JP2024035693A - ALDH2 activity-enhancing nucleic acid molecules and their uses - Google Patents

Abstract

[Problem] A nucleic acid molecule that increases the proportion of wild-type ALDH2 and improves ALDH2 activity by specifically knocking down a single nucleotide polymorphism, and a nucleic acid molecule containing the nucleic acid molecule for treating diseases related to ALDH2. A pharmaceutical composition is provided. [Solution] A nucleotide sequence complementary to a nucleotide sequence of 15 or more consecutive nucleotides in a target sequence of 25 or less consecutive nucleotides in a region indicated by a specific nucleotide sequence in the mutant ALDH2 gene is A nucleic acid molecule that contains a gene expression suppressing sequence. [Selection diagram] None

Description

The present invention provides a nucleic acid molecule that increases the proportion of wild-type ALDH2 and improves ALDH2 activity by specifically knocking down a single nucleotide polymorphism, and a nucleic acid molecule that includes the nucleic acid molecule and is used for treating diseases related to ALDH2. The present invention relates to a pharmaceutical composition of the invention.

ALDH2 has a dimer as its active unit, and exists in the body in the form of a tetramer in which two active units are coordinated. Although there are many genetic polymorphisms in ALDH2, only the rs671 mutant exhibits significant phenotypic differences. There are three genotypes of the ALDH2 gene: wild type homozygote, heterozygote, and mutant homozygote. The ALDH2 activity of the wild type homozygote is 100%, and the ALDH2 activity of the mutant homozygote is 100%. However, in the case of a heterozygote, it has been revealed that the activity is not 50% but theoretically decreases to about 15.6%. Since the rs671 mutant is a mutation in an amino acid involved in crosslinking of the dimer, which is the active unit, the activity disappears if even one of the rs671 mutants is contained in the dimer. Therefore, if one rs671 mutant exists in the tetramer, the activity in the tetramer decreases by up to 50%. Furthermore, when two rs671 mutants exist in a tetramer, the activity of the tetramer is completely abolished when each activity unit contains one rs671 mutant. Therefore, in a heterozygote, all combinations of tetramers will theoretically decrease to about 15.6%, taking into account enzyme activity, appearance frequency, and half-life. Although there are ALDH2 activators using low molecular weight compounds, there have been no reports of improved activity due to the RNAi effect.

The purpose of the present invention is to selectively cleave the mRNA of mutant ALDH2 by RNAi effect, increase the proportion of wild-type ALDH2, improve ALDH2 activity, and reduce formaldehyde By improving metabolic abnormalities, and by this action, a therapeutic and/or prophylactic agent for diseases related to ALDH2, such as ADD (Aldehyde degradation deficiency)/AMeD (Aplastic anemia, mental retardation, and dwarfism) syndrome and Fanconi anemia. It is to provide.

As a result of intensive research to achieve the above object, the present inventors discovered a nucleic acid having a gene sequence that specifically knocks down a single nucleotide polymorphism variant that causes attenuation of ALDH2 activity, and has developed the present invention. It was completed.

That is, the present invention is as follows.
[1] A nucleotide sequence that is complementary to a nucleotide sequence of 15 or more consecutive nucleotides in a target sequence of 25 or less consecutive nucleotides in the region indicated by the nucleotide sequence represented by SEQ ID NO: 1 in the mutant ALDH2 gene. , a nucleic acid molecule comprising a mutant ALDH2 gene expression suppressing sequence.
[2] The expression suppressing sequence is
(a) 15 or more consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 2n (n is an integer selected from 1 to 11) (in this sequence, each U may be T) array,
(b) A nucleotide sequence in which one or two nucleotides have been deleted, substituted, inserted, or added in the nucleotide sequence of (a), or (c) 90% or more identical to the nucleotide sequence of (a). is a nucleotide sequence having
The nucleic acid molecule according to [1], preferably having the nucleotide sequence of (a).
[3] The nucleic acid molecule according to [1] or [2], further comprising a nucleotide sequence complementary to the expression suppressing sequence.
[4] The complementary nucleotide sequence is
(d) Nucleotide sequence represented by SEQ ID NO: 2n+1 (n is the same as in (a) above) (however, the pairing of G and U is considered complementary),
(e) A nucleotide sequence in which one or two nucleotides have been deleted, substituted, inserted, or added in the nucleotide sequence of (d), or (f) 90% or more identical to the nucleotide sequence of (d). is a nucleotide sequence having
The nucleic acid molecule according to [3], preferably having the nucleotide sequence of (d).
[5] The nucleic acid molecule according to [3] or [4], comprising the nucleotide sequence represented by SEQ ID NO: 2n (n is an integer selected from 1 to 11) and the nucleotide sequence represented by SEQ ID NO: 2n+1. .
[6] The nucleic acid molecule according to any one of [3] to [5], which is siRNA against the mutant ALDH2 gene.
[7] The nucleic acid molecule according to [6], wherein the siRNA has a 3'-overhang on one or both strands.
[8] The nucleic acid according to [7], consisting of a nucleotide sequence represented by SEQ ID NO: 2m (m is an integer selected from 12 to 45) and a nucleotide sequence represented by SEQ ID NO: 2m+1 annealed to the sequence. molecule.
[9] The nucleic acid molecule according to [8], which has the structure of any one of the following (1) to (34).

[10] The following general formula:

[wherein, X, Y, X ₁ , Y ₁ , X ₂ , Y ₂ are each independently an optionally modified ribonucleotide residue or an optionally modified deoxyribonucleotide residue;
Z is a linker that connects the 2' or 5' position of the sugar moiety of (X) and the 2' or 3' position of the sugar moiety of (Y), or the base moiety of (X) and (Y) A linker that connects the base portion of;
The sequence T is a nucleotide sequence containing the expression suppressing sequence (Ta) of the mutant ALDH2 gene described in [1] or [2] above, and the sequence Q is a sequence (Qa) complementary to the expression suppressing sequence Ta. a nucleotide sequence comprising;
m ₁ and m ₂ are each independently an integer of 0 to 5; and n ₁ and n ₂ are each independently an integer of 0 to 5]
Nucleic acid molecule shown as .
[10-1] Linker Z is a non-nucleotide structure having an alkyl chain having an amide bond inside, or a structure that connects the base moiety of (X) and the base moiety of (Y), described in [10] nucleic acid molecule.
[10-2] In the formula, the following containing linker Z

The nucleic acid molecule according to [10] or [10-1], wherein the structure is selected from [Formula 1], [Formula 2], [Formula 3], or [Formula 4].

or

[In the formula, B and B' are each independently an atomic group having a nucleobase skeleton,
A ₁ and A ₁ ' are each independently -O-, -NR ^1a -, -S- or -CR ^1a R ^1b - (here, R ^1a and R ^1b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₂ and A ₂ ' are each independently -CR ^2a R ^2b -, -CO-, an alkynyl group, an alkenyl group, or a single bond (here, R ^2a and R ^2b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₃ and A ₃ ' are each independently -O-, -NR ^3a -, -S-, -CR ^3a R ^3b - or a single bond (here, R ^3a and R ^3b are each independently is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₄ and A ₄ ' are each independently -(CR ^4a R ^4b )n-, -(CR ^4a R ^4b )n-ring D- (here, ring D is an aryl group having 6 to 10 carbon atoms) , a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms, or a heterocycloalkyl group having 4 to 10 carbon atoms; R ^4a and R ^4b are each independently a hydrogen atom or a carbon number is an alkyl group of 1 to 10; n is an integer of 1 to 6) or a single bond,
A ₅ and A ₅ ' are each independently -NR ^5a - or a single bond (wherein R ^5a is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₆ and A ₆ ' are each independently -(CR ^6a R ^6b )n- or a single bond (here, R ^6a and R ^6b are each independently a hydrogen atom or a carbon number of 1 to 10). is an alkyl group; n is an integer from 1 to 6),
W ₁ is -(CR ¹ R ² )n-, -CO-, -(CR ¹ R ² )n-COO-(CR ¹ R ² )n-COO-(CR ¹ R ² )n, -(CR ¹ R ² ) n-O-(CR ¹ R ² CR ¹ R ² O) n-CH ₂ -, -(CR ¹ R ² ) n-ring D-(CR ¹ R ² ) n- or -(CR ¹ R ² )n-SS-(CR ¹ R ² )n- (wherein, ring D is an aryl group having 6 to 10 carbon atoms, a heteroaryl group having 2 to 10 carbon atoms, or a heteroaryl group having 4 to 10 carbon atoms. It is a cycloalkyl group or a heterocycloalkyl group having 4 to 10 carbon atoms; R ¹ and R ² are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; n is an integer of 1 to 6; be),
E ₂ and E ₂ ' are each independently -CR ^2a R ^2b -, -CO-, an alkynyl group, an alkenyl group, or a single bond (here, R ^2a and R ^2b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
E ₃ and E ₃ ' are each independently -O-, -NR ^3a -, -S-, -CR ^3a R ^3b - or a single bond (here, R ^3a and R ^3b are each independently is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
E ₄ and E ₄ ' are each independently -(CR ^4a R ^4b )n-, -(CR ^4a R ^4b )n-ring D- (here, ring D is an aryl group having 6 to 10 carbon atoms); , a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms, or a heterocycloalkyl group having 4 to 10 carbon atoms; R ^4a and R ^4b are each independently a hydrogen atom or a carbon number is an alkyl group of 1 to 10; n is an integer of 1 to 6) or a single bond,
E ₅ and E ₅ ' are each independently -NR ^5a - or a single bond (wherein R ^5a is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
E ₆ and E ₆ ' are each independently -(CR ^6a R ^6b )n- or a single bond (here, R ^6a and R ^6b are each independently a hydrogen atom or a carbon number of 1 to 10). is an alkyl group: n is an integer from 1 to 6)].
[10-3] In the formula, the following containing linker Z

The structure of [Formula 4]

The nucleic acid molecule according to [10], [10-1] or [10-2], wherein the definition of each symbol is the same as above.
[10-4] The nucleic acid molecule according to any one of [10], [10-1] to [10-3], which has the structure of any one of the following (35) to (74).

[11] A nucleotide sequence T containing the expression suppressing sequence (Ta) of the mutant ALDH2 gene described in [1] or [2] above, and a nucleotide sequence containing a sequence (Qa) complementary to the expression suppressing sequence Ta. Q in the 5' to 3' direction or 3' to 5' direction in the T-L-Q order via the linker L, and the expression suppressing sequence and its complementary sequence are duplicated within the molecule. Nucleic acid molecules linked in an orientation capable of forming chains.
[12] The nucleic acid molecule according to [11], wherein the linker L is a proline derivative linker represented by the following formula.

[13] The sequence T has an additional sequence Tb at the 5' end of the sequence Ta, and the sequence Q has an additional sequence Qb at the 3' end of the sequence Qa, and the sequences Tb and Qb are complementary. The nucleic acid molecule according to [11] or [12].
[14] The nucleic acid molecule according to any one of [11] to [13], wherein the sequence T has a 3'-overhang.
[15] The nucleic acid molecule according to [14], which has the structure of any one of the following (75) to (79).

[16] An expression vector that expresses the nucleic acid molecule according to any one of [1] to [15] and [10-1] to [10-4].
[17] A medicament comprising the nucleic acid molecule according to any one of [1] to [15] and [10-1] to [10-4] or the expression vector according to [16].
[18] The medicament according to [17], which suppresses mutant ALDH2 gene expression and improves ALDH2 activity.
[19] The medicament according to [17], which is used for the treatment or prevention of an ALDH2-related disease.
[20] The medicament according to [19], wherein the disease associated with ALDH2 is short birth height and weight, mild mental retardation, and aplastic anemia.
[21] The medicament according to [19], wherein the disease associated with ALDH2 is ADD syndrome and/or Fanconi anemia.

The nucleic acid molecule of the present invention can suppress the expression of the mutant ALDH2 gene, thereby effectively suppressing the expression of the mutant ALDH2 protein.

Unless otherwise specified, terms used herein can be used with the meanings commonly used in the art.
The terms and symbols used in the present invention are defined below.

"Protecting groups for amino groups" specifically include amide groups such as acetyl, trichloroacetyl, trifluoroacetyl, benzoyl, and N-phthalimide, and carbamate groups such as 9-fluoronylmethoxycarbonyl and t-butoxycarbonyl. can be mentioned.

"Hydroxyl group-protecting group" means a general hydroxyl-protecting group known to those skilled in the art that is introduced to prevent the reaction of hydroxyl groups; for example, Protective Groups in Organic Synthesis, published by John Wiley and Sons ( 1980), specifically, acyl protecting groups such as acetyl and benzoyl, alkyl protecting groups such as trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, and benzyl, and trimethylsilyl. , tert-butyldimethylsilyl, tert-butyldiphenylsilyl and the like.

"Electron-withdrawing group" refers to a group that attracts electrons more easily from the bonded atom side than a hydrogen atom, and specifically includes cyano, nitro, alkylsulfonyl (e.g., methylsulfonyl, ethylsulfonyl), halogen ( Examples include fluorine atom, chlorine atom, bromine atom, or iodine atom), arylsulfonyl (eg, phenylsulfonyl, naphthylsulfonyl), trihalomethyl (eg, trichloromethyl, trifluoromethyl), and the like.

Examples of "halogen" include fluorine atom, chlorine atom, bromine atom, and iodine atom.

"Alkyl group" (alkyl, alkyl chain) means a straight or branched alkyl group having 1 to 30 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, particularly preferably 1 to 4 carbon atoms, Specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl. , hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl and the like. Preferred examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl and the like. Examples of "alkyl groups having 1 to 10 carbon atoms" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl etc., preferably C _1-6 alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl). , 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl).

"Alkenyl group" (alkenyl) means a straight or branched alkenyl group having 2 to 30 carbon atoms, preferably 2 to 12 carbon atoms, more preferably 2 to 8 carbon atoms, and in the alkyl group, one or more Examples include those having a double bond. Specific examples include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 3-methyl-2-butenyl, and the like.

"Alkynyl group" (alkynyl) means a straight-chain or branched alkynyl group having 2 to 30 carbon atoms, preferably 2 to 12 carbon atoms, more preferably 2 to 8 carbon atoms, in which one or more triple Examples include those having a bond. Specific examples include ethynyl, propynyl, propargyl, butynyl, pentynyl, hexynyl, and the like. The alkynyl group may further have one or more double bonds.

"Alkoxy group" (alkoxy) means a straight or branched alkoxy group having 1 to 30 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, particularly preferably 1 to 4 carbon atoms, and specifically is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy, neopentyloxy, 2-pentyloxy, Examples include 3-pentyloxy, n-hexyloxy, 2-hexyloxy and the like.

"Aryl group" means an aryl group having 6 to 24 carbon atoms, preferably 6 to 10 carbon atoms, and includes monocyclic aromatic hydrocarbon groups such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2- Examples include polycyclic aromatic hydrocarbon groups such as anthryl, 9-anthryl, 1-phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, and 9-phenanthryl. Examples of the "aryl group having 6 to 10 carbon atoms" include those having 6 to 10 carbon atoms among the above-mentioned aryl groups, and specific examples thereof include phenyl, naphthyl, and the like.

"Heterocycloalkyl group" means a hecycloalkyl group having 6 to 24 carbon atoms, preferably 6 to 10 carbon atoms. Examples of the heterocycloalkyl group include those in which one or more carbon atoms forming the cyclic structure of the cycloalkyl group described below are substituted with a nitrogen atom, an oxygen atom, a sulfur atom, or the like. Specific examples include [1,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. . Examples of the "heterocycloalkyl group having 4 to 10 carbon atoms" include those having 4 to 10 carbon atoms among the above heterocycloalkyl groups, specifically pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, oxetanyl, and tetrahydrofuryl. , tetrahydropyranyl, thioxetanyl, tetrahydrothienyl, and tetrahydrothiopyranyl groups.

The term "aralkyl group" refers to an aralkyl group having 7 to 30 carbon atoms, preferably 7 to 11 carbon atoms, and specific examples include benzyl, 2-phenethyl, and naphthalenylmethyl.

"Cycloalkyl group" means a cycloalkyl group having 3 to 24 carbon atoms, preferably 3 to 15 carbon atoms, and specifically includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bridged ring. Examples thereof include formula hydrocarbon groups, spiro hydrocarbon groups, etc., and preferred examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and bridged cyclic hydrocarbon groups. Examples of the "bridged cyclic hydrocarbon group" include bicyclo[2.1.0]pentyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, and bicyclo[3.2.1] Examples include octyl, tricyclo[2.2.1.0]heptyl, bicyclo[3.3.1]nonyl, 1-adamantyl, 2-adamantyl, and the like. Examples of the "spiro hydrocarbon group" include spiro[3.4]octyl and the like. Examples of the "cycloalkyl group having 4 to 10 carbon atoms" include those having 4 to 10 carbon atoms among the above cycloalkyl groups, specifically cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. Can be mentioned.

"Cycloalkenyl group" means a cycloalkenyl group having 3 to 24 carbon atoms, preferably 3 to 7 carbon atoms, containing at least 1, preferably 1 or 2 double bonds, and specifically, cyclopropenyl , cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and the like. The cycloalkenyl group also includes bridged cyclic hydrocarbon groups and spirohydrocarbon groups having an unsaturated bond in the ring. Examples of "bridged cyclic hydrocarbon group having an unsaturated bond in the ring" include bicyclo[2.2.2]octenyl, bicyclo[3.2.1]octenyl, tricyclo[2.2.1.0] Examples include heptenyl. Examples of the "spiro hydrocarbon group having an unsaturated bond in the ring" include spiro[3.4]octenyl and the like.

"Cycloalkylalkyl group" means an alkyl group (described above) substituted with the cycloalkyl group, preferably a cycloalkylalkyl group having 4 to 30 carbon atoms, more preferably 4 to 11 carbon atoms. Specific examples include cyclopropylmethyl, 2-cyclobutylethyl, cyclopentylmethyl, 3-cyclopentylpropyl, cyclohexylmethyl, 2-cyclohexylethyl, and cycloheptylmethyl.

"Alkoxyalkyl group" means an alkyl group (described above) substituted with the alkoxy group, preferably a straight or branched alkoxyalkyl group having 2 to 30 carbon atoms, more preferably 2 to 12 carbon atoms. . Specific examples include methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl and t-butoxymethyl.

"Alkylene group" (alkylene chain) means a straight or branched alkylene group having 1 to 30 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, particularly preferably 1 to 4 carbon atoms, and specifically Examples include methylene, ethylene, and propylene.

A "heteroaryl group" includes, for example, a monocyclic aromatic heterocyclic group and a fused aromatic heterocyclic group. The heteroaryl is, for example, furyl (e.g. 2-furyl, 3-furyl), thienyl (e.g. 2-thienyl, 3-thienyl), pyrrolyl (e.g. 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), imidazolyl (e.g. 1-imidazolyl, 2-imidazolyl, 4-imidazolyl), pyrazolyl (e.g. 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl), triazolyl (e.g. 1,2,4-triazol-1-yl), 1,2,4-triazol-3-yl, 1,2,4-triazol-4-yl), tetrazolyl (e.g. 1-tetrazolyl, 2-tetrazolyl, 5-tetrazolyl), oxazolyl (e.g. 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), isoxazolyl (e.g. 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), thiazolyl (e.g. 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), thiadiazolyl, isothiazolyl (e.g. 3) -isothiazolyl, 4-isothiazolyl, 5-isothiazolyl), pyridyl (e.g. 2-pyridyl, 3-pyridyl, 4-pyridyl), pyridazinyl (e.g. 3-pyridazinyl, 4-pyridazinyl), pyrimidinyl (e.g. 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), furazanyl (e.g. 3-furazanyl), pyrazinyl (e.g. 2-pyrazinyl), oxadiazolyl (e.g. 1,3,4-oxadiazol-2-yl), benzofuryl (e.g. 2-benzo[b]furyl, 3-benzo[b]furyl, 4-benzo[b]furyl, 5-benzo[b]furyl, 6-benzo[b]furyl, 7-benzo[b]furyl), benzo Thienyl (e.g. 2-benzo[b]thienyl, 3-benzo[b]thienyl, 4-benzo[b]thienyl, 5-benzo[b]thienyl, 6-benzo[b]thienyl, 7-benzo[b] thienyl), benzimidazolyl (e.g. 1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl), dibenzofuryl, benzoxazolyl, benzothiazolyl, quinoxalinyl (e.g. 2-quinoxalinyl, 5-quinoxalinyl, 6-quinoxalinyl), cinnolinyl (e.g. 3-cinnolinyl, 4-cinnolinyl, 5-cinnolinyl, 6-cinnolinyl, 7-cinnolinyl, 8-cinnolinyl), quinazolinyl (e.g. 2-quinazolinyl, 4-quinazolinyl, 5-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl, 8-quinazolinyl), quinolyl (e.g. 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), phthalazinyl (e.g. 1-phthalazinyl, 5-phthalazinyl, 6-phthalazinyl), isoquinolyl (e.g. 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), prill, pteridinyl ( Examples: 2-pteridinyl, 4-pteridinyl, 6-pteridinyl, 7-pteridinyl), carbazolyl, phenanthridinyl, acridinyl (examples: 1-acridinyl, 2-acridinyl, 3-acridinyl, 4-acridinyl, 9-acridinyl) , indolyl (e.g. 1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), isoindolyl, phenazinyl (e.g. 1-phenazinyl, 2-phenazinyl) or pheno Examples include thiazinyl (eg, 1-phenothiazinyl, 2-phenothiazinyl, 3-phenothiazinyl, 4-phenothiazinyl), and the like.
Examples of the "heteroaryl group having 2 to 10 carbon atoms" include those having 2 to 10 carbon atoms among the above heteroaryl groups, and specifically, furyl, thienyl, pyrrolyl, oxazolyl, and triazolyl groups. , pyridyl group, quinolinyl group, etc.

In the present invention, examples of the "substituent" include those described in Substituent Group A below.
Substituent group A
(1) Halogen (fluorine atom, chlorine atom, bromine atom or iodine atom);
(2) alkyl group (described above);
(3) alkoxy group (described above);
(4) alkenyl group (described above);
(5) alkynyl group (described above);
(6) haloalkyl group (e.g., chloromethyl, fluoromethyl, dichloromethyl, difluoromethyl, dichlorofluoromethyl, trifluoromethyl, pentafluoroethyl, etc.);
(7) Aryl group (described above);
(8) heteroaryl group (described above);
(9) Aralkyl group (described above)
(10) cycloalkyl group (described above);
(11) cycloalkenyl group (described above);
(12) cycloalkylalkyl group (described above);
(13) cycloalkenylalkyl group (e.g., cyclopentenylethyl, cyclohexenylethyl, cyclohexenylbutyl, etc.);
(14) hydroxyalkyl group (e.g., hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, etc.);
(15) alkoxyalkyl group (described above);
(16) aminoalkyl group (e.g., aminomethyl, aminoethyl, aminopropyl, etc.);
(17) Heterocyclyl group (e.g., 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolidinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, pyrrolidinonyl, 1-imidazolinyl, 2-imidazolinyl, 4-imidazolinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, imidazolidinonyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 1-pyrazolidinyl, 3-pyrazolidinyl, 4-pyrazolidinyl, piperidinonyl, piperidino, 2-piperidinyl, 3-piperidinyl, 4- piperidinyl, 1-piperazinyl, 2-piperazinyl, piperazinonyl, 2-morpholinyl, 3-morpholinyl, morpholino, tetrahydropyranyl, tetrahydrofuranyl, etc.);
(18) heterocyclylalkenyl group (e.g., 2-piperidinylethenyl, etc.);
(19) heterocyclylalkyl group (e.g., piperidinylmethyl, piperazinylmethyl, etc.);
(20) heteroarylalkyl group (e.g., pyridylmethyl, quinolin-3-ylmethyl, etc.);
(21) Silyl group;
(22) Silyloxyalkyl group (e.g., silyloxymethyl, silyloxyethyl, etc.)
(23) mono-di- or trialkylsilyl groups (eg, methylsilyl, ethylsilyl, etc.); and (24) mono-, di- or trialkylsilyloxyalkyl groups (eg, trimethylsilyloxymethyl, etc.).

1. Nucleic acid molecule that suppresses the expression of the mutant ALDH2 gene The present invention provides a nucleic acid molecule that suppresses the expression of the mutant ALDH2 gene (hereinafter referred to as “the present invention (sometimes referred to as "nucleic acid molecule").

The nucleic acid molecule of the present invention contains a sequence complementary to a specific site of the mutant ALDH2 mRNA as a sequence that suppresses the expression of the mutant ALDH2 gene. The nucleotide sequences of human ALDH2 genomic DNA and mRNA are listed in the NCBI database as GenBank Accession No. It is registered as NG_012250.2 and NM_000690.4. The mutant ALDH2 gene targeted by the nucleic acid molecule of the present invention is a single nucleotide polymorphism observed in the human ALDH2 gene, specifically, guanine at base 42,076 (104th of exon 12) is adenine. The rs671 mutation is replaced by . Due to this mutation, glutamic acid, which is amino acid number 504 in precursor ALDH2 and amino acid number 487 in mature ALDH2, is replaced with lysine.
More specifically, the nucleic acid molecule of the present invention has SEQ ID NO: 1 in the mutant ALDH2 gene (rs671 mutation):

Contains a nucleotide sequence complementary to a nucleotide sequence of 15 or more consecutive nucleotides in a target sequence of 25 or less consecutive nucleotides in the region represented by the nucleotide sequence represented by, as an expression suppressing sequence of the mutant ALDH2 gene. It is a nucleic acid molecule. Here, the nucleotide sequence represented by SEQ ID NO: 1 is a sequence of a region including the rs671 mutation site of the mutant ALDH2 gene. Variations are shown in bold.

The sequence that suppresses gene expression of mutant ALDH2 (hereinafter also simply referred to as "expression suppression sequence") is a sequence that is complementary to the nucleotide sequence of a specific site of ALDH2 mRNA. Here, the term "complementary sequence" refers not only to a sequence that is completely complementary to the target sequence (that is, hybridizes without mismatch), but also to a sequence that is compatible with mutant ALDH2 RNA under physiological conditions in mammalian cells. The sequence may contain a mismatch of one or several nucleotides, preferably one or two nucleotides, as long as it can hybridize specifically. Examples include sequences having an identity of 90% or more, preferably 95% or more, 97% or more, 98% or more, 99% or more to the complementary strand sequence of the target nucleotide sequence in mutant ALDH2 RNA. . "Nucleotide sequence identity" in the present invention is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gaps; filtering = ON; Match score = 1; mismatch score = -3). Furthermore, complementarity in individual bases is not limited to forming Watson-Crick type base pairs with the target base, but also forming Hoogsteen type base pairs and Wobble base pairs. Also includes doing.

Alternatively, a "complementary nucleotide sequence" is a nucleotide sequence that hybridizes under stringent conditions to a target sequence. Here, "stringent conditions" refer to, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, such as 6×SSC (sodium chloride/sodium citrate). )/45°C, followed by one or more washes at 0.2×SSC/0.1% SDS/50-65°C, etc., but those skilled in the art will be able to use equivalent strings. Hybridization conditions that provide the desired compatibility can be selected as appropriate.

The nucleotide sequence of the mutant ALDH2 gene targeted by the expression suppressing sequence includes a nucleotide sequence of 25 or less consecutive nucleotides in the region represented by the nucleotide sequence represented by SEQ ID NO: 1. The expression suppressing sequence may be complementary to all of these target sequences, or may be complementary to a part of the target sequences, but Taking this into account, it is preferable to be complementary to a sequence of 15 or more consecutive nucleotides in each target sequence. In addition to the sequence of 15 or more consecutive nucleotides in each target sequence, the expression suppressing sequence can further include a sequence complementary to the nucleotide sequence of the mutant ALDH2 gene adjacent to the target sequence. . The upper limit of the length of the nucleotide sequence targeted by the expression suppressing sequence is not particularly limited, but considering the ease of synthesis, it may be, for example, 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less, and It is preferably a continuous partial nucleotide sequence of the mutant ALDH2 gene of 25 nucleotides or less. Therefore, the length of the nucleotide sequence targeted by the expression suppressing sequence is preferably a partial nucleotide sequence of 15 to 30 consecutive nucleotides, more preferably 15 to 25 consecutive nucleotides in the nucleotide sequence of the mutant ALDH2 gene. could be.

The nucleic acid molecule of the present invention may be RNA, DNA, or a DNA/RNA chimera as long as it can suppress gene expression of mutant ALDH2. Further, the nucleic acid molecule of the present invention may be a double-stranded nucleic acid or a single-stranded nucleic acid, as long as it can suppress gene expression of mutant ALDH2. In the case of double-stranded nucleic acids, they may be double-stranded DNA, double-stranded RNA, DNA:RNA hybrids, or hybrids of DNA/RNA chimeras and DNA, RNA, or DNA/RNA chimeras.

When the nucleic acid molecule of the present invention is a double-stranded nucleic acid, one strand is the expression suppressing sequence of the mutant ALDH2 gene, that is, a target of 25 consecutive nucleotides or less in the region represented by the nucleotide sequence represented by SEQ ID NO: 1. Contains a sequence complementary to a sequence of 15 or more consecutive nucleotides in the sequence (hereinafter, a strand containing a sequence that binds to the target mutant ALDH2 gene and suppresses gene expression is also referred to as a "guide strand"), and the other The chain contains at least a sequence complementary to the expression suppressing sequence (hereinafter, a chain containing a sequence complementary to the expression suppressing sequence is also referred to as a "passenger strand"). Here, the term "complementary sequence" has the same meaning as described above regarding the complementarity of the expression suppressing sequence to the nucleotide sequence of the mutant ALDH2 gene.

When the nucleic acid molecule of the present invention is a single-stranded nucleic acid, it has only the above-mentioned guide strand, and the guide strand and passenger strand are connected via an arbitrary linker, suppressing the expression of the mutant ALDH2 gene within the molecule. There are cases in which a sequence and a sequence complementary thereto can hybridize to form a duplex.

Examples of the constituent units of the nucleic acid molecule of the present invention include ribonucleotide residues and deoxyribonucleotide residues. These nucleotide residues may be modified or unmodified, for example. The nucleic acid molecules of the present invention can have improved nuclease resistance and improved stability, for example, by containing modified nucleotide residues. Further, the nucleic acid molecule of the present invention may further contain non-nucleotide residues in addition to the above-mentioned nucleotide residues, for example.

In the nucleic acid molecule of the present invention, the constituent units of regions other than the linker (guide strand and passenger strand) are preferably nucleotide residues. Each region is composed of, for example, the following residues (1) to (3).
(1) Unmodified nucleotide residues (2) Modified nucleotide residues (3) Unmodified nucleotide residues and modified nucleotide residues

The nucleic acid molecule of the present invention may be labeled with a labeling substance, for example. The labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes, and the like. Examples of the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dye, and Cy5 dye, and examples of the dye include Alexa dyes such as Alexa488. Examples of isotopes include stable isotopes and radioactive isotopes. Stable isotopes, for example, have less risk of exposure and do not require dedicated facilities, making them easier to handle and reducing costs. Furthermore, stable isotopes do not change the physical properties of the labeled compound, and have excellent properties as tracers. Examples of stable isotopes include ^2H , ^13C , ^15N , ^17O , ^18O , ^33S , ^34S and ^36S .

Nucleotide residues include sugars, bases and phosphates as constituents. Ribonucleotide residues have ribose residues as sugars and adenine (A), guanine (G), cytosine (C) and uracil (U) (which can also be replaced by thymine (T)) as bases. However, deoxyribonucleotide residues have deoxyribose residues as sugars, and adenine (dA), guanine (dG), cytosine (dC) and thymine (dT) as bases (can also be replaced by uracil (dU)). ).

A modified nucleotide residue may have any component of the nucleotide residue modified. In the present invention, "modification" may be, for example, substitution, addition, and/or elimination of the aforementioned constituent elements, substitution, addition, and/or elimination of atoms and/or functional groups in the aforementioned constituent elements. The modified nucleotide residue may be, for example, a naturally occurring modified nucleotide residue or an artificially modified nucleotide residue. For naturally occurring modified nucleotide residues, see, for example, Limbach et al. (1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22:2183-2196).

Examples of modification of nucleotide residues include modification of ribose-phosphate skeleton (hereinafter referred to as ribophosphate skeleton).

In the riboric acid skeleton, for example, a ribose residue can be modified. The ribose residue can, for example, modify the 2'-position carbon, and specifically, for example, modify the hydroxyl group bonded to the 2'-position carbon with a hydrogen atom, a halogen atom such as fluorine, or an -O-alkyl group (e.g., -O-Me group), -O-acyl group (e.g. -O-COMe group), and amino group, preferably selected from the group consisting of hydrogen atom, methoxy group, and fluorine atom can be substituted with an atom or group. By replacing the hydroxyl group at the 2'-position carbon with hydrogen, the ribose residue can be replaced with deoxyribose. The ribose residue may be substituted with a stereoisomer, for example, with an arabinose residue.

The ribophosphate backbone may be replaced, for example, by a non-ribophosphate backbone with a non-ribose residue and/or a non-phosphate. Examples of the non-ribophosphoric acid skeleton include uncharged riboric acid skeletons. Examples of substitutes for nucleotides substituted with non-ribophosphate backbones include morpholino, cyclobutyl, pyrrolidine, and the like. Other examples of the above-mentioned substitutes include, for example, artificial nucleic acid monomer residues. Specific examples include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), and ENA (2'-O,4'-C-Ethylene bridged Nucleic Acid), with PNA being preferred.

In the ribophosphate skeleton, the phosphate group can also be modified. In the ribophosphate backbone, the phosphate group closest to the sugar residue is called the alpha phosphate group. The alpha phosphate group is negatively charged, and its charge is evenly distributed over the two oxygen atoms that are not attached to the sugar residue. Among the four oxygen atoms in the α-phosphate group, the two oxygen atoms that are not bonded to sugar residues in the phosphodiester bond between nucleotide residues are hereinafter also referred to as "non-linking oxygen". . On the other hand, two oxygen atoms bonded to a sugar residue in a phosphodiester bond between nucleotide residues are hereinafter referred to as "linking oxygen." The α-phosphate group is preferably modified, for example, to become uncharged or to have an asymmetric charge distribution in non-bonded oxygen.

A phosphate group may, for example, replace a non-bonding oxygen. Non-bonded oxygen is, for example, any of S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen) and OR (R is an alkyl or aryl group). It is preferably substituted with S. For example, both non-bonding oxygens are preferably substituted, more preferably both are substituted with S. Such modified phosphate groups include, for example, phosphorothioates, phosphorodithioates, phosphoroselenates, boranophosphates, boranophosphate esters, phosphonate hydrogens, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. Among these, phosphorodithioate in which the two non-bonding oxygens are both replaced with S is preferred.

A phosphate group may, for example, replace a bound oxygen. The bound oxygen can be substituted, for example, by any of the S (sulfur), C (carbon) and N (nitrogen) atoms; such modified phosphate groups include, for example, bridged phosphoroamidates substituted with N. , S-substituted bridged phosphorothioate, and C-substituted bridged methylene phosphonate. For example, the substitution of bound oxygen is preferably performed at at least one of the 5' terminal nucleotide residue and the 3' terminal nucleotide residue of the nucleic acid molecule of the present invention, and in the case of the 5' side, substitution by C is preferable; In the latter case, substitution by N is preferred.

The phosphate group may be substituted, for example, by a phosphorus-free linker. Examples of linkers include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethyl Examples include hydrazo and methyleneoxymethylimino, and preferred examples include methylenecarbonylamino and methylenemethylimino groups.

The nucleic acid molecule of the present invention may be modified, for example, at least one of the nucleotide residues at the 3' end and the 5' end. The modification is as described above, and is preferably performed on the terminal phosphate group. The entire phosphate group may be modified, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be replaced or deleted.

Examples of modification of terminal nucleotide residues include addition of other molecules. Other molecules include, for example, functional molecules such as labeling substances and protective groups. Examples of the protecting group include S (sulfur), Si (silicon), B (boron), and ester-containing groups. The functional molecules such as the labeling substances can be used, for example, to detect the nucleic acid molecules of the present invention.

Other molecules may be attached to the phosphate groups of nucleotide residues, or via spacers to phosphate groups or sugar residues. The terminal atom of the spacer can be added to or replaced with, for example, the bound oxygen of a phosphate group, or O, N, S or C of a sugar residue. The binding site of the sugar residue is preferably, for example, C at the 3' position or C at the 5' position, or an atom bonded to these. A spacer can also be added to or substituted, for example, at the terminal atom of a nucleotide substitute, such as the PNA.

Spacers are not particularly limited, and include, for example, -(CH ₂ ) _n -, -(CH ₂ ) _n N-, -(CH ₂ ) _n O-, -(CH ₂ ) _n S-, O(CH ₂ CH ₂ Examples include O) _n CH ₂ CH ₂ OH, abasic sugars, amides, carboxy, amines, oxyamines, oximines, thioethers, disulfides, thioureas, sulfonamides, morpholinos, and biotin reagents and fluorescein reagents. In the above formula, n is a positive integer, and preferably n=3 or 6.

In addition to these, molecules added to the terminal include, for example, dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphirin), polycyclic aromatic group hydrocarbons (e.g. phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g. cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)col acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] ₂ , polyaminos, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption enhancers (e.g. aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g. imidazole, bisimidazole, histamine, imidazole) cluster, acridine-imidazole complex, Eu ³⁺ complex of tetraazamacrocycle), etc.

The 5' end of the nucleic acid molecule of the present invention may be modified, for example, with a phosphate group or a phosphate group analog. Phosphate groups are, for example, 5' monophosphate ((HO) ₂ (O)PO-5'), 5' diphosphate ((HO) ₂ (O)POP(HO)(O)-O-5 '), 5' triphosphate ((HO) ₂ (O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or non-methylated), Methylation, 7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-adenosine cap (Appp), optional modification or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5' monothiophosphate (phosphorothioate: (HO ) ₂ (S)PO-5'), 5'-dithiophosphoric acid (phosphorodithioate: (HO)(HS)(S)PO-5'), 5'-phosphorothiolic acid ((HO) ₂ (O ) PS-5'), sulfur-substituted mono-, di- and triphosphates (e.g. 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoric acid ruamidate ((HO) ₂ (O)P-NH-5', (HO)(NH ₂ )(O)PO-5'), 5'-alkylphosphonic acid (e.g. RP(OH)(O) -O-5', (OH) ₂ (O)P-5'-CH ₂ , R is alkyl (e.g. methyl, ethyl, isopropyl, propyl, etc.)), 5'-alkyl ether phosphonic acid (e.g. RP( OH)(O)-O-5', R is an alkyl ether (eg, methoxymethyl, ethoxymethyl, etc.).

Among the nucleotide residues, the bases are not particularly limited. The base may be a natural base or a non-natural base. For example, common bases, modified analogs thereof, etc. can be used.

Examples of the base include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil and thymine. Other bases include inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and the like. Bases include, for example, alkyl derivatives such as 2-aminoadenine, 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6- Azouracil, 6-azocytosine and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil; 8-halination, amination, thiol 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidine; N-2, N- 6, and O-6 substituted purines (including 2-aminopropyladenine); 5-propynyluracil and 5-propynylcytosine; dihydrouracil; 3-deaza-5-azacytosine; 2-aminopurine; 5-alkyluracil; 7 -Alkylguanine; 5-alkylcytosine; 7-deazaadenine; N6,N6-dimethyladenine; 2,6-diaminopurine; 5-amino-allyl-uracil; N3-methyluracil; substituted 1,2,4-triazole; 2 -Pyridinone; 5-nitroindole; 3-nitropyrrole; 5-methoxyuracil; uracil-5-oxyacetic acid; 5-methoxycarbonylmethyluracil; 5-methyl-2-thiouracil; 5-methoxycarbonylmethyl-2-thiouracil; 5-Methylaminomethyl-2-thiouracil; 3-(3-amino-3-carboxypropyl)uracil; 3-methylcytosine; 5-methylcytosine; N4-acetylcytosine; 2-thiocytosine; N6-methyladenine; N6- It can be isopentyladenine; 2-methylthio-N6-isopentenyladenine; N-methylguanine; O-alkylated base, etc. Purines and pyrimidines also include, for example, U.S. Pat. (Englisch et al.), Angewandte Chemie, International Edition, 1991, vol. 30, p. 613.

In addition to these, modified nucleotide residues may include, for example, residues lacking a base, ie, an abasic riboric acid backbone. Modified nucleotide residues may also be used, for example, in U.S. Provisional Application No. 60/465,665 (filed on April 25, 2003) and International Application No. PCT/US04/07070 (filed on March 8, 2004). ) can be used, and the present invention can make use of these documents.

The method for synthesizing the nucleic acid molecule of the present invention is not particularly limited, and conventionally known methods can be employed. Examples of the synthesis method include a genetic engineering method, a chemical synthesis method, and the like. Genetic engineering methods include, for example, in vitro transcription synthesis methods, methods using vectors, and methods using PCR cassettes. The vector is not particularly limited, and includes non-viral vectors such as plasmids, viral vectors, and the like. The chemical synthesis method is not particularly limited, and examples thereof include the phosphoramidite method and the H-phosphonate method. For the chemical synthesis method, for example, a commercially available automatic nucleic acid synthesizer can be used. The chemical synthesis method generally uses amidites. The amidite is not particularly limited, and commercially available amidites include, for example, RNA Phosphoramidites (2'-O-TBDMSi, trade name, Sansenri Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, etc. can be given.

Examples of the nucleic acid molecules of the present invention include siRNA against the mutant ALDH2 gene, antisense nucleic acids against the mutant ALDH2 gene, and the like. Furthermore, examples of the nucleic acid molecule of the present invention include single-stranded nucleic acid molecules capable of forming a double strand in which a guide strand and a passenger strand complementary thereto are connected via a linker.

(i) siRNA against mutant ALDH2 gene
siRNA against the mutant ALDH2 gene includes a sequence complementary to all or part of the target sequence of 25 consecutive nucleotides or less in the region shown by the nucleotide sequence represented by SEQ ID NO: 1 in the mutant ALDH2 gene. A double-stranded oligo RNA consisting of a guide strand and a passenger strand containing a sequence complementary to the guide strand, which is incorporated into the RISC complex and has a complementary sequence to the mRNA encoded by the mutant ALDH2 gene in the guide strand. refers to a nucleic acid that cleaves the mRNA and suppresses gene expression by forming a double strand with the target sequence in the mRNA. Here, "complementary sequence" has the same meaning as above.

The length of the siRNA for the mutant ALDH2 gene is such that the guide strand contains a sequence complementary to all or part of the target sequence of 25 consecutive nucleotides or less in the region indicated by the nucleotide sequence represented by SEQ ID NO: 1. Although not particularly limited as long as it contains, the nucleotide sequence targeted by siRNA can in principle be 15 to 50 nucleotides, preferably 19 to 30 nucleotides, more preferably 19 to 27 nucleotides, particularly preferably 19 to 21 nucleotides. . The guide strand and passenger strand may also have additional nucleotides at the 5' or 3' ends. The length of the additional nucleotides is usually about 2 to 4 nucleotides, and the total length of the siRNA is 19 nucleotides or more. The additional nucleotide may be DNA or RNA, although DNA may be used to improve the stability of the nucleic acid. Sequences of such additional nucleotides include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3'. Examples include, but are not limited to, sequences such as ', uuuuu-3'.

The siRNA against the mutant ALDH2 gene is complementary to all or part of a target sequence of 25 consecutive nucleotides or less in the region represented by the nucleotide sequence represented by SEQ ID NO: 1, as a sequence that suppresses the expression of the mutant ALDH2 gene. However, in a preferred embodiment, the expression suppressing sequence includes any of the following nucleotide sequences (SEQ ID NO: 2n (n is an integer selected from 1 to 11; , U may be T), and a passenger strand complementary thereto (preferably SEQ ID NO: 2n+1 (n is an integer selected from 1 to 11; however, in the sequence, U may be T). Examples include nucleic acid molecules consisting of the following:

The siRNA against the mutant ALDH2 gene may have a 3'-overhang on one or both strands. When having an overhang, the length of the overhang is not particularly limited, and the lower limit is, for example, 1 base length, the upper limit is, for example, 4 bases long, 3 bases long, and the range is, for example, 1 base length. ~4 bases in length, 1 to 3 bases in length, and 1 to 2 bases in length.

The arrangement of the overhang is not particularly limited, and may be any of A, U, G, C, and T. Examples of the overhang arrangement include TT, UU, CU, GC, UA, AA, CC, UG, CG, AU, etc. from the 3' side. The overhang can add resistance to RNA degrading enzymes by making it TT or UU, for example.

In a preferred embodiment, the expression suppressing sequence having a 3'-overhang is one of the following nucleotide sequences (SEQ ID NO: 2m (m is an integer from 12 to 45; provided that in the sequence, U is T) a guide strand having a 3'-overhang complementary thereto (preferably SEQ ID NO: 2m+1 (m is an integer from 12 to 45; provided that in the sequence, U is T and Examples include nucleic acid molecules consisting of

The method for synthesizing siRNA for the mutant ALDH2 gene is not particularly limited, and conventionally known methods for producing nucleic acids can be employed. As a synthesis method, for example, a nucleic acid containing the above-mentioned complementary sequence and a nucleic acid having a complementary sequence thereto are each synthesized using an automatic DNA/RNA synthesizer, and then incubated at about 90 to about 95°C in an appropriate annealing buffer. Examples include a method of preparing by denaturing for about 1 minute and then annealing at about 30 to about 70°C for about 1 to about 8 hours. Alternatively, it can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it using dicer. Nucleotide residues constituting siRNA may also be modified in the same manner as above in order to improve stability, specific activity, etc.

(ii) Antisense nucleic acid against the mutant ALDH2 gene Antisense nucleic acid against the mutant ALDH2 gene refers to a target of 25 consecutive nucleotides or less in the region represented by the nucleotide sequence represented by SEQ ID NO: 1 in the mutant ALDH2 gene. All or part of the sequence, preferably a sequence complementary to a nucleotide sequence of 15 or more consecutive nucleotides in the nucleotide sequence, and binds to the target sequence in ALDH2 RNA by forming a specific double strand. refers to a nucleic acid that has the effect of suppressing gene expression. Here, "complementary sequence" has the same meaning as above.

The length of the antisense nucleic acid for the mutant ALDH2 gene is not particularly limited, but may be, for example, 10 to 100 nucleotides, preferably 15 to 40 nucleotides, and more preferably 15 to 30 nucleotides.

In a preferred embodiment, the antisense nucleic acid for the mutant ALDH2 gene has the above SEQ ID NO: 2n (n is an integer from 1 to 11; however, U may be T in the sequence) as an expression suppressing sequence. It contains a nucleotide sequence represented by any of the following.

The antisense nucleic acid for the mutant ALDH2 gene may be of the gapmer type. A gapmer-type antisense nucleic acid is a nucleic acid having DNA and nucleic acids to which modifications or crosslinks have been introduced on both sides. With the DNA strand as the main chain, RNA complementary to the main chain forms a hetero double-stranded nucleic acid, and the RNA is degraded by RNAase H. O-methylation at the 2' position of the sugar moiety improves the stability of the antisense nucleic acid and increases its binding affinity to the target. Furthermore, replacing phosphate bonds with phosphorothioate bonds increases the nuclease resistance of antisense nucleic acids.

The method for synthesizing the antisense nucleic acid for the mutant ALDH2 gene is not particularly limited, and conventionally known methods for producing nucleic acids can be employed. Examples of the synthesis method include a method in which nucleic acids containing the complementary sequences are synthesized using an automatic DNA/RNA synthesizer, respectively. Furthermore, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by conventionally known techniques.

(iii) Single-stranded nucleic acid molecule for mutant ALDH2 gene In the present invention, "single-stranded nucleic acid molecule for mutant ALDH2 gene" refers to the nucleotide sequence represented by SEQ ID NO: 1 in mutant ALDH2 gene. A guide strand sequence T containing a sequence Ta complementary to all or part of a target sequence of 25 consecutive nucleotides or less in a region, and a passenger strand sequence Q containing a sequence Qa complementary to Ta are linked via a linker L. ALDH2, in which the sequence Ta and the sequence Qa are connected in the 5' to 3' direction or in the 3' to 5' direction in the T-L-Q order, and in an orientation that allows the sequence Ta and the sequence Qa to form a double strand within the molecule. A nucleic acid molecule that suppresses gene expression. Here, "complementary sequence" has the same meaning as above.

The sequence Ta is not particularly limited as long as it includes a sequence complementary to all or part of the target sequence of 25 consecutive nucleotides or less in the region shown by the nucleotide sequence represented by SEQ ID NO: 1 of the mutant ALDH2 gene. , the nucleotide sequence targeted by Ta may in principle be 15-49 nucleotides, preferably 19-30 nucleotides, more preferably 19-27 nucleotides, particularly preferably 19-21 nucleotides.

The single-stranded nucleic acid molecule for the mutant ALDH2 gene has, as an expression suppressing sequence, a sequence complementary to all or part of the target sequence of 25 consecutive nucleotides or less in the region represented by the nucleotide sequence represented by SEQ ID NO: 1. is included in the guide strand sequence T as the sequence Ta, but in a preferred embodiment, the above SEQ ID NO: 2n (n is an integer from 1 to 11; however, in the sequence, U is T and A guide strand sequence T containing a nucleotide sequence represented by any one of the following nucleotide sequences (preferably SEQ ID NO: 2n+1 (n is an integer from 1 to 11; however, in the sequence, U may be T) and a passenger strand sequence Q.
In another embodiment, the sequence Ta includes the nucleotide sequence represented by SEQ ID NO: 2n (n is the same as above) (in which each U may be T), and A guide strand sequence T containing a nucleotide sequence of 15 or more consecutive nucleotides in a target sequence of 25 or less consecutive nucleotides in the region indicated by the nucleotide sequence represented by SEQ ID NO: 1, and a sequence Qa complementary to the sequence Ta. Examples include nucleic acid molecules containing a passenger strand sequence Q (preferably a sequence completely complementary to sequence Qa (however, the pairing of G and U is considered complementary)).
In a particularly preferred embodiment, the single-stranded nucleic acid molecule for the mutant ALDH2 gene has the nucleotide sequence represented by SEQ ID NO: 2n (n is an integer selected from 1 to 11) as the sequence Ta, and the nucleotide sequence represented by SEQ ID NO: 2n (n is an integer selected from 1 to 11) as the sequence Qa. 2n+1 nucleotide sequence.

The guide strand sequence T may, for example, consist only of the sequence Ta, or may further include an additional sequence Tb. In the latter case, the additional sequence Tb does not need to be complementary to the nucleotide sequence of the mutant ALDH2 gene. The additional sequence Tb may be added to either the 5' end or the 3' end of Ta, or may be added to both ends (Tb and Tb'). Preferably, it is added to the end of Ta that is connected to linker L. The length of the sequence Tb (Tb') is, for example, 1 to 35 nucleotides, preferably 1 to 25 nucleotides, more preferably 1 to 11 nucleotides, particularly preferably 1, 2, 3 , 4, 5 or 6 nucleotides.

The passenger strand sequence Q is not particularly limited as long as it includes a sequence Qa complementary to the sequence Ta, and for example, it may consist only of the sequence Qa or may further include an additional sequence Qb. In the latter case, the additional sequence Qb does not need to be complementary to the additional sequence Tb, but it is desirable that it be, and in particular, the additional sequences Tb and Qb are linked to the linker L of Ta and Qa, respectively. When added to the side ends, it is more desirable that Tb and Qb are complementary. The additional sequence Qb may be added to either the 5' end or the 3' end of Qa, or may be added to both ends (Qb and Qb'). Preferably, it is added to the end of Qa that is connected to linker L. The length of the sequence Yb (Yb') is, for example, 1 to 35 nucleotides, preferably 1 to 25 nucleotides, more preferably 1 to 11 nucleotides, particularly preferably 1, 2, 3 , 4, 5 or 6 nucleotides.

The guide strand sequence T and the passenger strand sequence Q may further have an overhang at the end not connected to the linker L. The overhang is preferably added to the 3' end of the guide and passenger strand sequences, the 5' end of which is linked to the linker L.

The length of the overhang is not particularly limited, and the lower limit is, for example, 1 nucleotide length, the upper limit is, for example, 4 nucleotide length, 3 nucleotide length, and the range is, for example, 1 to 4 nucleotide length, 1 nucleotide length. ~3 nucleotides long, 1-2 nucleotides long.

The arrangement of the overhang is not particularly limited, and may be any of A, U, G, C, and T. Examples of the overhang arrangement include TT, UU, CU, GC, UA, AA, CC, UG, CG, AU, etc. from the 5' side. By setting the overhang to, for example, TT or UU, resistance to RNA degrading enzymes can be added.

In a preferred embodiment, the single-stranded nucleic acid molecule for the mutant ALDH2 gene has a nucleotide sequence T and a nucleotide sequence Q in the 5' to 3' direction or in the 3' to 5' direction via a linker L. The sequences Ta and Qa are connected in the order -LQ and in an orientation that allows them to form a double strand within the molecule. Linker L may be composed of, for example, nucleotide residues, non-nucleotide residues, nucleotide residues and non-nucleotide residues. Nucleotide residues include ribonucleotide residues and deoxyribonucleotide residues.

When linker L is composed of nucleotide residues, the sense region and antisense region base pair with each other within one molecule to form a stem structure, and at the same time, the nucleotide sequence of linker L forms a loop structure. The whole molecule forms a hairpin-shaped stem-loop structure, and the single-stranded nucleic acid molecule for the mutant ALDH2 gene can also be called shRNA (small hairpin RNA or short hairpin RNA). The length of the linker L is not particularly limited, but preferably has a length that allows the sequences Ta and Qa to form a double strand within the molecule, for example. The lower limit of the number of bases in the linker L is, for example, 1 base, 2 bases, or 3 bases, and the upper limit is, for example, 100 bases, 80 bases, or 50 bases. Specific examples of the number of bases in each linker region include 1 to 50 bases, 1 to 30 bases, 1 to 20 bases, 1 to 10 bases, 1 to 7 bases, 1 to 4 bases, etc. is not limited to. The linker L preferably has a structure that does not cause self-annealing.

The linker L composed of non-nucleotide residues, or the linker L composed of nucleotide residues and non-nucleotide residues, is represented by the following formula (I), for example.

In formula (I), for example, X ^1a and X ^1b are both hydrogen atoms or together form =O, =S or =NH; X ^2a and X ^2b are both hydrogen atoms or taken together to form =O, =S or =NH;
R ³ is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on ring A,
L ¹ is an alkylene chain consisting of m _L carbon atoms, where the hydrogen atoms on the alkylene carbon atoms are OH, OR ^a , NH ₂ , NHR ^a , NR ^a R ^b , SH, or SR ^a or L ¹ is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom, provided that Y ¹ is NH, In the case of O or S, the L ¹ atom bonded to Y ¹ is carbon, the L ¹ atom bonded to OR ¹ is carbon, and the oxygen atoms are not adjacent to each other;
L ² is an alkylene chain consisting of n _L carbon atoms, where the hydrogen atoms on the alkylene carbon atoms are OH, OR ^c , NH ₂ , NHR ^c , NR ^c R ^d , SH or SR ^c It may be substituted or unsubstituted, or ^L2 is a polyether chain in which one or more carbon atoms of the alkylene chain is substituted with an oxygen atom, provided that ^Y2 is NH, O Or in the case of S, the L ² atom bonded to Y ² is carbon, the L ² atom bonded to OR ² is carbon, and the oxygen atoms are not adjacent to each other;
R ^a , R ^b , R ^c and R ^d are each independently a substituent or a protecting group;
l is 1 or 2;
m _L is an integer in the range of 0 to 30;
n _L is an integer in the range of 0 to 30;
In ring A, one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen, or sulfur,
Ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond,
Nucleotide sequence T and nucleotide sequence Q are linked to a non-nucleotide structure via -OR ¹ - or -OR ² -, respectively, where R ¹ and R ² may be present or absent. Often, when present, R ¹ and R ² are each independently a nucleotide residue or a structure of formula (I).

In formula (I), X ^1a and X ^1b are, for example, both hydrogen atoms or together form =O, =S or =NH. The same applies to X ^2a and X ^2b .

In formula (I), Y ¹ and Y ² are each independently a single bond, CH ₂ , NH, O or S.

In the formula (I), l is 1 or 2 in ring A. When l=1, ring A is a 5-membered ring, for example, a pyrrolidine skeleton. Examples of the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton, and examples thereof include divalent structures. When l=2, ring A is a 6-membered ring, for example, a piperidine skeleton. In ring A, one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen or sulfur. Further, ring A may include a carbon-carbon double bond or a carbon-nitrogen double bond within ring A. Ring A may be, for example, either L-type or D-type.

In formula (I), R ³ is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on ring A. When R ³ is a substituent, the number of substituents R ³ may be one or more, or none may exist, and if there are more than one, they may be the same or different.

The substituent R ³ is, for example, halogen, OH, OR ⁴ , NH ₂ , NHR ⁴ , NR ⁴ R ⁵ , SH, SR ⁴ or an oxo group (=O).

R ⁴ and R ⁵ are, for example, each independently a substituent or a protecting group, and may be the same or different. Examples of the substituent include those described in Substituent Group A above, such as halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, and cycloalkenylalkyl. , hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl, heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl, and the like. The same applies hereafter. The substituent R ³ may be any of the substituents listed above.

The protecting group is, for example, a functional group that converts a highly reactive functional group into an inactive one, and includes known protecting groups. Regarding the protecting group, for example, the description in the literature (JFW McOmie, "Protecting Groups in Organic Chemistry" PrenumPress, London and New York, 1973) can be referred to. The protecting group is not particularly limited, and examples include tert-butyldimethylsilyl group (TBDMS), bis(2-acetoxyethyloxy)methyl group (ACE), triisopropylsilyloxymethyl group (TOM), 1- (2- (cyanoethoxy) ethyl group (CEE), 2-cyanoethoxymethyl group (CEM), tolylsulfonylethoxymethyl group (TEM), dimethoxytrityl group (DMTr), and the like. When R ³ is OR ⁴ , the protecting group is not particularly limited and includes, for example, a TBDMS group, an ACE group, a TOM group, a CEE group, a CEM group, and a TEM group. Other examples include silyl-containing groups. The same applies hereafter.

In formula (I), L ¹ is an alkylene chain consisting of m _L carbon atoms. The hydrogen atom on the alkylene carbon atom may be substituted or unsubstituted with, for example, OH, OR ^a , NH ₂ , NHR ^a , NR ^a R ^b , SH, or SR ^a . Alternatively, L ¹ may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom. The polyether chain is, for example, polyethylene glycol. Note that when Y ¹ is NH, O, or S, the atom of L ¹ bonded to Y ¹ is carbon, the atom of L ¹ bonded to OR ¹ is carbon, and the oxygen atoms are not adjacent to each other. That is, for example, when Y ¹ is O, its oxygen atom and the oxygen atom of L ¹ are not adjacent, and the oxygen atom of OR ¹ and the oxygen atom of L ¹ are not adjacent.

In formula (I), L ² is an alkylene chain consisting of n _L carbon atoms. The hydrogen atoms on the alkylene carbon atoms may be substituted or unsubstituted, for example with OH, OR ^c , NH ₂ , NHR ^c , NR ^c R ^d , SH or SR ^c . Alternatively, L ² may be a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with an oxygen atom. Note that when Y ² is NH, O, or S, the L ² atom bonded to Y ² is carbon, the L ² atom bonded to OR ² is carbon, and oxygen atoms are not adjacent to each other. That is, for example, when Y ² is O, its oxygen atom and the oxygen atom of L ² are not adjacent, and the oxygen atom of OR ² and the oxygen atom of L ² are not adjacent.

m _L of L ¹ and n _L of L ² are not particularly limited, and the lower limit of each is, for example, 0, and the upper limit is also not particularly limited. n _L and m _L can be appropriately set, for example, depending on the desired length of the non-nucleotide structure. For example, n _L and m _L are each preferably from 0 to 30, more preferably from 0 to 20, and even more preferably from 0 to 15, from the viewpoint of production cost, yield, etc. n _L and m _L may be the same (n _L = m _L ) or different. n _L +m _L is, for example, 0 to 30, preferably 0 to 20, more preferably 0 to 15.

R ^a , R ^b , R ^c and R ^d are each independently a substituent or a protecting group, for example. The substituents and protecting groups are, for example, the same as those described above.

In formula (I), hydrogen atoms may be substituted with halogens such as Cl, Br, F and I, for example, each independently.

Nucleotide sequence T and nucleotide sequence Q are linked to a non-nucleotide structure, for example, via -OR ¹ - or -OR ² -, respectively. Here, R ¹ and R ² may or may not exist. When R ¹ and R ² are present, R ¹ and R ² are each independently a nucleotide residue or a structure of formula (I). When R ¹ and/or R ² are nucleotide residues, the structure of linker L may include, for example, a non-nucleotide residue consisting of the structure of formula (I) excluding the nucleotide residues R ¹ and/or R ² , and a nucleotide residue. It is formed from a group. When R ¹ and/or R ² have the structure of formula (I), the non-nucleotide structure is, for example, a structure in which two or more non-nucleotide residues having the structure of formula (I) are linked. The structure of formula (I) may contain, for example, 1, 2, 3 or 4. In this way, when a plurality of structures of formula (I) are included, the structures of formula (I) may be connected directly, for example, or may be bonded via nucleotide residues. On the other hand, if R ¹ and R ² are absent, the non-nucleotide structure is formed solely from non-nucleotide residues consisting of, for example, the structure of formula (I).

The combination of bonds between the nucleotide sequence T and the nucleotide sequence Q and -OR ¹ - and -OR ² - is not particularly limited, and includes, for example, any of the following conditions.
Condition (1)
The nucleotide sequence T is bonded to the structure of formula (I) via -OR ² -, and the nucleotide sequence Q is bonded to the structure of formula (I) via -OR ¹ -.
Condition (2)
The nucleotide sequence T is bonded to the structure of formula (I) via -OR ¹ -, and the nucleotide sequence Q is bonded to the structure of formula (I) via -OR ² -.

The structure of formula (I) can be exemplified by the following formulas (I-1) to (I-9), and in the following formula, n _L and m _L are the same as in formula (I). In the following formula, q is an integer from 0 to 10.

In formulas (I-1) to (I-9), n _L , m _L and q are not particularly limited and are as described above.
As specific examples, in formula (I-1), n _L =8, in formula (I-2), n _L =3, in formula (I-3), n _L =4 or 8, and in formula (I-4 ), n _L =7 or 8, in formula (I-5), n _L =3 and m _L =4, in formula (I-6), n _L =8 and m _L =4, formula (I- 7), n _L = 8 and m _L = 4, n _L = 5 and m _L = 4 in formula (I-8), and q = 1 and m _L = 4 in formula (I-9). It will be done. An example of formula (I-4) (n _L =8) is replaced with the following formula (I-4a), and an example of formula (I-8) (n _L =5, m _L =4) is replaced with the following formula (I-4a). -8a).

Further, a proline derivative linker represented by the following formula, which is an example of formula (I-8) (n _L =5, m _L =4), is shown below.

Examples of the single-stranded nucleic acid molecule for the mutant ALDH2 gene include the following nucleic acid molecules. L is a linker represented by the above structure, and spacer 1 and spacer 2 are preferably complementary.

5'-(SEQ ID NO: 2n)-(Spacer 1)-L-(Spacer 2)-(SEQ ID NO: 2n+1)-UU-3' (n is an integer from 1 to 11. UU may be tt)

In a particularly preferred embodiment, single-stranded nucleic acid molecules for the mutant ALDH2 gene include those having the following structure.

As a single-stranded nucleic acid molecule for the mutant ALDH2 gene, not only a hairpin-shaped nucleic acid molecule represented by T-L-Q but also linkers are added to both ends of a guide strand containing an expression suppressing sequence, and a guide strand is added to both ends of the guide strand containing an expression suppressing sequence. A single-stranded nucleic acid molecule having a dumbbell-shaped structure in which a nucleotide sequence complementary to one part of the strand and a nucleotide sequence complementary to the remaining part of the guide strand are bound together (for example, Japanese Patent No. 4968811, Japanese Patent No. 4965745, etc.) are also included.

Furthermore, the single-stranded nucleic acid molecule for the mutant ALDH2 gene may be a nucleic acid molecule having an L-type structure (L-type single-stranded nucleic acid molecule). L-type single-stranded nucleic acid molecules include (i) L-type nucleic acid molecules obtained by cross-linking the antisense strand and the sense strand between 2' and 3' or between 2' and 5' of the sugar moiety of the nucleoside; a single-stranded nucleic acid molecule, (ii) an L-type single-stranded nucleic acid molecule using an alkyl chain having an amide bond inside as a linker, (iii) an L-type single-stranded nucleic acid molecule in which nucleoside bases are directly linked; and (iv) an L-type single chain obtained by replacing 1' of the sugar moiety of the nucleoside with H and cross-linking the antisense strand and the sense strand between 2' and 2' of the sugar moiety of the nucleoside. stranded nucleic acid molecules.
The L-type single-stranded nucleic acid molecule has the following general formula:

[wherein X, Y, X ₁ , Y ₁ , X ₂ , Y ₂ are each independently an optionally modified ribonucleotide residue or an optionally modified deoxyribonucleotide residue,
T and Q are a sequence consisting of 14 to 30 consecutive optionally modified ribonucleotide residues complementary to the target nucleic acid sequence and a ribonucleotide sequence complementary thereto (one is complementary to the target nucleic acid sequence). If a sequence is complementary to one sequence, the other is a complementary sequence to it),
Z is a linker that connects the 2' or 5' position of the sugar moiety of (X) and the 2' or 3' position of the sugar moiety of (Y), or a linker that connects the base moiety of (X) and the 2' or 3' position of the sugar moiety of (Y). It is a linker that connects the base part,
m ₁ and m ₂ are each independently an integer of 0 to 5;
n ₁ and n ₂ are each independently an integer of 0 to 5]
A nucleic acid molecule represented by:
In a preferred embodiment, in the nucleic acid molecule of the present invention

The structure of

or

It is.
In the above formula,
A ₁ and A ₁ ' are each independently -O-, -NR ^1a -, -S- or -CR ^1a R ^1b - (here, R ^1a and R ^1b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₂ and A ₂ ' are each independently -CR ^2a R ^2b -, -CO-, an alkynyl group, an alkenyl group, or a single bond (here, R ^2a and R ^2b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₃ and A ₃ ' are each independently -O-, -NR ^3a -, -S-, -CR ^3a R ^3b - or a single bond (here, R ^3a and R ^3b are each independently is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₄ and A ₄ ' are each independently -(CR ^4a R ^4b )n-, -(CR ^4a R ^4b )n-ring D- (here, ring D is an aryl group having 6 to 10 carbon atoms) , a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms, or a heterocycloalkyl group having 4 to 10 carbon atoms, and R ^4a and R ^4b are each independently a hydrogen atom or a carbon number is an alkyl group of 1 to 10; n is an integer of 1 to 6) or a single bond,
A ₅ and A ₅ ' are each independently -NR ^5a - or a single bond (wherein R ^5a is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
A ₆ and A ₆ ' are each independently -(CR ^6a R ^6b )n- or a single bond (here, R ^6a and R ^6b are each independently a hydrogen atom or a carbon number of 1 to 10). is an alkyl group; n is an integer from 1 to 6),
W ₁ is -(CR ¹ R ² )n-, -CO-, -(CR ¹ R ² )n-COO-(CR ¹ R ² )n-COO-(CR ¹ R ² )n, -(CR ¹ R ² ) n-O-(CR ¹ R ² CR ¹ R ² O) n-CH ₂ -, -(CR ¹ R ² ) n-ring D-(CR ¹ R ² ) n- or -(CR ¹ R ² )n-SS-(CR ¹ R ² )n- (wherein, ring D is an aryl group having 6 to 10 carbon atoms, a heteroaryl group having 2 to 10 carbon atoms, or a heteroaryl group having 4 to 10 carbon atoms. It is a cycloalkyl group or a heterocycloalkyl group having 4 to 10 carbon atoms; R ¹ and R ² are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and n is an integer of 1 to 6. ),
E ₂ and E ₂ ' are each independently -CR ^2a R ^2b -, -CO-, an alkynyl group, an alkenyl group, or a single bond (here, R ^2a and R ^2b are each independently a hydrogen atom). or an alkyl group having 1 to 10 carbon atoms),
E ₃ and E ₃ ' are each independently -O-, -NR ^3a -, -S-, -CR ^3a R ^3b - or a single bond (here, R ^3a and R ^3b are each independently is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
E ₄ and E ₄ ' are each independently -(CR ^4a R ^4b )n-, -(CR ^4a R ^4b )n-ring D- (here, ring D is an aryl group having 6 to 10 carbon atoms); , a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms, or a heterocycloalkyl group having 4 to 10 carbon atoms; R ^4a and R ^4b are each independently a hydrogen atom or a carbon number is an alkyl group of 1 to 10; n is an integer of 1 to 6) or a single bond,
E ₅ and E ₅ ' are each independently -NR ^5a - or a single bond (wherein R ^5a is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms),
E ₆ and E ₆ ' are each independently -(CR ^6a R ^6b )n- or a single bond (here, R ^6a and R ^6b are each independently a hydrogen atom or a carbon number of 1 to 10). is an alkyl group: n is an integer from 1 to 6).

In the above formula, "alkyl group having 1 to 10 carbon atoms", "aryl group having 6 to 10 carbon atoms", "heteroaryl group having 2 to 10 carbon atoms", "cycloalkyl group having 4 to 10 carbon atoms", and The "heterocycloalkyl group having 4 to 10 carbon atoms" may be substituted at a substitutable position, and examples of the substituent include those described in Substituent Group A above.

Here, the L-type single-stranded nucleic acid molecule includes all of the target sequence of 25 consecutive nucleotides or less in the region represented by the nucleotide sequence represented by SEQ ID NO: 1, as a sequence that suppresses the expression of the mutant ALDH2 gene. Alternatively, the antisense strand contains a partially complementary sequence, but in a preferred embodiment, the expression suppressing sequence is one of the following nucleotide sequences (SEQ ID NO: 2k (k is an integer selected from 93 to 132)). ; However, in the sequence, U may be T) and a complementary sense strand (preferably SEQ ID NO: 2k-1 (k is an integer selected from 93 to 132; however, In the sequence, U may be T).

Upper row: sense strand, lower row: antisense strand

In a particularly preferred embodiment, the L-type single-stranded nucleic acid molecule for the mutant ALDH2 gene includes one having the following structure.

The method for synthesizing a single-stranded nucleic acid molecule for the mutant ALDH2 gene is not particularly limited, and conventionally known methods for producing nucleic acids can be employed. Examples of the synthesis method include a genetic engineering method, a chemical synthesis method, and the like. Genetic engineering methods include, for example, in vitro transcription synthesis methods, methods using vectors, and methods using PCR cassettes. Vectors are not particularly limited, and include non-viral vectors such as plasmids, viral vectors, and the like. The chemical synthesis method is not particularly limited, and examples include the phosphoramidite method and the H-phosphonate method. For the chemical synthesis method, for example, a commercially available automatic nucleic acid synthesizer can be used. In the chemical synthesis method, amidites are generally used. The amidite is not particularly limited, and commercially available amidites include, for example, RNA Phosphoramidites (2'-O-TBDMSi, trade name, Sansenri Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, etc. can give.

When the single-stranded nucleic acid molecule for the mutant ALDH2 gene is composed only of natural unmodified ribonucleotide residues, it is used as a precursor of the nucleic acid molecule in the form of a vector encoding the nucleic acid molecule in an expressible state. It can also be provided. The expression vector is characterized in that it contains a DNA encoding a single-stranded nucleic acid molecule for the mutant ALDH2 gene under the control of a promoter that is functional in the target cell, and other configurations are not limited at all. The vector into which the DNA is inserted is not particularly limited, and common vectors can be used, such as viral vectors and non-viral vectors. Examples of non-viral vectors include plasmid vectors. By introducing the expression vector into a target cell (a mammalian cell capable of expressing the mutant ALDH2 gene) using a gene introduction method known per se, the expression of the mutant ALDH2 gene within the cell is suppressed. be able to.

2. Mutant ALDH2 Gene Expression Suppressor/Treatment/Preventive Agent for Diseases Caused by ALDH2 Deficiency As described above, the nucleic acid molecule of the present invention can suppress the expression of the mutant ALDH2 gene. Therefore, the nucleic acid molecule of the present invention is effective in treating and preventing the onset of diseases caused by ALDH2 deficiency by suppressing the expression of mutant ALDH2. Diseases caused by ALDH2 deficiency include short stature and low weight after birth, mild mental developmental delay, and aplastic anemia. Furthermore, a relationship between ADD syndrome, Fanconi anemia, and ALDH2 deficiency has also been reported.

The medicament of the present invention may use an effective amount of the nucleic acid molecule of the present invention alone, or may be formulated as a pharmaceutical composition together with any carrier, for example, a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and Aerosil, citric acid, Flavoring agents such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinyl pyrrolid, dispersing agents such as surfactants, water, Examples include diluents such as physiological saline, base wax, etc., but are not limited thereto.

In order to promote the introduction of the nucleic acid molecule of the present invention into target cells, the medicament of the present invention can further contain a reagent for nucleic acid introduction. The nucleic acid introduction reagents include atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly(ethyleneimine) (PEI). Cationic lipids such as, for example, can be used.

In a preferred embodiment, the medicament of the present invention may be a pharmaceutical composition in which the nucleic acid molecule of the present invention is encapsulated in liposomes. Liposomes are microscopic closed vesicles that have an internal phase surrounded by one or more lipid bilayers, and can typically retain water-soluble substances in the internal phase and lipid-soluble substances within the lipid bilayer. As used herein, "encapsulation" refers to the nucleic acid molecule of the present invention that may be retained in the internal phase of a liposome or within a lipid bilayer. The liposome used in the present invention may be a monolayer or a multilayer, and the particle size can be appropriately selected within the range of, for example, 10 to 1000 nm, preferably 50 to 300 nm. Considering deliverability to the target tissue, the particle size may be, for example, 200 nm or less, preferably 100 nm or less.

Methods for encapsulating water-soluble compounds such as nucleic acids in liposomes include the lipid film method (vortex method), reversed-phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method. The method is not limited, and any known method can be selected as appropriate.

In another preferred embodiment, the medicament of the invention may be a pharmaceutical composition administered as a conjugate of the nucleic acid molecule of the invention with a ligand. An example is a nucleic acid molecule to which GalNAc (N-acetylgalactosamine), which is a type of sugar chain, is added. GalNAc strongly binds to the asialoglycoprotein receptor expressed specifically on the cell surface of hepatic parenchymal cells and is endocytosed, and this receptor undergoes active recycling during endocytosis and exocytosis. Therefore, nucleic acid molecules are efficiently drawn into hepatic parenchymal cells.

The medicament of the present invention is administered orally or parenterally to mammals (e.g., humans, cats, ferrets, mink, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys). Although it is possible to administer the drug parenterally, it is preferable to administer it parenterally.

Formulations suitable for parenteral administration (e.g., intravenous, subcutaneous, intramuscular, local, intraperitoneal, etc.) include aqueous and non-aqueous isotonic sterile injection solutions. may contain antioxidants, buffers, bacteriostatic agents, tonicity agents, and the like. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, and the like. The formulation can be packaged in units or multiple doses in containers such as ampoules and vials. Alternatively, the active ingredient and the pharmaceutically acceptable carrier can be lyophilized and stored by dissolving or suspending them in a suitable sterile vehicle immediately before use.
Other formulations suitable for parenteral administration include sprays and the like.

The content of the nucleic acid molecule of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.

The dosage of the medicament of the present invention varies depending on the purpose of administration, the method of administration, the type and severity of the target disease, and the circumstances of the subject (sex, age, body weight, etc.). For example, when administering systemically to adults, Usually, the single dose of the nucleic acid molecule of the present invention is preferably 2 nmol/kg or more and 50 nmol/kg or less, and when locally administered, it is preferably 1 pmol/kg or more and 10 nmol/kg or less. It is desirable to administer such a dose 1 to 10 times, more preferably 5 to 10 times.

The medicament of the present invention can be used, for example, in combination with other medicaments that have been reported to have therapeutic effects on diseases caused by ALDH2 deficiency. These concomitant drugs can be formulated together with the medicament of the present invention and administered as a single preparation, or alternatively, they can be formulated separately from the medicament of the present invention and administered by the same or different route as the medicament of the present invention. , they can be administered simultaneously or at staggered intervals. Further, the dosage of these combined drugs may be the amount normally used when the drugs are administered alone, or may be reduced from the amount normally used.

EXAMPLES The present invention will be explained in more detail by showing examples below, but the present invention is not limited to these examples at all.

Example 1: Synthesis of double-stranded RNA (siRNA) siRNA shown in Table 9 was synthesized based on the phosphoramidite method.

The definition of each symbol in the formula is as follows:
Underline: Variation of single nucleotide polymorphism Underline in italics: Mismatch Underline (wavy line): Bond between nucleotides is phosphorothioate bond □: 2'-Ome RNA
Lower case: 2'-F RNA

Example 2: Synthesis of L-type single-stranded nucleic acid molecule The nucleic acid molecules shown in Table 10 were synthesized using a nucleic acid synthesizer (trade name: ABI 3900 DNA Synthesizer, Applied Biosystems) based on the phosphoramidite method. EMM amidite (WO/2013/027843) or TBDMS amidite (Proligo) was used as the RNA amidite, and AEM amidite or AEC amidite was used as the 2′-position crosslinking amidite. Amidite deprotection was performed according to a conventional method. The synthesized nucleic acid molecules were purified by HPLC.

The definition of each symbol in the upper row is the Q array and the lower row is the T array formula is as follows:
Underline: Variation of single nucleotide polymorphism Underline in italics: Mismatch Underline (wavy line): Bond between nucleotides is phosphorothioate bond □: 2'-Ome RNA
Lower case: 2'-F RNA
Underlined fine print: Wobble pair
The line connecting the bases corresponds to -Z-, meaning that it can bind to a complementary strand at that site.

Example 3: Synthesis of single-stranded nucleic acid molecules Each single-stranded nucleic acid molecule shown in Table 11 was synthesized on the 3' side using a nucleic acid synthesizer (trade name: ABI 3900 DNA Synthesizer, Applied Biosystems) based on the phosphoramidite method. Synthesis was carried out toward the 5' side. For the synthesis, EMM amidite (WO/2013/027843) was used as RNA amidite. Furthermore, L-proline amidite (WO/2012/017919) was used for the linker region. Amidite deprotection followed the method described in WO/2013/027843. The synthesized single-stranded nucleic acid molecule was purified by HPLC.
The following L-proline amidite (hereinafter referred to as P) was used for the linker region of the single-stranded nucleic acid molecule of the present invention.

The definition of each symbol in the formula is as follows:
Underline: Variation of single nucleotide polymorphism Underline in italics: Mismatch Underline (wavy line): Bond between nucleotides is phosphorothioate bond □: 2'-Ome RNA
Lower case: 2'-F RNA
P: Proline derivative linker represented by the above formula

Example 4: ALDH2 mRNA KD activity evaluation (SNP selective RT-qPCR)
JHH-7 cells (distributed from the JCRB cell bank) were detached from the culture dish using a trypsin-EDTA solution, the number of cells was counted, and then William' containing 10% FBS was added to 2.5 x 10 ⁵ cells/mL. Adjusted with S E Medium (Thermo Fisher Scientific). Next, 99 μL of a mixture solution adjusted to 1 μL of Lipofectamine RNAiMAX (Invitrogen) was dispensed into each well of a 24-well plate to 98 μL of Opti-MEM (Invitrogen). Add 1 μL of each nucleic acid molecule (5 μM) prepared in Examples 1 to 3 to the wells, leave it for 15 minutes at room temperature, then add 400 μL of the cell suspension with adjusted cell number to the wells and start culturing. did.
After 24 hours of culture, total RNA was extracted and purified using RNeasy Mini Kit (QIAGEN). Incidentally, total RNA was extracted and purified using the RNeasy Mini Kit according to the product protocol. The concentration of the collected total RNA was measured using NanoDrop One (Thermo Fisher Scientific). After measurement, total RNA was diluted with water for injection (Otsuka Pharmaceutical) to a concentration of 10 ng/μL, and this was used as a template for reverse transcription reaction. PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio) was used for the reverse transcription reaction. The reaction mix was adjusted according to Table 12, and 5 μL of each reaction mix and 5 μL of the template were added to a 96-well PCR plate. After spinning down, the reaction was started using a thermal cycler. The reaction conditions are shown in Table 13.

After the reaction was completed, 140 μL of water for injection was added to each well. After stirring, it was spun down and stored at -30°C. For qPCR, Brilliant III Ultra-Fast SYBR (registered trademark) Green QPCR Master Mix (Agilent Technologies) was used. The reaction mix was adjusted according to Table 14. The sequence of the Forward Primer is 5'-AACAATTCCACGTACGGGCT-3' (SEQ ID NO: 182), and the sequence of the Reverse Primer for detecting wild-type ALDH2 is 5'-TGACTGTGACAGTTTTCTCTTC-3' (SEQ ID NO: 183). The sequence of the Reverse Primer for detecting mutant ALDH2 was 5'-TGACTGTGACAGTTTTCTCTTT-3' (SEQ ID NO: 184).

14 μL each of the adjusted reaction mix was added to a 96-well PCR plate, and 6 μL each of cDNA was added. After spinning down, the reaction was started using a real-time quantitative PCR system. The reaction conditions are shown in Table 15.

After the reaction was completed, the relative expression level of ALDH2 (wild type, mutant type) with respect to Mock was calculated from the Ct value of each sample by the ΔΔCt method. Note that the relative expression level was standardized using HPRT1.
The results are shown in Table 16.

Example 5: Measurement of ALDH2 enzyme activity Mitochondrial Aldehyde Dehydrogenase (ALDH2) Activity Assay Kit (abcam) was used to measure ALDH2 enzyme activity. cComplete ^™ Protease Inhibitor Cocktail was added to the 1× Extraction Buffer in the kit as a protease inhibitor.
JHH-7 cells were detached from the culture dish using a trypsin-EDTA solution, the number of cells was counted, and then adjusted to 2.5×10 ⁵ cells/mL with William's E Medium containing 10% FBS. Next, 1,980 μL of a mixed solution adjusted to 1,960 μL of Opti-MEM and 20 μL of Lipofectamine RNAiMAX per dish was dispensed into a 10 cm dish. Add 20 μL of each nucleic acid molecule (5 μM) prepared in Examples 1 to 3 to the dish, leave it for 15 minutes at room temperature, then add 8 mL of the cell suspension with adjusted cell number to the dish and culture. It started.
After 3 days of culture, JHH-7 cells were detached from the culture dish using a trypsin-EDTA solution, collected into a 1.5 mL tube, and centrifuged (1,000×g, 5 min, RT). The supernatant was removed, washed with PBS(-), and centrifuged (1,000×g, 5 min, RT). The supernatant was removed again, washed with PBS(-), and centrifuged (1,000×g, 5 min, RT). The supernatant was removed, and 50 μL of 1× Extraction Buffer containing protease inhibitor was added. After standing on ice for 20 minutes, it was centrifuged (16,000 x g, 20 min, 4°C). The supernatant was transferred to a new 1.5 mL tube and used as a sample. Some of the samples had total protein concentration determined by protein quantification. Subsequent operations followed the product protocol. Absorbance (450 nm) was measured every 10 minutes for 60 minutes, six times in total, using a microplate reader (Multiskan GO, Thermo Fisher Scientific). The slope of the approximate curve was calculated from the obtained absorbance, and this was divided by the protein concentration to obtain ALDH2 activity per unit protein, and the relative activity to Mock was determined. The results are shown in Table 16.

The present invention provides a nucleic acid molecule that can effectively suppress the expression of a mutant ALDH2 gene, and a medicament containing the nucleic acid molecule. The drug is extremely effective as a treatment and/or preventive agent for ADD (Aldehyde degradation deficiency)/AMeD (Aplastic anemia, mental retardation, and dwarfism) syndrome and Fanconi anemia by improving ALDH2 activity and improving formaldehyde metabolic abnormalities. Useful.

Claims (22)

A nucleotide sequence that is complementary to a nucleotide sequence of 15 or more consecutive nucleotides in a target sequence of 25 or less consecutive nucleotides in the region indicated by the nucleotide sequence represented by SEQ ID NO: 1 in the mutant ALDH2 gene is A nucleic acid molecule containing an ALDH2 gene expression suppressing sequence. (a) 15 or more consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 2n (n is an integer selected from 1 to 11) (in this sequence, each U may be T) array,
(b) A nucleotide sequence in which one or two nucleotides have been deleted, substituted, inserted, or added in the nucleotide sequence of (a), or (c) 90% or more identical to the nucleotide sequence of (a). is a nucleotide sequence having
The nucleic acid molecule according to claim 1. The nucleic acid molecule according to claim 2, further comprising a nucleotide sequence complementary to the expression suppressing sequence. The complementary nucleotide sequence is
(d) Nucleotide sequence represented by SEQ ID NO: 2n+1 (n is the same as in (a) above) (however, the pairing of G and U is considered complementary),
(e) A nucleotide sequence in which one or two nucleotides have been deleted, substituted, inserted, or added in the nucleotide sequence of (d), or (f) 90% or more identical to the nucleotide sequence of (d). is a nucleotide sequence having
The nucleic acid molecule according to claim 3. The nucleic acid molecule according to claim 4, comprising a nucleotide sequence represented by SEQ ID NO: 2n (n is an integer selected from 1 to 11) and a nucleotide sequence represented by SEQ ID NO: 2n+1. The nucleic acid molecule according to claim 2, which is siRNA against the mutant ALDH2 gene. 7. The nucleic acid molecule of claim 6, wherein the siRNA has a 3'-overhang on one or both strands. The nucleic acid molecule according to claim 7, comprising a nucleotide sequence represented by SEQ ID NO: 2m (m is an integer selected from 12 to 45) and a nucleotide sequence represented by SEQ ID NO: 2m+1 annealed to the sequence. The nucleic acid molecule according to claim 8, which has the structure of any one of the following (1) to (34).




During the ceremony,
Underlined are single nucleotide polymorphism mutations,
Mismatches are underlined in italics.
The underline (wavy line) indicates that the bond between nucleotides is a phosphorothioate bond.
The boxed nucleotides are 2'-OmeRNAs.
Nucleotides marked in lowercase letters indicate 2'-F RNA.
Each is shown below. General formula below:

[wherein, X, Y, X ₁ , Y ₁ , X ₂ , Y ₂ are each independently an optionally modified ribonucleotide residue or an optionally modified deoxyribonucleotide residue;
Z is a linker that connects the 2' or 5' position of the sugar moiety of (X) and the 2' or 3' position of the sugar moiety of (Y), or the base moiety of (X) and (Y) A linker that connects the base portion of;
Sequence T is a nucleotide sequence containing an expression suppressing sequence (Ta) of the mutant ALDH2 gene according to claim 1 or 2, and sequence Q is a nucleotide sequence containing a sequence (Qa) complementary to the expression suppressing sequence Ta. And;
m ₁ and m ₂ are each independently an integer of 0 to 5; and n ₁ and n ₂ are each independently an integer of 0 to 5]
Nucleic acid molecule shown as . The nucleic acid molecule according to claim 10, which has the structure of any one of the following (35) to (74).




During the ceremony,
Underlined are single nucleotide polymorphism mutations,
Mismatches are underlined in italics.
The underline (wavy line) indicates that the bond between nucleotides is a phosphorothioate bond.
The boxed nucleotides are 2'-OmeRNAs.
Nucleotides marked in lowercase letters indicate 2'-F RNA.
The underlined fine print indicates Wobble pair.
The line connecting the bases is a part corresponding to -Z-, and it is possible to bind to a complementary strand at this site.
each meaning. A nucleotide sequence T containing an expression suppressing sequence (Ta) of the mutant ALDH2 gene according to claim 2 and a nucleotide sequence Q containing a sequence (Qa) complementary to the expression suppressing sequence Ta are linked via a linker L. The expression suppressing sequence and its complementary sequence are linked in the 5' to 3' direction or 3' to 5' direction in the T-L-Q order, and in an orientation that allows the expression suppressing sequence and its complementary sequence to form a double strand within the molecule. Nucleic acid molecules. The nucleic acid molecule according to claim 12, wherein the linker L is a proline derivative linker represented by the following formula.
The sequence T has an additional sequence Tb at the 5' end of the sequence Ta, and the sequence Q has an additional sequence Qb at the 3' end of the sequence Qa, and the sequences Tb and Qb are complementary. The nucleic acid molecule according to item 12. 15. The nucleic acid molecule of claim 14, wherein the sequence T has a 3'-overhang. The nucleic acid molecule according to claim 15, having the structure of any one of the following (75) to (79).

During the ceremony,
Underlined are single nucleotide polymorphism mutations,
The italic underline indicates a mismatch, and the underline (wavy line) indicates that the bond between nucleotides is a phosphorothioate bond.
The boxed nucleotides are 2'-OmeRNAs.
Nucleotides marked in lowercase letters indicate 2'-F RNA.
P is a proline derivative linker,
Each is shown below. An expression vector expressing the nucleic acid molecule according to any one of claims 1 to 16. A medicament comprising the nucleic acid molecule according to any one of claims 1 to 6 or the expression vector according to claim 17. The medicament according to claim 18, which suppresses mutant ALDH2 gene expression and improves ALDH2 activity. The medicament according to claim 18, which is used for the treatment or prevention of a disease associated with ALDH2. 20. The medicament according to claim 19, wherein the disease associated with ALDH2 is short stature and low birth weight, mild mental retardation, and aplastic anemia. 20. The medicament according to claim 19, wherein the disease associated with ALDH2 is ADD syndrome and/or Fanconi anemia.