18) and Big Dye Terminator v3.1 Cycle Sequence Kit, and was analyzed by Applied Biosystem 3500 Genetic Species (Genetic Science). From the chromatographic chart obtained by sequencing, the editing ratio (%) was calculated from the peak height ratio (G / (G + A)) of the target site (A311).
- the editing-inducing activity was exhibited in the same manner as the compound of Reference Example 1.
- the compound of Reference Example 2 (del03_22) has a stem-loop sequence of Z-type DNA, and is adapted from the gRNA design method described in WO2016 / 097212 according to the Rluc_sRNA sequence. It can be seen that del03_22 has a weaker editing-inducing activity than the compound of the example (del03_21) having the same Z-type DNA stem sequence.
- the editing-inducing activity was similar to that of the compound of Reference Example 1.
- RNA was extracted from cells cultured in 24-well Plate using Sepazol RNA I Super G (Nakarai), subjected to DNase treatment using Recombinant DNase I (TaKaRa), and then phenol. / Purified by chloroform extraction and ethanol precipitation.
- a reverse transcriptase reaction was performed using PrimeScript II Reverse Transcriptase (TaKaRa), Total RNA 0.5 ⁇ g, and 0.25 ⁇ M Oligo (dT) 17 (SEQ ID NO: 19) to amplify the cDNA.
- 2nd PCR (30 cycles (denaturation: 98 ° C., 10 seconds) using PrimeStar GXL DNA polymerase (TaKaRa), Rluc_F01 primer (SEQ ID NO: 20), Rluc_R01 primer (SEQ ID NO: 22) using cDNA obtained by diluting 1st PCR product 200 times as a template.
- C Luciferase Reporter Assay Method
- a cell extract was obtained using 100 ⁇ L of Passive Lysis Buffer (Promega) on cells cultured on a 24-well plate. 100 ⁇ L of LARII was added to 20 ⁇ L of the obtained cell extract, and 60 seconds later, the luminescence intensity of Firefly luciferase (Fluc) was measured by GloMax (R) 20/20 Luminometer (Promega). Immediately after that, 100 ⁇ L of Stop & Glo Reagent was added, and 60 seconds later, the luminescence intensity of Renilla luciferase (Rluc) was measured. The emission intensity was normalized by Fluc.
- FIG. 6C shows the editing ratio (%) when the compound of Reference Example 1 (del03_01) and the compound of Example (del03_26) were used.
- the editing rate was about 20%.
- the compound of Reference Example 1 (del03_01) was transfected, no editing-inducing activity was observed.
- the compound of Example (del03_26) was transfected, high edit-inducing activity was observed.
- the results of the luciferase reporter assay using the compound of Reference Example 1 (del03_01) and the compound of Example (del03_26) are shown in FIG. 6D.
- the editing ratio (%) when the compounds of Examples (del03_26, del03_30 to del03_33) are used is shown in FIG. 7A.
- the compound of the example (del03_30 to del03_32) was transfected, the same level of editing-inducing activity as that of transfecting the compound of the above-mentioned example (del03_26) was observed.
- the results of the luciferase reporter assay using the compounds of Examples (del03_26, del03_30 to del03_33) are shown in FIG. 7B.
- the compounds of the examples (del03_30 to del03_32) were transfected, the same high luciferase activity as that of the above-mentioned compounds of the examples (del03_26) was observed.
- the editing ratio (%) when the compounds of Examples (del03_26, del03_34 to del03_41) are used is shown in FIG. 8A.
- the editing-inducing activity equal to or higher than that of the above-mentioned compound of the example (del03_26) was observed.
- the results of the luciferase reporter assay using the compounds of Examples (del03_26, del03_30 to del03_33) are shown in FIG. 8B.
- the luciferase activity was as high as that of the compound of the above-mentioned example (del03_26).
- the plasmid was added so as to have a molar ratio of 3: 1 and a ligation reaction was carried out using DNA Ligation Kit ⁇ Mighty Mix> (TaKaRa).
- the obtained ligation sample was transformed into DH5 ⁇ and cultured overnight at 37 ° C. using an LB agar plate.
- the selected colonies were cultured overnight at 37 ° C. in LB liquid medium, and plasmids were extracted using QIAprep Spin Miniprep Kit (QIAGEN).
- the obtained plasmid 100 ng was used in a Big Dye Terminator v3.1 Cycle Sequence Kit (Thermo Fisher Scientific), 0.165 ⁇ M pUC19_seqFW primer (SEQ ID NO: 25) and 0.165 ⁇ M pUC19_sequRV primer (SEQ ID NO: 25) and 0.165 ⁇ M pUC19_sequRV primer.
- sequence analysis was performed by Applied Biosystems 3500 Genetic Analyser (Thermo Fisher Scientific).
- PCR denaturation: 98 ° C, 10 seconds, annealing: 55 ° C, 15
- T7_pUC19_FW SEQ ID NO: 27
- M13_RV primer SEQ ID NO: 28
- PrimeStar GXL DNA Polymerase TaKaRa
- In vitro transcription was performed using AmpliScribe T7 Kit (Epicenter Biotechnology), and cut-out purification was performed using a 5% polyacrylamide gel containing 8M urea.
- Test Example 4 Evaluation of Editing Inducibility for RNA Having GAPDH Gene Sequence
- A The RNA synthesis operation was carried out in the same manner as the method described in Test Example 3 (A) RNA synthesis. However, the oligo DNA described below was used.
- gRNA Target Editing Guide RNA
- GFAP_del03 (SEQ ID NO: 38) is an annealing reaction (annealing buffer (10 mM Tri-HCl (10 mM Tri-HCl)) using oligo DNAs of GFAP_del03_RV (SEQ ID NO: 34) and T7proGGG (SEQ ID NO: 29).
- annealing buffer (10 mM Tri-HCl (10 mM Tri-HCl)
- oligo DNAs of GFAP_del03_RV (SEQ ID NO: 34)
- T7proGGG SEQ ID NO: 29
- 5'AS_GFAP_gRNA (SEQ ID NO: 37) using 5'AS_GFAP_FW (SEQ ID NO: 35), 5'AS_ADg_RV oligo DNA (SEQ ID NO: 36) and DNA polymerase I, Large (Klenow) Fragment (NEB) at 25 ° C. for 30 minutes.
- the reaction was carried out to prepare a template DNA. Then, it was purified by phenol / chloroform extraction and ethanol precipitation. In vitro transcription was performed using 1.0 ⁇ g of each template DNA and AmpliScribe T7 Kit (Epicenter Biotechnology), and excision and purification were performed using an 8% polyacrylamide gel containing 8M urea.
- Annealing reaction Annealing buffer (10 mM Tri-HCl (pH 7) was used to combine 0.3 ⁇ M Rluc_sRNA with 0.9 ⁇ M compounds (gRNA) of Examples and Reference Examples. .6), 150 mM NaCl), heated at 80 ° C. for 3 min and cooled to 25 ° C. over 15 min.
- Example 51 to 66 The oligonucleotide compounds of Examples 51 to 57 were obtained by introducing modified nucleotides as shown below based on the oligonucleotide sequence of Reference Example 1. Further, the oligonucleotide compounds of Examples 58 to 63 have first oligonucleotides (ASR) of various lengths corresponding to hGAPDH, and are obtained by introducing modified nucleotides as shown below. The oligonucleotide compounds of Examples 64 to 66 have a first oligonucleotide (ASR) having the sequence shown below and are obtained by introducing a modified nucleotide as shown below. These oligonucleotide compounds were synthesized by the phosphoramidite method in the same manner as in Examples 1 to 50.
- ASR first oligonucleotide
- Molecular weight in the table indicates the measured value by negative ion ESI mass spectrometry.
- sequence in the "sequence" in the table, uppercase letters are RNA, lowercase letters are DNA, N (M) is 2'-O-methylation of D-ribofuranose, and N (F) is 2'-deoxy-2'of D-ribofuranose.
- N (L) is 2'-O, 4'-C-methyleneation of D-ribofuranose, N (E) is 2'-O, 4'-C-ethylene of D-ribofuranose Shows the change.
- the 5'end and 3'end of the oligonucleotide are hydroxyl groups in which a hydrogen atom is bonded to an oxygen atom in the chemical formula.
- Test Example 7 Evaluation of editing inducibility for Rluc_sRNA (2) (A) Annealing reaction, in vitro editing reaction, and editing analysis method The same procedure as in Test Example 1 or Test Example 6 was carried out.
- hADAR2 recombinant hADAR2 was added to 5 nM Rluc_sRNA-gRNA complex so as to be 12.5 nM, and the mixture was incubated at 37 ° C. for 1 hour.
- hADAR1 p110 recombinant hADAR1 p110 was added to a 5 nM Rluc_sRNA-gRNA complex to 250 nM, and the mixture was incubated at 37 ° C. for 30 minutes.
- FIGS. 11A and 12A results of Editing Analysis
- FIGS. 11B and 12B results of Editing Analysis
- FIGS. 11A and 12A results of hADAR2 are shown in FIGS. 11A and 12A
- the results of hADAR1 p110 are shown in FIGS. 11B and 12B.
- FIG. 11A even when a modified nucleic acid was introduced as in the compounds of Examples (del03_32, 39, 42, 43, 44 and 45), the editing-inducing activity by ADAR2 was exhibited as in the compound of Reference Example 1.
- FIG. 11B even when a modified nucleic acid was introduced as in the compounds of Examples (del 03_32, 39, 43 and 45), the editing-inducing activity by ADAR1 p110 was shown as in the compound of Reference Example 1.
- FIG. 13B The results of the luciferase reporter assay using the compounds of Examples (del03_26, and del03_34 to del03_40) are shown in FIG. 13B.
- the compounds of the example del03_26 and del03_38 to del03_40
- a clearly higher luciferase activity was observed than when only the ADAR2, ADAR1 p110, or ADAR1 p150 plasmid was transfected.
- the compounds of Examples (del03_34 to del03_37) were used, a clearly higher luciferase activity was observed than when the ADAR2 plasmid alone was transfected.
- the editing ratio (%) when the compounds of Examples (del03_26 and del03_32, 39, 43 and 45) were used is shown in FIG. 14A.
- the editing rate was about 60%, which was clearly higher than when the plasmid expressing ADAR2 alone was transfected.
- the editing rate is about 10% to 40%, which is clearer than when only the ADAR1 p110 or ADAR1 p150 plasmid is transfected. it was high.
- FIG. 14B The results of the luciferase reporter assay using the compounds of Examples (del03_26 and del03_32, 39, 43 and 45) are shown in FIG. 14B.
- the compounds of Examples (del03_26 and del03_32, 39, 43 and 45) were used, a clearly higher luciferase activity was observed than when only the ADAR2, ADAR1 p110 or ADAR1 p150 plasmid was transfected.
- the editing ratio (%) when the compounds of Examples (del03_26 and del03_42, 44, 46, 47 and 48) were used is shown in FIG. 15A.
- the editing rate was about 30% to 60%, which was clearly higher than when the plasmid expressing ADAR2 alone was transfected.
- the editing rate was about 10% to 30%, and the rate was It was clearly higher than when only the ADAR1 p110 or p150 plasmid was transfected.
- FIG. 15B The results of the luciferase reporter assay using the compounds of Examples (del03_26 and del03_42, 44, 46, 47 and 48) are shown in FIG. 15B.
- the compounds of the Examples (del03_26, and del03_46 and 47) were used, significantly higher luciferase activity was observed than when only the ADAR2, ADAR1 p110, or p150 plasmids were transfected.
- a plasmid expressing ADAR1 p110 or ADAR1 p150 was transfected using the compounds of Examples (del03_42, 44 and 48)
- the ratio was that only the ADAR1 p110 or ADAR1 p150 plasmid was transfected.
- Test Example 9 Evaluation of editing inducibility for RNA having a GAPDH gene sequence (2)
- A RNA synthesis and evaluation of editing inducibility
- hADAR1 p110 described in Test Example 6 was used in addition to hADAR2.
- recombinant hADAR2 was added to 5 nM Rluc_sRNA-gRNA complex so as to be 12.5 nM, and the mixture was incubated at 37 ° C. for 1 hour.
- hADAR1 p110 recombinant hADAR1 p110 was added to the 5 nM Rluc_sRNA-gRNA complex to 250 nM, and the mixture was incubated at 37 ° C. for 30 minutes.
- RNA having a GAPDH gene sequence in cultured cells
- A Cell culture HEK293 cells were subcultured into a 24-well plate at 5.0 ⁇ 10 4 cells / well. The cells were cultured for 48 hours. Examples of plasmids expressing 500 ng of ADAR using Lipofectamine 3000 (Thermo) (pcDNA3.1 (-) Hygro_ADAR2, pcDNA3.1 (-) Hygro_ADAR1 p110, or pcDNA3.1 (-) Hygro_ADAR1 p150), 50 nM The example compound, gRNA, was Transfected. As a control, cultured cells not transfected with gRNA, which is a compound of Example or Reference Example, were used.
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Abstract
Description
(1-1)標的RNAを特定する第一オリゴヌクレオチドと、前記第一オリゴヌクレオチドの3’側に連結する第二オリゴヌクレオチドと、前記第二オリゴヌクレオチドと相補対を形成し得る第三オリゴヌクレオチドと、前記第二オリゴヌクレオチドと第三オリゴヌクレオチドとを連結する第一連結部とを含み、前記第一オリゴヌクレオチドは、前記標的RNA中のアデノシン残基に対応する標的対応ヌクレオチド残基と、前記標的対応ヌクレオチド残基の5’側に連結し、前記標的RNAに相補的な塩基配列を有する10から30残基のオリゴヌクレオチドと、前記標的対応ヌクレオチド残基の3’側に連結し、前記標的RNAに相補的な塩基配列を有する3から6残基のオリゴヌクレオチドとからなり、前記第二オリゴヌクレオチドの残基数は5から8であり、前記第三オリゴヌクレオチドの残基数は5から8であり、前記標的対応ヌクレオチド残基、並びにその3’側及び5’側の各1残基からなるカウンタ領域から選択される少なくとも1残基は、天然型リボヌクレオチド残基以外のヌクレオチド残基であり、前記標的RNAに対する部位特異的編集を誘導するオリゴヌクレオチド、又はその薬学的に許容される塩。
前記第一オリゴヌクレオチドの3’側に連結する第二オリゴヌクレオチドと、
前記第二オリゴヌクレオチドと相補対を形成し得る第三オリゴヌクレオチドと、
前記第二オリゴヌクレオチドと第三オリゴヌクレオチドとを連結する第一連結部とを含み、前記第一オリゴヌクレオチドは、前記標的RNA中のアデノシン残基に対応する標的対応ヌクレオチド残基と、前記標的対応ヌクレオチド残基の5’側に連結し、前記標的RNAに相補的な塩基配列を有する10から30残基のオリゴヌクレオチドと、前記標的対応ヌクレオチド残基の3’側に連結し、前記標的RNAに相補的な塩基配列を有する3から6残基のオリゴヌクレオチドとからなり、前記第二オリゴヌクレオチドの残基数は5から8であり、前記第三オリゴヌクレオチドの残基数は5から8であり、前記第二オリゴヌクレオチド及び第三オリゴヌクレオチドは、全て2’-O-アルキルリボヌクレオチドからなり、前記標的対応ヌクレオチド残基、並びにその3’側及び5’側の各1残基からなるカウンタ領域から選択される少なくとも1残基は、天然型リボヌクレオチド残基以外のヌクレオチド残基であり、全てのリン酸ジエステル結合がホスホロチオエート結合であり、前記標的RNAに対する部位特異的編集を誘導するオリゴヌクレオチド、又はその薬学的に許容される塩。
前記第一オリゴヌクレオチドの3’側に連結する第二オリゴヌクレオチドと、
前記第二オリゴヌクレオチドと相補対を形成し得る第三オリゴヌクレオチドと、
前記第二オリゴヌクレオチドと第三オリゴヌクレオチドとを連結する第一連結部とを含み、前記第一オリゴヌクレオチドは、前記標的RNA中のアデノシン残基に対応する標的対応ヌクレオチド残基と、前記標的対応ヌクレオチド残基の5’側に連結し、前記標的RNAに相補的な塩基配列を有する10から30残基のオリゴヌクレオチドと、前記標的対応ヌクレオチド残基の3’側に連結し、前記標的RNAに相補的な塩基配列を有する3から6残基のオリゴヌクレオチドとからなり、前記第二オリゴヌクレオチドの残基数は5から8であり、前記第三オリゴヌクレオチドの残基数は5から8であり、前記第二オリゴヌクレオチド及び第三オリゴヌクレオチドは、全て2’-O-アルキルリボヌクレオチドからなり、前記標的対応ヌクレオチド残基、並びにその3’側及び5’側の各1残基からなるカウンタ領域から選択される少なくとも1残基は、天然型リボヌクレオチド残基以外のヌクレオチド残基であり、全てのリン酸ジエステル結合の修飾がホスホロチオエート結合であり、前記標的RNAに対する部位特異的編集を誘導するオリゴヌクレオチド、又はその薬学的に許容される塩。
(4-51) 疾患が遺伝性疾患である(4-50)に記載の方法。
(4-52) 遺伝性疾患が、標的RNAにおけるアデノシン残基をイノシン残基に変換することにより治療されうる疾患である(4-51)に記載の方法。
(4-53) 遺伝性疾患が、遺伝子におけるグアノシン残基からアデノシン残基への変異を原因とする遺伝性疾患である(4-51)に記載の方法。
(4-54) 温血動物がヒトである(4-50)から(4-53)のいずれかに記載の方法。
D-リボフラノースの2’位と4’位間が架橋された架橋型リボヌクレオチド(例えば、2’-O,4’-C-エチレン化、2’-O,4’-C-メチレン化、2’-O,4’-C-プロピレン化、2’-O,4’-C-テトラメチレン化、2’-O,4’-C-ペンタメチレン化、2’-S,4’-C-メチレン化、D-リボフラノースの2’-デオキシ-2’-C,4’-C-メチレンオキシメチレン化体、S-cEt(2’,4’-constrained エチル)、AmNA等);
3’-デオキシ-3’-アミノ-2’-デオキシ-D-リボフラノース;3’-デオキシ-3’-アミノ-2’-デオキシ-2’-フルオロ-D-リボフラノース;2’-デオキシ-2’-フルオロ-D-リボフラノースなどを挙げることができる。
疾患の処置方法は、上記治療剤を対象に投与する工程を含む。明細書において用いられる「処置」とは、疾患について施される何らかの処置であればよく、例えば、疾患の治療、改善、進行の抑制(悪化の防止)、予防等(好適には、治療又は予防)が挙げられる。処置の対象は、ヒトを含む温血動物であってよく、非ヒト温血動物であってもよい。
標的RNAの部位特異的編集方法は、標的RNAと、標的RNAに対する部位特異的編集を誘導するオリゴヌクレオチドである標的編集ガイドRNAとを、アデノシンデアミナーゼの存在下に、接触させる工程を含む。標的編集ガイドRNAが標的RNAと部分的に二本鎖を形成し、アデノシンデアミナーゼをリクルートすることで、標的RNAが含むアデノシン残基を部位特異的にイノシン残基に変換することができる。標的RNAの部位特異的編集方法は、標的編集ガイドRNAを準備する工程を更に含んでいてもよい。
表1に示す配列を有し、すべてが天然型RNA残基からなるオリゴヌクレオチド(以下、del03_01ともいう)を、ホスホロアミダイト法(例えば、Nucleic Acids Research, 12, 4539 (1984)、Nature Communications 6, Article number: 6317 (2015)参照)を用いて合成を行った。得られた化合物は、負イオンESI質量分析により同定した(実測値:11194.40)。
実施例1から46のオリゴヌクレオチド化合物は参考例1のオリゴヌクレオチドの配列を元にして、以下に示すように修飾ヌクレオチドを導入して得られたものである。また実施例47から50のオリゴヌクレオチド化合物は、以下に示す配列の第一オリゴヌクレオチド(ASR)を有し、以下に示すように修飾ヌクレオチドを導入して得られたものである。これらのオリゴヌクレオチド化合物は参考例1と同様にホスホロアミダイト法で合成した。なお、配列中、「18」の部分を合成する際には、DMT Hexaethylene Glycol phosphoramidite(ChemGene、カタログ番号:CLP-9765)を用いた。「9」の部分を合成する際には、DMT-Triethoxy-glycol phosphoramidite(ChemGene、カタログ番号:CLP-1113)を用いた。「6」の部分を合成する際には、DMT-hexane-Diol phosphoramidite(ChemGene、カタログ番号:CLP-1120)を用いた。「3」の部分を合成する際には、DMT-propane-Diol phosphoramidite(ChemGene、カタログ番号:CLP-9908)を用いた。「2’-O-メチルヌクレオシド」の部分は、Nucleic Acids Research 17, 3373 (1989)に記載されたホスホロアミダイト体を用いて合成した。「DNA」の部分は、Nucleic Acids Research 11, 4539 (1984)に記載されたホスホロアミダイト体を用いて合成した。「2’-O,4’-C-メチレンヌクレオシド」の部分は、国際公開第99/14226号に記載されたホスホロアミダイト体を用いて合成した。「2’-デオキシ-2’-フルオロヌクレオシド」の部分は、J.Med.Chem.,36,831(1993)に記載されたホスホロアミダイト体を用いて合成した。
表5に、塩基配列と化学修飾の状態を示すオリゴヌクレオチド化合物を、参考例1と同様にホスホロアミダイト法で合成した。表中の記号は上記と同様である。
(A)Rluc_sRNAの合成
psiCHECKTM-2 Vector(プロメガ社)から、常法により転写して調製したRluc_WT RNA(配列番号7)を0.2nMになるように調製した。組み換えhADAR2(酵母発現系を用いて合成・精製 Macbeth, MR. and Bass, BL. Methods Enzymol. 424, 319-331 (2007), Fukuda, M. et al. Sci. Rep. srep41478 (2017))を終濃度1μMとなるように加え、editing reaction buffer(20mM HEPES-KOH(pH7.5),2mM MgCl2,100mM NaCl,0.5mM DTT,0.01% TritonX-100,5%glycerol)中において37℃で2時間インキュベートすることにより、in vitro編集反応を行なった。
0.3μMのRluc_sRNAと、0.9μMの実施例及び参考例の化合物(以下、gRNAと表記することがある)とをアニーリングbuffer(10mM Tri-HCl(pH7.6),150mM NaCl)中で、80℃,3min加熱し、15minかけて25℃まで冷却した。
アニーリング反応によりRluc_sRNAがgRNAと完全に複合体を形成したと仮定し、以下のRluc_sRNA-gRNA複合体濃度を計算した。編集反応は、editing reaction buffer(20mM HEPES-KOH(pH7.5),2mM MgCl2,100mM NaCl,0.5mM DTT,0.01% TritonX-100,5% glycerol)中、5nMRluc_sRNA-gRNA複合体に対して、組み換えhADAR2を12.5nM又は6.25nM加え、37℃で1時間インキュベートすることで行なった。
編集反応後のRluc_sRNAをフェノール/クロロホルム抽出、エタノール沈殿により精製し、Primescript Reverse Transcriptase II(TaKaRa)を用いて、Rluc_sRNA_R01プライマー(配列番号18)によりcDNAを合成した。その後、Rluc_sRNA_F01プライマー(配列番号18)及びRluc_sRNA_R01プライマー(配列番号15)、並びにPrimeStar GXL DNA Polymerase(TaKaRa)を用いてPCR(変性:98℃,10秒、アニーリング:55℃,15秒、伸長:68℃,20秒)により、cDNAを増幅した。得られたcDNAの配列解析はRluc_sRNA_F01プライマー(配列番号18)、Big Dye Terminator v3.1 Cycle Sequence Kitを用いてシーケンシング反応を行ない、Applied Biosystem 3500 Genetic Analyzer(Thermo Fisher Scientific)により解析した。シーケンシングにより得られたクロマトチャートより、標的部位(A311)のピーク高比(G/(G+A))から編集割合(%)を算出した。
参考例1の化合物及び実施例の化合物を用いた場合の編集割合(%)を図1A及び図1Bに示した。参考例1の化合物では、90%を超える編集割合を示した。実施例の化合物(del03_02からdel03_07)のように修飾核酸を導入した場合でも参考例1の化合物と同様に編集誘導活性を示した。また、標的編集部位と対になるCにホスホロチオエート結合を導入した化合物(del03_08)、ADAR誘導領域のループをヘキサエチエングルコールからなるリンカーに置換した化合物(del03_09)も、参考例1の化合物と同程度の編集誘導活性を示した。
また、参考例2の化合物(del03_22)は、Z型DNAのステムループ配列を持つものであってWO2016/097212に記載されているgRNAの設計方法をRluc_sRNA配列に合わせて適応したものである。del03_22は、同じZ型DNAのステム配列を持つ実施例の化合物(del03_21)と比較して編集誘導活性が弱いことがわかる。一方、実施例の化合物(del03_Am1からdel03_Am7)のように修飾核酸を導入した場合でも参考例1の化合物と同程度の編集誘導活性を示した。
(A)細胞培養
HeLa細胞を24-well Plateに5.0×105cells/wellとなるように継代し、48時間培養した。Lipofectamine 3000(Thermo)を用いて50ngのpsiCHECK2TM_Rluc_K41R_W104X、350ngのpcDNA3.1(-)Hygro_ADAR2、10nMの実施例又は参考例の化合物であるgRNAをTransfectionした。コントロールとして、gRNAを発現するプラスミド(特願2017-234341号の参考例2に記載)の100ngをTransfectionして用いた。
24-well Plateで培養した細胞から、セパゾール RNA I Super G(ナカライ)を用いてtotal RNAを抽出し、Recombinant DNase I(TaKaRa)を用いてDNase処理を行った後、フェノール/クロロホルム抽出、エタノール沈殿により精製した。PrimeScript II Reverse Transcriptase(TaKaRa)、Total RNA 0.5μg、0.25μM Oligo(dT)17(配列番号19)を用いた逆転写反応を行ない、cDNAを増幅した。PrimeStar GXL DNA polymerase(TaKaRa)、Rluc_F01プライマー(配列番号20)、3’-Adpプライマー(配列番号21)を用いて1stPCR(30サイクル(変性:98℃,10秒、アニーリング:55℃,15秒、伸長:68℃,60秒))を行った。1stPCR産物を200倍希釈したcDNAを鋳型に、PrimeStar GXL DNA polymerase(TaKaRa)、Rluc_F01プライマー(配列番号20)、Rluc_R01プライマー(配列番号22)を用いて2ndPCR(30サイクル(変性:98℃,10秒、アニーリング:55℃,15秒、伸長:68℃ 60秒))を行い、Rlucフラグメントを増幅した。Big Dye Terminator v3.1 Cycle Sequence Kit(Thermo Fisher Scientific)、0.165μM Rluc_sRNA_F01プライマー(配列番号18)を用いてシーケンシング反応を行ない、Applied Biosystems 3500 Genetic Analyzer(Thermo Fisher Scientific)により解析した。シーケンシングにより得られたクロマトチャートより、標的部位(A311)のピーク高比(G/(G+A))から編集割合(%)を算出した。
Dual-Luciferase Reporter Assay System(Promega)を使用した。24-well Plateで培養した細胞に100 μLのPassive Lysis Buffer(Promega)を用いて細胞抽出液を得た。得られた細胞抽出液20μLにLARII 100μLを添加し、60秒後、GloMax(R)20/20 Luminometer(Promega)によりFirefly luciferase(Fluc)の発光強度を測定した。その後すぐに、Stop&Glo Reagentを100μL添加し、60秒後にRenilla luciferase(Rluc)の発光強度を測定した。発光強度は、Flucによりノーマライズした。
実施例の化合物(del03_24からdel03_26)を用いた場合の編集割合(%)を図6Aに示した。プラスミドをトランスフェクトした場合では、10から20%程度の編集割合を示す。実施例の化合物(del03_25及びdel03_26)のようなホスホロチオエート結合の数を増加した修飾核酸をトランスフェクトした場合は、編集誘導活性が認められ、その割合は、プラスミドをトランスフェクトした場合よりも高かった。
(A)RNAの合成
ACTB_FW(配列番号23)、ACTB_RV(配列番号24)のオリゴDNA、及びNEBuffer 2(NEB)を用いてアニーリング反応(80℃,3min加熱し、15minかけて25℃まで冷却)を行ない、インサートを作製した。pUC19をEcoRI(TaKaRa)及びHindIII(TaKaRa)を用いて37℃で1時間反応を行なった後、フェノール/クロロホルム抽出、エタノール沈殿により精製した。インサート:プラスミドが3:1のモル比となるように加え、DNA Ligation Kit<Mighty Mix>(TaKaRa)を用いてライゲーション反応を行なった。得られたライゲーションサンプルをDH5αに形質転換し、LB寒天プレートを用いて37℃で一晩培養した。選別したコロニーを、LB液体培地を用いて37℃で一晩培養し、QIAprep Spin Miniprep Kit(QIAGEN)を用いてプラスミドを抽出した。得られたプラスミド100ngをBig Dye Terminator v3.1 Cycle Sequence Kit(Thermo Fisher Scientific)、0.165μMのpUC19_seqFWプライマー(配列番号25)及び0.165μMのpUC19_seqRVプライマー(配列番号26)を用いてシーケンシング反応を行ない、Applied Biosystems 3500 Genetic Analyzer(Thermo Fisher Scientific)により配列解析を行なった。正しく作製できたプラスミドを鋳型に、T7_pUC19_FW(配列番号27)及びM13_RVプライマー(配列番号28)、PrimeStar GXL DNA Polymerase(TaKaRa)を用いてPCR(変性:98℃,10秒、アニーリング:55℃,15秒、伸長:68℃,20秒)により、鋳型DNAを作製し、フェノール/クロロホルム抽出、エタノール沈殿により精製した。AmpliScribe T7 Kit(Epicentre Biotechnologies)を用いてin vitro転写を行ない、8M尿素を含む5%ポリアクリルアミドゲルを用いて切り出し精製を行なった。
実施例化合物(ACTB_26)の編集誘導能評価の方法は、試験例1の(B)-(D)と同様の方法を用いた。但し、プライマーは、以下に記載したものを用いた。実施例の化合物(ACTB_26)を用いた場合の編集割合(%)を図9A及び図9Bに示した。その結果、実施例の化合物(ACTB_26)は、50%程度の編集割合を示した。
(A)RNAの合成
操作は、試験例3の(A)RNAの合成に記載した方法と同様に行った。但し、オリゴDNAは、以下に記載したものを用いた。
実施例化合物(GAPDH_26)の編集誘導能評価の方法は、試験例3の「(B)編集誘導能評価とその結果」と同様の方法を用いた。実施例の化合物(GAPDH_26)を用いた場合の編集割合(%)を図9A及び図9Bに示した。その結果、実施例の化合物(GAPDH_26)は、90%程度の編集割合を示した。
(A)標的RNAの合成
操作は、試験例3の(A)RNAの合成に記載した方法と同様に行った。但し、オリゴDNAは、以下に記載したものを用いた。
GFAP_del03(配列番号38)は、GFAP_del03_RV(配列番号34)とT7proGGG(配列番号29)のオリゴDNAを用いてアニーリング反応(アニーリングbuffer(10mM Tri-HCl(pH7.6),150mM NaCl)中で、80℃,3min加熱し、15minかけて25℃まで冷却)を行なうことでin vitro転写の鋳型DNAを作製した。5’AS_GFAP_gRNA(配列番号37)は、5’AS_GFAP_FW(配列番号35)、5’AS_ADg_RVオリゴDNA(配列番号36)及びDNA Polymerase I,Large(Klenow)Fragment(NEB)を用いて25℃で30分反応を行ない、鋳型DNAを作製した。その後、フェノール/クロロホルム抽出、エタノール沈殿により精製した。それぞれの鋳型DNA 1.0μgとAmpliScribe T7 Kit(Epicentre Biotechnologies)を用いてin vitro転写を行ない、8M尿素を含む8%ポリアクリルアミドゲルを用いて切り出し精製を行った。
実施例化合物(GFAP_26)の編集誘導能評価の方法は、試験例3の「(B)編集誘導能評価とその結果」と同様の方法を用いて行った。
(A)アニーリング反応
0.3μMのRluc_sRNAと0.9μMの実施例及び参考例の化合物(gRNA)とをアニーリングbuffer(10mM Tri-HCl(pH7.6),150mM NaCl)中で、80℃、3min加熱し、15minかけて25℃まで冷却した。
アニーリング反応によりRluc_sRNAがgRNAと完全に複合体を形成したと仮定し、以下のRluc_sRNA-gRNA複合体濃度を計算した。編集反応は、editing reaction buffer(20mM HEPES-KOH(pH7.5),2mM MgCl2,100mM NaCl,0.5mM DTT,0.01% TritonX-100,5% glycerol)中、5nM Rluc_sRNA-gRNA複合体に対して、組み換えhADAR1 p110(酵母発現系を用いて合成・精製した。Macbeth,MR.and Bass,BL. Methods Enzymol. 424, 319-331 (2007), Fukuda, M. et al. Sci. Rep. srep41478 (2017)を参照)を終濃度250nMとなるように加え、37℃で30分インキュベートすることで行なった。
編集反応後のRluc_sRNAをフェノール/クロロホルム抽出、エタノール沈殿により精製し、Primescript Reverse Transcriptase II(TaKaRa)を用いて、Rluc_sRNA_R01プライマーによりcDNAを合成した。その後、Rluc_sRNA_F01プライマー(配列番号18)およびRluc_sRNA_R01プライマー(配列番号15)、PrimeStar GXL DNA Polymerase(TaKaRa)を用いてPCR(変性:98℃、10秒、アニーリング:55℃、15秒、伸長:68℃、20秒)により、cDNAを増幅した。得られたcDNAの配列解析はRluc_sRNA_F01プライマー(配列番号18)、Big Dye Terminator v3.1 Cycle Sequence Kitを用いてシーケンシング反応を行ない、Applied Biosystem 3500 Genetic Analyzerにより解析した。シーケンシングにより得られたクロマトチャートより、標的部位(A311)のピーク高比(G/(G+A))から編集割合(%)を算出した。
実施例の化合物(del03_21、del03_26及びdel03_32)を用いた場合の編集割合(%)を図10に示した。図中、ADg(-)は、gRNAを加えない場合を示す。実施例の化合物(del03_21)は、参考例1(del03_01)の化合物及び参考例2の化合物(del03_22)と同程度のhADAR1 p110による編集誘導活性を示した。また、実施例の化合物(del03_26及びdel03_32)のように修飾核酸を導入した場合でもhADAR1 p110による編集誘導活性を示した。
実施例51から57のオリゴヌクレオチド化合物は参考例1のオリゴヌクレオチドの配列を元にして、以下に示すように修飾ヌクレオチドを導入して得られたものである。また実施例58から63のオリゴヌクレオチド化合物は、hGAPDHに対応する種々の長さの第一オリゴヌクレオチド(ASR)を有し、以下に示すように修飾ヌクレオチドを導入して得られたものである。実施例64から66のオリゴヌクレオチド化合物は、以下に示す配列の第一オリゴヌクレオチド(ASR)を有し、以下に示すように修飾ヌクレオチドを導入して得られたものである。これらのオリゴヌクレオチド化合物は実施例1から50と同様にホスホロアミダイト法で合成した。
(A)アニーリング反応、in vitro編集反応、及び編集解析方法
試験例1又は試験例6と同様に行った。ここで、hADAR2の場合は、5nMのRluc_sRNA-gRNA複合体に対して、組み換えhADAR2を12.5nMになるように加え、37℃で1時間インキュベートすることで行なった。また、hADAR1 p110の場合は、5nMのRluc_sRNA-gRNA複合体に対して、組み換えhADAR1 p110を250nMになるように加え、37℃で30分インキュベートすることで行なった。
図11A及び図12AにhADAR2の結果を、図11B及び図12BにhADAR1 p110の結果を示す。図11Aに示すように、実施例の化合物(del03_32、39、42、43、44及び45)のように修飾核酸を導入した場合でも参考例1の化合物と同様にADAR2による編集誘導活性を示した。また、図11Bに示すように、実施例の化合物(del03_32、39、43及び45)のように修飾核酸を導入した場合でも参考例1の化合物と同様にADAR1 p110による編集誘導活性を示した。一方、実施例の化合物(del03_42及び44)のような修飾核酸を導入した場合は、参考例1の化合物と比較して、ADAR1 p110による編集誘導活性は、著しく低下した。上記のことから、実施例の化合物(del03_42及び44)は、ADAR2に選択的に作用することが明らかとなった。
(A)細胞培養
HeLa細胞を24-well Plateに5.0×105cells/wellとなるように継代し、48時間培養した。Lipofectamine 3000(Thermo)を用いて50ngのpsiCHECK2TM_Rluc_K41R_W104X、350ngのADARを発現するプラスミド(pcDNA3.1(-)Hygro_ADAR2、pcDNA3.1(-)Hygro_ADAR1 p110、または、pcDNA3.1(-)Hygro_ADAR1 p150)、20nMの実施例又は参考例の化合物であるgRNAをTransfectionした。コントロールとして、実施例又は参考例の化合物であるgRNAの代わりにpSuper_neoをTransfectionしたものを用いた 。
試験例2と同様に行った。
実施例の化合物(del03_26、及びdel03_34からdel03_40)を用いた場合の編集割合(%)を図13Aに示した。ADAR1 p110、またはADAR1 p150を発現するプラスミドをトランスフェクトした場合では、10%から40%程度の編集割合を示し、その割合は、ADAR1 p110、または、ADAR1 p150プラスミドのみをトランスフェクトした場合よりも明らかに高かった。
(A)RNAの合成、及び編集誘導能評価
hADAR2に加えて、試験例6に記載のhADAR1 p110を用いたこと以外は試験例4と同様に行った。ここで、hADAR2の場合は、5nMのRluc_sRNA-gRNA複合体に対して、組み換えhADAR2を12.5nMになるように加え、37℃で1時間インキュベートすることで行なった。また、hADAR1 p110の場合は、5nM Rluc_sRNA-gRNA複合体に対して、組み換えhADAR1 p110を250nMになるように加え、37℃で30分インキュベートすることで行なった。
実施例化合物(GAPDH_26、及びGAPDH_49から51)の編集誘導能評価の方法は、試験例4記載の(B)編集誘導能評価とその結果と同様の方法を用いた。実施例の化合物(GAPDH_26、及びGAPDH_49から51)を用いた場合の編集割合(%)を図16A及び図16Bに示した。その結果、実施例の化合物(GAPDH_26、及び、GAPDH_49から51)は、hADAR2、及び、hADAR1 p110を用いた場合において、高い編集割合を示した。
(A)細胞培養
HEK293細胞を24-well Plateに5.0×104cells/wellとなるように継代し、48時間培養した。Lipofectamine 3000(Thermo)を用いて500ngのADARを発現するプラスミド(pcDNA3.1(-)Hygro_ADAR2、pcDNA3.1(-)Hygro_ADAR1 p110、またはpcDNA3.1(-)Hygro_ADAR1 p150)、50nMの実施例又は参考例の化合物であるgRNAをTransfectionした。コントロールとして、実施例又は参考例の化合物であるgRNAをTransfectionしていない培養細胞を用いた。
実施例化合物(GAPDH_26、及びGAPDH_49から51)の編集誘導能評価の方法は、試験例4の(B)編集誘導能評価とその結果と同様の方法を用いた。実施例の化合物(GAPDH_26、及びGAPDH_49から51)を用いた場合の編集割合(%)を図16Cに示した。その結果、実施例の化合物(GAPDH_26、及び、GAPDH_49から51)は、hADAR2、hADAR1 p110、及びhADAR1 p150を用いた場合において、高い編集割合を示した。
Claims (34)
- 標的RNAを特定する第一オリゴヌクレオチドと、
前記第一オリゴヌクレオチドの3’側に連結する第二オリゴヌクレオチドと、
前記第二オリゴヌクレオチドと相補対を形成し得る第三オリゴヌクレオチドと、
前記第二オリゴヌクレオチドと第三オリゴヌクレオチドとを連結する第一連結部とを含み、
前記第一オリゴヌクレオチドは、前記標的RNA中のアデノシン残基に対応する標的対応ヌクレオチド残基と、
前記標的対応ヌクレオチド残基の5’側に連結し、前記標的RNAに相補的な塩基配列を有する10から30残基のオリゴヌクレオチドと、
前記標的対応ヌクレオチド残基の3’側に連結し、前記標的RNAに相補的な塩基配列を有する3から6残基のオリゴヌクレオチドとからなり、
前記第二オリゴヌクレオチドの残基数は5から8であり、
前記第三オリゴヌクレオチドの残基数は5から8であり、
前記標的対応ヌクレオチド残基、並びにその3’側及び5’側の各1残基からなるカウンタ領域から選択される少なくとも1残基は、天然型リボヌクレオチド残基以外のヌクレオチド残基であり、
前記標的RNAに対する部位特異的編集を誘導するオリゴヌクレオチド、又はその薬学的に許容される塩。 - 前記第一連結部は、4又は5残基のオリゴヌクレオチド、及び1から8のアルキレンオキシ単位からなるポリアルキレンオキシ基からなる群から選択される少なくとも1種を含む請求項1に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第一オリゴヌクレオチドと第二オリゴヌクレオチドの間に、アルキレンオキシ単位を含む第二連結部を含む請求項1又は2に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第一オリゴヌクレオチドは、ホスホロチオエート結合を含む請求項1から3のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第一オリゴヌクレオチドは、2’-O-アルキルリボヌクレオチド残基、2’-デオキシ-2’-フルオロリボヌクレオチド残基、架橋型ヌクレオチド残基、及び2’-デオキシリボヌクレオチドからなる群から選択される少なくとも1種の修飾ヌクレオチド残基を含む請求項1から4のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記カウンタ領域は、ホスホロチオエート結合を含む請求項1から5のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基が、ホスホロチオエート結合を含む請求項1から6のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基の3’側及び5’側の各1残基のうち少なくとも1残基は、2’-O-アルキルリボヌクレオチド残基、及び2’-デオキシ-2’-フルオロリボヌクレオチド残基からなる群から選択される少なくとも1種の修飾ヌクレオチド残基である請求項1から7のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第二オリゴヌクレオチド及び第三オリゴヌクレオチドの少なくとも一方は、2’-O-アルキルリボヌクレオチド残基、2’-デオキシ-2’-フルオロリボヌクレオチド残基、及び2’-デオキシリボヌクレオチド残基からなる群から選択される少なくとも1種の修飾ヌクレオチド残基を含む請求項1から8のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基の5’側に連結するオリゴヌクレオチドが、2’-デオキシ-2’-フルオロヌクレオチド残基と2’-O-アルキルリボヌクレオチド残基とが交互に連結する塩基配列を有する請求項1から9のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基の5’側に連結するオリゴヌクレオチドにおいて、前記標的対応ヌクレオチドから5’方向に数えて3番目のヌクレオチド残基が2’-デオキシ-2’-フルオロヌクレオチド残基である請求項10に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基の5’側に連結するオリゴヌクレオチドが、架橋型ヌクレオチド残基と2’-O-アルキルリボヌクレオチド残基とが交互に連結する塩基配列を有する請求項1から9のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基の5’側に連結するオリゴヌクレオチドにおいて、前記標的対応ヌクレオチドから5’方向に数えて3番目のヌクレオチド残基が架橋型ヌクレオチド残基である請求項12に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基の5’側に連結するオリゴヌクレオチドが、2’-デオキシ-2’-フルオロヌクレオチド残基と架橋型ヌクレオチド残基とが交互に連結する塩基配列を有する請求項1から9のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記標的対応ヌクレオチド残基の5’側に連結するオリゴヌクレオチドにおいて、前記標的対応ヌクレオチドから5’方向に数えて3番目のヌクレオチド残基が2’-デオキシ-2’-フルオロヌクレオチド残基である請求項14に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第一オリゴヌクレオチドにおいて、前記標的対応ヌクレオチド残基の3’側に連結するオリゴヌクレオチドが、2’-O-アルキルリボヌクレオチド残基が連結する塩基配列を有する請求項1から15のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第一オリゴヌクレオチドにおいて、前記標的対応ヌクレオチド残基の3’側に連結するオリゴヌクレオチドが、2’-O-アルキルリボヌクレオチド残基と架橋型ヌクレオチド残基とが交互に連結する塩基配列を有する請求項1から15のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第一オリゴヌクレオチドにおいて、前記標的対応ヌクレオチド残基の3’側に連結するオリゴヌクレオチドが、4から6残基からなる請求項1から17のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩
- 前記第二オリゴヌクレオチド及び第三オリゴヌクレオチドが、2’-O-アルキルリボヌクレオチド残基が連結する塩基配列を有する請求項1から18のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記第一オリゴヌクレオチド、第二オリゴヌクレオチド及び第三オリゴヌクレオチドのそれぞれは、ヌクレオチド残基がホスホロチオエート結合で連結されてなる請求項1から19のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 前記部位特異的編集は、アデノシンデアミナーゼによる酵素反応に起因する請求項1から20のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 請求項1から21のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩を含有する医薬。
- 請求項1から21のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩を含有する遺伝性疾患治療剤。
- 請求項1から21のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩を活性成分として含有する医薬組成物。
- 遺伝性疾患の予防、又は治療のための請求項24に記載の医薬組成物。
- 遺伝性疾患が、標的RNAにおけるアデノシン残基をイノシン残基に変換することにより治療されうる疾患である請求項25に記載の医薬組成物。
- 遺伝性疾患が、遺伝子におけるグアノシン残基からアデノシン残基への変異を原因とする遺伝性疾患である請求項25に記載の医薬組成物。
- 疾患の予防、又は治療のための医薬を製造するための請求項1から21のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩の使用。
- 疾患の予防、又は治療における使用のための請求項1から請求項21のいずれか1項に記載のオリゴヌクレオチド、又はその薬学的に許容される塩。
- 請求項1から請求項29のいずれか1項に記載のオリゴヌクレオチド、又はその薬理上許容される塩の薬理学的有効量を温血動物に投与することによる疾患の予防、又は治療のための方法。
- 疾患が遺伝性疾患である請求項30に記載の方法。
- 遺伝性疾患が、標的RNAにおけるアデノシン残基をイノシン残基に変換することにより治療されうる疾患である請求項31に記載の方法。
- 遺伝性疾患が、遺伝子におけるグアノシン残基からアデノシン残基への変異を原因とする遺伝性疾患である請求項31に記載の方法。
- 温血動物がヒトである請求項30から請求項33のいずれか1項に記載の方法。
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EP (1) | EP3981436A4 (ja) |
JP (1) | JPWO2020246560A1 (ja) |
KR (1) | KR20220016876A (ja) |
CN (1) | CN113939592A (ja) |
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CA (1) | CA3140438A1 (ja) |
IL (1) | IL288633A (ja) |
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WO2021182474A1 (ja) * | 2020-03-12 | 2021-09-16 | 株式会社Frest | オリゴヌクレオチド及び標的rnaの部位特異的編集方法 |
WO2023152371A1 (en) | 2022-02-14 | 2023-08-17 | Proqr Therapeutics Ii B.V. | Guide oligonucleotides for nucleic acid editing in the treatment of hypercholesterolemia |
US11827880B2 (en) | 2019-12-02 | 2023-11-28 | Shape Therapeutics Inc. | Therapeutic editing |
WO2024013360A1 (en) | 2022-07-15 | 2024-01-18 | Proqr Therapeutics Ii B.V. | Chemically modified oligonucleotides for adar-mediated rna editing |
WO2024013361A1 (en) | 2022-07-15 | 2024-01-18 | Proqr Therapeutics Ii B.V. | Oligonucleotides for adar-mediated rna editing and use thereof |
WO2024084048A1 (en) | 2022-10-21 | 2024-04-25 | Proqr Therapeutics Ii B.V. | Heteroduplex rna editing oligonucleotide complexes |
WO2024110565A1 (en) | 2022-11-24 | 2024-05-30 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of hereditary hfe-hemochromatosis |
WO2024115635A1 (en) | 2022-12-01 | 2024-06-06 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of aldehyde dehydrogenase 2 deficiency |
WO2024121373A1 (en) | 2022-12-09 | 2024-06-13 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of cardiovascular disease |
US12018257B2 (en) | 2016-06-22 | 2024-06-25 | Proqr Therapeutics Ii B.V. | Single-stranded RNA-editing oligonucleotides |
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WO2024114908A1 (en) * | 2022-11-30 | 2024-06-06 | Eberhard Karls Universität Tübingen | Chemically modified antisense oligonucleotides (asos) and compositions comprising the same for rna editing |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998054198A1 (en) | 1997-05-30 | 1998-12-03 | Hybridon, Inc. | Novel sulfur transfer reagents for oligonucleotide synthesis |
WO1999014226A2 (en) | 1997-09-12 | 1999-03-25 | Exiqon A/S | Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues |
WO2016097212A1 (en) | 2014-12-17 | 2016-06-23 | Proqr Therapeutics Ii B.V. | Targeted rna editing |
WO2017010556A1 (ja) | 2015-07-14 | 2017-01-19 | 学校法人福岡大学 | 部位特異的rna変異導入方法およびそれに使用する標的編集ガイドrnaならびに標的rna-標的編集ガイドrna複合体 |
WO2019111957A1 (ja) * | 2017-12-06 | 2019-06-13 | 学校法人福岡大学 | オリゴヌクレオチド、その製造方法及び標的rnaの部位特異的編集方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3475424A1 (en) * | 2016-06-22 | 2019-05-01 | ProQR Therapeutics II B.V. | Single-stranded rna-editing oligonucleotides |
DK3507366T3 (da) * | 2016-09-01 | 2020-11-02 | Proqr Therapeutics Ii Bv | Kemisk modificeret, enkeltstrengede rna-modificerende oligonukleotider |
-
2020
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- 2020-06-04 JP JP2021524908A patent/JPWO2020246560A1/ja active Pending
- 2020-06-04 US US17/616,430 patent/US20230061732A1/en active Pending
- 2020-06-04 BR BR112021024373A patent/BR112021024373A2/pt unknown
- 2020-06-04 AU AU2020287787A patent/AU2020287787A1/en active Pending
- 2020-06-04 WO PCT/JP2020/022200 patent/WO2020246560A1/ja unknown
- 2020-06-04 CN CN202080041457.0A patent/CN113939592A/zh active Pending
-
2021
- 2021-12-02 IL IL288633A patent/IL288633A/en unknown
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998054198A1 (en) | 1997-05-30 | 1998-12-03 | Hybridon, Inc. | Novel sulfur transfer reagents for oligonucleotide synthesis |
WO1999014226A2 (en) | 1997-09-12 | 1999-03-25 | Exiqon A/S | Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues |
WO2016097212A1 (en) | 2014-12-17 | 2016-06-23 | Proqr Therapeutics Ii B.V. | Targeted rna editing |
JP2017537618A (ja) * | 2014-12-17 | 2017-12-21 | プロキューアール・セラピューティクス・セカンド・ベスローテン・フェンノートシャップProQR Therapeutics II B.V. | 標的化rna編集 |
WO2017010556A1 (ja) | 2015-07-14 | 2017-01-19 | 学校法人福岡大学 | 部位特異的rna変異導入方法およびそれに使用する標的編集ガイドrnaならびに標的rna-標的編集ガイドrna複合体 |
WO2019111957A1 (ja) * | 2017-12-06 | 2019-06-13 | 学校法人福岡大学 | オリゴヌクレオチド、その製造方法及び標的rnaの部位特異的編集方法 |
Non-Patent Citations (23)
Title |
---|
BIOORGANIC & MEDICAL CHEMISTRY LETTERS, vol. 8, 1998, pages 2359 |
BIOPHYS. J., vol. 113, 2017, pages 257 - 267 |
FUKUDA MASATORA, UMENO HIROMITSU, NOSE KANAKO, NISHITARUMIZU AZUSA, NOGUCHI RYOMA, NAKAGAWA HIROYUKI: "Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing", SCI. REP., vol. 7, no. 1, 2 February 2017 (2017-02-02), pages 1 - 13, XP055770188 * |
FUKUDA, M. ET AL., SCI. REP., 2017 |
FUUKUDA MASATORA: "Development of RNA editing technology using RNA modification mechanism", EXPERIMENTAL MEDICINE TNA RNA, vol. 36, no. 19, 2018, pages 3246 - 3251, XP009521250 * |
HIDAKA KOTA : "2P-0634 (1PW-19-6) Development of A-to-I RNA editing technology using the short chain guide RNA", 42ND ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN, DECEMBER 3-6, 2019; FUKUOKA, vol. 42, 19 November 2019 (2019-11-19), JP, pages 2P-0634, XP009532457 * |
HIDAKA KOTA; KANAKO NOSE; YOKEI TOMITA, MASATORA FUKUDA: "P-18: Design and functional evaluation of the short-type quide RNA for site-directed A-to-I RNA editing", ABSTRACTS OF THE 21ST ANNUAL MEETING OF THE RNA SOCIETY OF JAPAN; JULY 17-19, 2019, 17 July 2019 (2019-07-17), JP, pages 79, XP009532460 * |
J. AM. CHEM. SOC., vol. 112, 1990, pages 1253 |
J. MED. CHEM., vol. 36, 1993, pages 831 |
MACBETH, MR.BASS, BL., METHODS ENZYMOL., vol. 424, 2007, pages 319 - 331 |
MACBETH, MRBASS, BL: "synthesized and purified by using a yeast expression system", METHODS ENZYMOL., vol. 424, 2007, pages 319 - 331 |
MASATORA FUKUDA; KANAKO NOSE: "The Latest genetic modification and regulation technology based on RNA editing", RNA EXPERIMENTAL MEDICINE, vol. 36, no. 9, 1 June 2018 (2018-06-01), pages 1522 - 1528, XP009521262 * |
NATURE BIOTECHNOLOGY, vol. 37, 2019, pages 133 - 138 |
NATURE COMMUNICATIONS, vol. 6, no. 6317, 2015 |
NOSE KANAKO ET AL.: "Development of nucleic acid drug based on RNA editing", JOURNAL OF THE NUCLEIC ACIDS THERAPEUTICS SOCIETY OF JAPAN, vol. 23, no. 1, 31 May 2019 (2019-05-31), JP, pages 32 - 39, XP009532462 * |
NOSE KANAKO, KOTA HIDAKA, YOHEI TOMIA, MASATORY FUKUDA: "P-050: Site-directed A-to-I RNA editing by using the short types of functional RNA", LECTURE ABSTRACT OF THE 5TH ANNUAL MEETING OF THE NUCLEIC ACIDS THERAPEUTICS SOCIETY OF JAPAN, vol. 5, 1 July 2019 (2019-07-01), JP, pages 130, XP009532461 * |
NUCLEIC ACIDS RESEARCH, vol. 11, 1984, pages 4539 |
NUCLEIC ACIDS RESEARCH, vol. 17, 1989, pages 3373 |
ORG. LETT., vol. 114, 2009, pages 967 |
See also references of EP3981436A4 |
STAFFORST, T. ET AL.: "An RNA-Deaminase Conjugate Selectively Repairs Point Mutations", ANGEW. CHEM. INT. ED., vol. 51, no. 44, 2012, pages 11166 - 11169, XP002765253 * |
TETRAHEDRON LETTERS, vol. 32, 1991, pages 3005 |
VOGEL, P. ET AL.: "Improving site-directed RNA editing in vitro and in cell culture by chemical modification of the guideRNA", ANGEW. CHEM. INT. ED., vol. 53, 2014, pages 6267 - 6271, XP002765255 * |
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US12018257B2 (en) | 2016-06-22 | 2024-06-25 | Proqr Therapeutics Ii B.V. | Single-stranded RNA-editing oligonucleotides |
US11827880B2 (en) | 2019-12-02 | 2023-11-28 | Shape Therapeutics Inc. | Therapeutic editing |
WO2021182474A1 (ja) * | 2020-03-12 | 2021-09-16 | 株式会社Frest | オリゴヌクレオチド及び標的rnaの部位特異的編集方法 |
WO2023152371A1 (en) | 2022-02-14 | 2023-08-17 | Proqr Therapeutics Ii B.V. | Guide oligonucleotides for nucleic acid editing in the treatment of hypercholesterolemia |
WO2024013360A1 (en) | 2022-07-15 | 2024-01-18 | Proqr Therapeutics Ii B.V. | Chemically modified oligonucleotides for adar-mediated rna editing |
WO2024013361A1 (en) | 2022-07-15 | 2024-01-18 | Proqr Therapeutics Ii B.V. | Oligonucleotides for adar-mediated rna editing and use thereof |
WO2024084048A1 (en) | 2022-10-21 | 2024-04-25 | Proqr Therapeutics Ii B.V. | Heteroduplex rna editing oligonucleotide complexes |
WO2024110565A1 (en) | 2022-11-24 | 2024-05-30 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of hereditary hfe-hemochromatosis |
WO2024115635A1 (en) | 2022-12-01 | 2024-06-06 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of aldehyde dehydrogenase 2 deficiency |
WO2024121373A1 (en) | 2022-12-09 | 2024-06-13 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of cardiovascular disease |
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CA3140438A1 (en) | 2020-12-10 |
US20230061732A1 (en) | 2023-03-02 |
EP3981436A1 (en) | 2022-04-13 |
BR112021024373A2 (pt) | 2022-01-18 |
CN113939592A (zh) | 2022-01-14 |
KR20220016876A (ko) | 2022-02-10 |
AU2020287787A1 (en) | 2022-01-20 |
TW202113076A (zh) | 2021-04-01 |
JPWO2020246560A1 (ja) | 2020-12-10 |
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IL288633A (en) | 2022-02-01 |
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