Method for designing oligonucleotide having reduced central toxicity.

Abstract

The present invention provides a method for designing an oligonucleotide having a reduced central toxicity, the method being characterized by including a step for substituting a carbon atom at the 5' position of a sugar portion of at least one nucleoside that constitutes an oligonucleotide having a phosphorothioate modification with a structure represented by formula I (in the formula, R6 and R7 are each independently a hydrogen atom, a halogen atom or a methyl group) or a structure represented by formula I' (in the formula, R4 and R5 are each independently a hydrogen atom, a methyl group or an ethyl group (excluding a case where R4 and R5 are both hydrogen atoms)).

Description

Method for designing oligonucleotides with reduced central toxicity

[0001] The present invention relates to a method for designing oligonucleotides with reduced central nervous system toxicity, oligonucleotides synthesized based on the design results, and therapeutic agents containing the oligonucleotides.

[0002] Antisense oligonucleotides exert their effects by hybridizing to target nucleic acid sequences and suppressing gene expression. Various artificial nucleic acids have been developed and introduced to improve the nuclease resistance of antisense oligonucleotides and their binding affinity and specificity to target nucleic acids, and various formulations have been brought to market. However, a new problem has recently emerged: how to avoid the potential toxicity of artificial nucleic acids.

[0003] The toxicity of antisense oligonucleotides can be categorized into two types: "toxicity due to hybridization with RNA (off-target toxicity)" and "toxicity due to binding to intracellular and extracellular proteins, metal ions, etc., that is not dependent on RNA hybridization (non-off-target toxicity)." Various approaches have been adopted to avoid these toxicities.

[0004] For example, Patent Document 1 discloses that non-off-target toxicity can be avoided by appropriate chemical modification of the base or sugar moiety of the nucleic acid. Patent Document 2 also discloses that off-target toxicity can be avoided by modifying the carbonyl group at the 2' position of the base (thymine) and bridging the 2'-4' positions of the sugar moiety, thereby suppressing and avoiding the formation of non-Watson-Crick base pairs during hybridization with mRNA. Furthermore, while phosphorothioate modifications between nucleosides are generally known to cause hepatotoxicity (e.g., Non-Patent Document 1), Patent Document 3 discloses that replacing phosphorothioate nucleic acids with artificial nucleic acids having a cyclopropane structure at the 5' position can reduce hepatotoxicity while maintaining activity. However, toxicity assessments related to antisense oligonucleotide toxicity reduction technologies to date have primarily focused on hepatotoxicity, and little insight has been gained into toxicity assessments in other tissues. For example, while central nervous system toxicity is an important endpoint in pharmaceutical safety testing, little is known about the central nervous system toxicity of antisense oligonucleotides.

[0005] As a finding focusing on the relationship between antisense oligonucleotides and central nervous system toxicity, particularly the reduction of central nervous system toxicity, Patent Document 4 discloses the correlation between the sequence of antisense oligonucleotides and central nervous system toxicity (oscillation of intracellular free calcium concentration in neurons). Furthermore, to the best of the inventors' knowledge, there is only one document evaluating the central nervous system toxicity due to phosphorothioate modifications in antisense oligonucleotides (Non-Patent Document 2). Non-Patent Document 2 discloses that central nervous system toxicity can be reduced by replacing phosphorothioate bonds in the wing portions of phosphorothioate-modified gapmer oligonucleotides with phosphodiester bonds.

[0006] International Publication No. WO 2018 / 155450 International Publication No. WO 2018 / 155451 International Publication No. WO 2020 / 158910 International Publication No. WO 2016 / 127000

[0007] Migawa MT et al., Nucleic Acids Res, 20;47(11):5465-5479 (2019)Moazami MP et al., bioRxiv, doi: https: / / doi.org / 10.1101 / 2021.02.14.431096 (2021)

[0008] However, Non-Patent Document 2 discloses that antisense oligonucleotides (ASOs) from which some phosphorothioate modifications have been removed and from which stability-related modifications have been added to the 2'-position of the sugar moiety may have reduced antisense activity compared to ASOs from which the phosphorothioate modifications have not been removed. The reduced antisense activity is thought to be due to the reduced stability of the ASO resulting from the removal of the phosphorothioate modifications.

[0009] Therefore, an object of the present invention is to provide a method for designing an ASO that can reduce central nervous system toxicity compared to the original ASO and preferably maintain or enhance antisense activity by modifying a site other than the 2' position of the sugar moiety of the ASO, and preferably further removing a portion of the phosphorothioate modification of the ASO, and to provide an ASO synthesized based on the design results.

[0010] The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they focused on modification of the 5' position of the sugar moiety, rather than the commonly used modification of the 2' or 4' position of the sugar moiety (e.g., Non-Patent Document 2) in nucleic acid modification. Specifically, as disclosed in Patent Document 3, they came up with the idea that by introducing a specific structure, such as a cyclopropane structure, into the 5' position of the sugar moiety of a phosphorothioate-modified nucleoside, it may be possible to reduce central nervous system toxicity mediated by inhibition of nonspecific protein binding while maintaining antisense activity. Patent Document 3 discloses that ASOs in which a modification is introduced into the 5' position of the sugar moiety of a nucleoside and the phosphorothioate bond near the modification is replaced with a phosphodiester bond reduce hepatotoxicity. However, because the mechanisms by which ASO-induced hepatotoxicity and central nervous system toxicity are thought to be significantly different, the above idea was completely original and not described or suggested in Patent Document 3 or prior art documents. Based on this idea, the present inventors conducted further extensive research and completed the present invention.

[0011]

[0013] That is, the present invention is as follows: [1] A method for designing an oligonucleotide with reduced central nervous system toxicity, comprising: (1) replacing the 5' carbon atom of the sugar moiety of at least one nucleoside constituting an oligonucleotide having a phosphorothioate modification with a nucleotide of the following formula I:

[0012]

[0013] (In the formula, R ６ and R ７ are each independently a hydrogen atom, a halogen atom, or a methyl group), or a group represented by the following formula I'

[0014]

[0015] (In the formula, R ４ and R ５ are each independently a hydrogen atom, a methyl group, or an ethyl group (provided that R ４ and R ５and (3) a phosphorothioate bond in the oligonucleotide having a structure represented by the formula (I) (except when both are hydrogen atoms), and optionally (2) a step of replacing at least one phosphorothioate bond in the oligonucleotide with a phosphodiester bond. [2] The method of [1], wherein the oligonucleotide is a gapmer-type oligonucleotide. [3] The method of [2], wherein at least one of the nucleosides substituted in step (1) is located in the gap region. [4] The method of [2] or [3], wherein at least one of the phosphorothioate bonds substituted in step (2) is located in the gap region. [5] The method of any one of [2] to [4], wherein at least one nucleoside constituting the 3' wing region and the 5' wing region is a bridged nucleoside. [6] A method for evaluating the degree of reduction in central toxicity of an oligonucleotide having a phosphorothioate modification, comprising the following steps (1) to (3): (1) a step of preparing an oligonucleotide having a phosphorothioate modification, (2) a step of preparing an oligonucleotide designed by the method described in any one of [1] to [5], and (3) a step of comparing the central toxicity of the oligonucleotide prepared in step (2) with the central toxicity of the oligonucleotide prepared in step (1). [7] An oligonucleotide having a phosphorothioate bond with reduced central toxicity, wherein the carbon atom at the 5' position of the sugar moiety of at least one nucleoside constituting the oligonucleotide is represented by the following formula I:

[0016]

[0017] (In the formula, R ６ and R ７ are each independently a hydrogen atom, a halogen atom, or a methyl group), or a group represented by the following formula I'

[0018]

[0019] (In the formula, R ４ and R ５ are each independently a hydrogen atom, a methyl group, or an ethyl group (provided that R ４ and R ５and I' are both hydrogen atoms), and optionally at least one internucleoside bond is a phosphodiester bond. [8] The oligonucleotide according to [7], which is a gapmer-type oligonucleotide. [9] The oligonucleotide according to [8], in which at least one nucleoside having a structure represented by formula I or I' is located in the gap region.

[10] The oligonucleotide according to [8] or [9], in which at least one of the phosphodiester bonds is located in the gap region.

[11] The oligonucleotide according to any one of [8] to

[10] , in which at least one nucleoside constituting the 3' wing region and the 5' wing region is a bridged nucleoside.

[12] A reagent for controlling gene expression, comprising the oligonucleotide according to any one of [7] to

[11] .

[13] A therapeutic drug for a disease, comprising the oligonucleotide according to any one of [7] to

[11] .

[14] A method for producing an oligonucleotide with low central nervous system toxicity, comprising: (1) designing an oligonucleotide by the method described in any one of [1] to [5], and (2) synthesizing the oligonucleotide designed in step (1).

[15] A method for regulating gene expression, comprising administering the oligonucleotide described in any one of [7] to

[11] .

[16] A method for treating a disease in a mammal, comprising administering to the mammal an effective amount of the oligonucleotide described in any one of [7] to

[11] .

[17] The oligonucleotide described in any one of [7] to

[11] for use in treating a disease.

[18] Use of the oligonucleotide described in any one of [7] to

[11] for producing a therapeutic drug for a disease.

[0020] The present invention makes it possible to design and produce safer antisense oligonucleotides with reduced central nervous system toxicity by, for example, replacing the phosphorothioate-modified nucleotides used in many antisense products with nucleotides modified at the 5' position of the sugar moiety.

[0021] Figure 1 shows the results of behavioral score measurements when an antisense oligonucleotide (LX-A4285) was intracerebroventricularly administered to mice. Figure 2 shows the results of evaluating the antisense effect when antisense oligonucleotides (LX-A0070, LX-A4285, LX-A5106, LX-A5108, and LX-A5113) were administered to mice with human neuroblastoma cells. Figure 3 shows the results of behavioral score measurements when antisense oligonucleotides (LX-A4285, LX-A5108, and LX-A5113) were administered to mice with intracerebroventricularly administered.

[0022] 1. Method for Designing Oligonucleotides with Reduced Central Toxicity The present invention was completed based on the discovery that oligonucleotides with reduced central toxicity can be designed by using nucleosides having a 5'-cyclopropane structure as modified nucleotides instead of phosphorothioate-modified nucleotides.

[0023] Specifically, the present invention provides a method for designing an oligonucleotide with reduced central nervous system toxicity, comprising the steps of: (1) modifying the 5′ carbon atom of the sugar moiety of at least one nucleoside constituting an oligonucleotide having a phosphorothioate modification (hereinafter, sometimes referred to as a “PS-modified nucleotide”) by a compound represented by the following formula I:

[0024]

[0025] (In the formula, R ６ and R ７ are each independently a hydrogen atom, a halogen atom, or a methyl group), or a group represented by the following formula I'

[0026]

[0027] (In the formula, R ４ and R ５ are each independently a hydrogen atom, a methyl group, or an ethyl group (provided that R ４ and R ５ and R are both hydrogen atoms) (hereinafter, this may be referred to as the "design method of the present invention"). ６and R ７ and are both hydrogen atoms. In the present specification, the oligonucleotide also includes pharmacologically acceptable salts of the oligonucleotide.

[0028] From the viewpoint of further reducing central toxicity, the design method of the present invention may include the step (2) of substituting at least one phosphorothioate bond in the oligonucleotide with a phosphodiester bond.

[0029] In another aspect, the present invention provides a method for reducing central nervous system toxicity of an oligonucleotide, comprising the steps of: (1′) modifying the 5′-position carbon atom of the sugar moiety of at least one nucleoside constituting a PS-modified nucleotide by a compound represented by the following formula I:

[0030]

[0031] (In the formula, R ６ and R ７ are each independently a hydrogen atom, a halogen atom, or a methyl group), or a group represented by the following formula I'

[0032]

[0033] (In the formula, R ４ and R ５ are each independently a hydrogen atom, a methyl group, or an ethyl group (provided that R ４ and R ５ and R are both hydrogen atoms) (hereinafter, this may be referred to as the "toxicity reduction method of the present invention"). ６ and R ７ are both hydrogen atoms.

[0034] The toxicity reduction method of the present invention may include the step of: (2') substituting at least one phosphorothioate bond in the oligonucleotide with a phosphodiester bond. Hereinafter, when matters apply to both the design method of the present invention and the toxicity reduction method of the present invention, these methods may be collectively referred to simply as the "method of the present invention."

[0035] In the present invention, "oligonucleotides having phosphorothioate modifications" refers to oligonucleotides in which at least one internucleoside bond is a phosphorothioate bond (i.e., the oxygen atom of the internucleoside phosphate group is replaced with a sulfur atom). Phosphorothioate modifications are generally used to improve the in vivo stability, particularly enzyme resistance, of natural oligonucleotides such as DNA and RNA. However, these phosphorothioate-modified oligonucleotides commonly have the problem of being toxic. The method of the present invention was developed as a result of research focused on central nervous system toxicity, one of the toxicities of phosphorothioate-modified oligonucleotides, and aimed at reducing this toxicity.

[0036] "Central toxicity" in the present invention is also referred to as "central neurotoxicity" and refers to toxic findings derived from the central nervous system identified by changes in general condition and histopathological changes in the brain in rodents, non-human primates, and humans. For example, in rodents, changes in general condition include reversible, mild symptoms such as irritability, decreased locomotor activity, bradypnea, and ptosis, as well as severe symptoms such as convulsions and death. Specifically, "central toxicity" in the present invention can be evaluated by behavioral scoring using a modified Irwin method (Irwin S., Psychopharmacologia. 1968; 13(3): 222-257).

[0037] In the present invention, "reduced central toxicity" means that the central toxicity of the oligonucleotide (i.e., the designed oligonucleotide or the oligonucleotide with reduced toxicity) (hereinafter, sometimes referred to as the "oligonucleotide after carrying out the present invention") obtained after carrying out step (1) or (1') of the method of the present invention is lower or is expected to be lower than the central toxicity of the PS-modified nucleotide (hereinafter, sometimes referred to as the "target oligonucleotide") that is the target (designed) of the method of the present invention. Specifically, as described in the Examples below, using the modified Irwin method, it can be evaluated that central toxicity has been reduced when the difference between the median behavioral score in the group administered with the target oligonucleotide and the median behavioral score in the group administered with the oligonucleotide after carrying out the present invention is 1 or more (e.g., 1, 5, 10, 20 or more), and the degree of central toxicity can also be evaluated based on such a value.

[0038] In step (1) of the design method of the present invention, it is not necessary to actually synthesize the oligonucleotide; it is sufficient to imagine it in one's mind. However, typically, the imagined oligonucleotide is embodied in a program (e.g., oligonucleotide design software, graphic design tool, office software, etc.) that runs on a computer or on paper. Therefore, for example, as described in Example 1 below, designing oligonucleotides that are expected to have reduced central nervous system toxicity and compiling the oligonucleotides in a table format also constitutes the implementation of the design method of the present invention.

[0039] The target oligonucleotide may be one that suppresses the function (post-transcriptional modification, translation, etc.) of a target mRNA (herein, mRNA includes pre-mRNA) containing a sequence complementary to the sequence of the oligonucleotide (hereinafter, sometimes referred to as an "inhibitory ASO"), or one that enhances the function (hereinafter, sometimes referred to as an "enhancing ASO"). Inhibitory ASOs typically form a double-stranded region with the target mRNA, and the double-stranded region is cleaved by ribonuclease H (RNase H), thereby suppressing the function of the target mRNA. On the other hand, enhancing ASOs typically form a double-stranded region with a splicing-promoting sequence of pre-mRNA, masking the sequence and skipping splicing, thereby enhancing (restoring) the function of the mRNA (which results in an increase in the amount of functional protein present in the cell). Alternatively, the function of mRNA can be enhanced by forming a double-stranded region with the binding sequence of an enzyme contained in the target mRNA that degrades the mRNA, masking the sequence and suppressing degradation of the mRNA.

[0040] From the viewpoint of in vivo stability and efficient mRNA cleavage, the inhibitory ASO is preferably a gapmer oligonucleotide. In the present invention, a "gapmer oligonucleotide" refers to a chimeric antisense oligonucleotide comprising an internal region (sometimes referred to herein as a "gap region") having multiple (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) nucleotides recognized by RNase H, sandwiched between external regions (sometimes referred to herein as the 3'-wing region and the 5'-wing region) having at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides modified to confer resistance to ribonucleases. At least one nucleoside constituting each of the 3'-wing region and the 5'-wing region is preferably a bridged nucleoside.

[0041] From the viewpoint of in vivo stability, the enhanced ASO preferably has at least one nucleoside modified to confer resistance to RNase. Such an ASO may have all nucleoside residues modified, or may have only some nucleoside residues modified (i.e., a mixmer oligonucleotide). Such a modified nucleoside is preferably a bridged nucleoside.

[0042] Examples of nucleosides constituting the subject oligonucleotide include nucleosides represented by the following formula II.

[0043]

[0044] (wherein, Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from group α, wherein the group α consists of a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom; and R ８ is a hydrogen atom and R ９ is a hydrogen atom, a halogen atom, or a linear alkoxy group having 1 to 6 carbon atoms which may be substituted with a linear alkoxy group having 1 to 6 carbon atoms.

[0045] Also, R ８ and R ９ together give the following formula:

[0046]

[0047] (In the formula, R ２１is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched or cyclic, an alkenyl group having 2 to 6 carbon atoms which may be branched or cyclic, an aryl group having 3 to 10 carbon atoms which may have one or more optional substituents selected from the α group and which may contain a heteroatom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may have one or more optional substituents selected from the α group and which may contain a heteroatom, or a protecting group for an amino group in nucleic acid synthesis; R ２２ and R ２３ are each independently a hydrogen atom; an alkyl group having 1 to 6 carbon atoms which may be substituted with an aryl group having 3 to 12 carbon atoms which may contain a heteroatom and which may be branched or cyclic; or an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a heteroatom; or R ２２ and R ２３ are combined, -(CH ２ ) ｑ - wherein q is an integer from 2 to 5; R ２４ and R ２５ are each independently a group selected from the group consisting of a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 6 carbon atoms which may be branched or cyclic; an alkoxy group having 1 to 6 carbon atoms which may be branched or cyclic; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or ２４ and R ２５ are combined, = C(R ３６ ) R ３７ [In the formula, R ３６ and R ３７ each independently represents a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, a linear or branched chain alkoxy group having 1 to 6 carbon atoms, a linear or branched chain alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a linear or branched chain alkylamino group having 1 to 6 carbon atoms; R ２６ and R ２７are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched or cyclic, an alkoxy group having 1 to 6 carbon atoms which may be branched or cyclic, or a linear or branched alkylthio group having 1 to 6 carbon atoms; R ２８ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched or cyclic, an alkoxy group having 1 to 6 carbon atoms which may be branched or cyclic, or a linear or branched alkylthio group having 1 to 6 carbon atoms; R ２９ is a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms which may be branched or cyclic, an alkoxy group having 1 to 6 carbon atoms which may be branched or cyclic, an amino group, or an amino group protected by a protecting group for nucleic acid synthesis; R ３１ , R ３２ , and R ３３ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched or cyclic, or a protecting group for an amino group in nucleic acid synthesis; R ３４ and R ３５ are each independently a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms which may be branched or cyclic, an alkoxy group having 1 to 6 carbon atoms which may be branched or cyclic, an amino group, or an amino group protected with a protecting group for nucleic acid synthesis; m is an integer of 0 to 2; n is an integer of 0 to 1; p is an integer of 0 to 1; X １ is an oxygen atom, a sulfur atom, or an amino group; and X ２ represents a divalent group represented by the formula: wherein R is an oxygen atom or a sulfur atom.) A nucleoside having such a structure is sometimes referred to as a bridged nucleoside.

[0048] More specifically, examples of the bridged nucleoside include nucleosides having the following structural formula:

[0049]

[0050] (In the formula, R represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may be branched or cyclic, an alkenyl group having 2 to 7 carbon atoms which may be branched or cyclic, an aryl group having 3 to 12 carbon atoms which may contain a heteroatom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a heteroatom, or a protecting group for an amino group in nucleic acid synthesis. Preferably, R represents a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, and more preferably, R represents a hydrogen atom or a methyl group. The definition of Base is the same as that in Formula II above.)

[0051] The nucleosides constituting the subject oligonucleotide may be natural nucleosides, and examples of such natural nucleosides include adenosine, N6-methyladenosine, guanosine, uridine, 5-methyluridine, cytidine, deoxyadenosine, N6-methyl-2'-deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, and 5-methyl-2'-deoxycytidine.

[0052] The length of the target oligonucleotide is not particularly limited as long as it has antisense activity, but is typically 10 to 50 nucleotides in length, preferably 10 to 30 nucleotides in length, more preferably 13 to 30 nucleotides in length, and even more preferably 15 to 20 nucleotides in length.

[0053] At least one nucleoside constituting the oligonucleotide after carrying out the present invention can be represented by the following formula III or III'.

[0054]

[0055] (In the formula, R ６ , R ７ , R ８ , R ９ The definitions of Base are the same as those in Formula I and Formula II above. ６ and R ７ are both hydrogen atoms.)

[0056]

[0057] (In the formula, R ４ , R ５ , R ８ , R ９ The definitions of Base are the same as those in Formula I' and Formula II above.

[0058] The nucleoside having the structure represented by formula I or formula I' as a result of step (1) or (1') of the method of the present invention may be located at any position in the target PS-modified nucleotide, but it is preferred that at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleoside is located in the gap region.

[0059] The phosphorothioate bond to be replaced as a result of step (2) or (2') of the method of the present invention may be present between any nucleosides of the target PS-modified nucleotide, but it is preferred that at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) internucleoside phosphorothioate bond is located in the gap region. In other words, it is preferred that in step (2) or (2') of the method of the present invention, the phosphorothioate bond located in the gap region of the target PS-modified nucleotide is replaced with a phosphodiester bond.

[0060] Furthermore, the phosphorothioate bond that is substituted as a result of step (2) or (2') of the method of the present invention may be a bond adjacent to the 5' or 3' side of the nucleoside substituted in step (1) or (1') of the method of the present invention, or may be a bond that is not adjacent to it. In other words, the nucleotide residues that are the target of step (1) or (1') and step (2) or (2') of the present invention can each be selected arbitrarily.

[0061] In the method of the present invention, in addition to substitution of the 5' carbon atom of the sugar moiety of a nucleoside or substitution of a phosphorothioate bond, further modifications may be made. Examples of such modifications include, but are not limited to, 2'-O-methoxyethyl modification of the sugar moiety, 2'-O-methyl modification of the sugar moiety, 2'-fluoro modification of the sugar moiety, cross-linking between the 2' and 4' positions of the sugar moiety (substitution with a bridged nucleoside), substitution of a bridged nucleoside with another bridged nucleotide, methylation or acetylation of the base (base moiety), and base substitution. When substituting the base, it is preferable to substitute a base that can form a base pair with the base that is the target of the base before substitution (i.e., that forms a base pair with the base). The base pair may be not only a Watson-Crick base pair, but also a Hoogsteen base pair or a wobble base pair. Examples of bridged nucleosides include nucleosides having the following structural formula:

[0062]

[0063] (In the formula, R represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may be branched or cyclic, an alkenyl group having 2 to 7 carbon atoms which may be branched or cyclic, an aryl group having 3 to 12 carbon atoms which may contain a heteroatom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a heteroatom, or a protecting group for an amino group in nucleic acid synthesis. Preferably, R represents a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, and more preferably, R represents a hydrogen atom or a methyl group. The definition of Base is the same as that in Formula II above.)

[0064] By carrying out step (1) or (1') of the method of the present invention on PS-modified nucleotides, an oligonucleotide having reduced central nervous system toxicity compared to PS-modified nucleotides can be designed, or the central nervous system toxicity of PS-modified nucleotides can be reduced. However, it is also possible to actually confirm that the central nervous system toxicity of the oligonucleotides is reduced after carrying out the present invention, and it is also possible to evaluate the degree of reduction in the central nervous system toxicity of the oligonucleotides after carrying out the present invention.

[0065] Therefore, in yet another aspect of the present invention, there is provided a method for evaluating the degree of reduction in central toxicity of a PS-modified nucleoside, comprising the following steps (I) to (III): (I) preparing a PS-modified nucleotide; (II) preparing an oligonucleotide designed by the design method of the present invention; and (III) comparing the central toxicity of the oligonucleotide prepared in step (II) with the central toxicity of the oligonucleotide prepared in step (I) (hereinafter, this may be referred to as the "evaluation method of the present invention").

[0066] The PS-modified nucleotides prepared in step (I) of the evaluation method of the present invention can be obtained by purchasing commercially available products, by producing them by known synthesis methods, or by having others produce them by outsourcing synthesis, etc. The oligonucleotides prepared in step (II) of the evaluation method of the present invention can be obtained by producing them by known synthesis methods described below based on the structure of an oligonucleotide designed by the design method of the present invention, or by having others produce them by outsourcing synthesis, etc.

[0067] Step (II) of the evaluation method of the present invention can be performed using the method of calculating scores by the above-mentioned modified Irwin method or other known methods. Step (III) of the evaluation method of the present invention can be performed by comparing the above scores, etc.

[0068] Oligonucleotides designed by the methods of the present invention not only exhibit reduced central nervous system toxicity, but also maintain antisense activity. In the present invention, "maintaining antisense activity" means that the antisense activity of an oligonucleotide after carrying out the present invention is maintained or is higher than that of a target oligonucleotide. Such antisense activity can be evaluated in the case of inhibitory ASOs, for example, by contacting SH-SY5Y cells with the same amount of each antisense oligonucleotide, recovering mRNA from the cells after 24 hours, and comparing the amount of mRNA, as described in the Examples below. If there is no statistically significant difference or a decrease in the amount of target mRNA in a group contacted with an oligonucleotide after carrying out the present invention compared to a group contacted with a target oligonucleotide, it can be evaluated that antisense activity has been maintained. On the other hand, in the case of enhancing ASOs, the same amount of each antisense oligonucleotide can be contacted with SH-SY5Y cells, recovering the protein encoded by the target mRNA from the cells after 24 hours, and comparing the amount of functional protein among the proteins.

[0069] Therefore, the method of the present invention may further include a step of evaluating the antisense activity of the oligonucleotide after carrying out the present invention, which can be carried out using the above-mentioned method of measuring the amount of the target mRNA or the protein encoded by the mRNA, or other known methods.

[0070] 2. Method for Producing Low Central Toxicity Oligonucleotides In another aspect, the present invention provides a method for producing low central toxicity oligonucleotides (hereinafter, sometimes referred to as "the method of the present invention"), comprising: (1) designing an oligonucleotide by the design method of the present invention; and (2) synthesizing the oligonucleotide designed in step (1).

[0071] In the present invention, " low-toxicity oligonucleotide " refers to the oligonucleotide that has or is expected to have low central toxicity compared with target oligonucleotide.Specifically, as described in the examples below, by using the above-mentioned Irwin's modified method, when the difference between the median behavioral score of the group that receives target oligonucleotide and the median behavioral score of the group that receives oligonucleotide after the present invention is 1 or more (for example, 1, 5, 10, 20 or more), it can be evaluated as the oligonucleotide with low central toxicity, and this value can also be used to evaluate the degree of central toxicity.

[0072] The method for synthesizing the oligonucleotide in step (2) of the production method of the present invention is not particularly limited, and known methods can be used. Examples of such known methods include chemical synthesis methods such as the phosphoramidite method and the H-phosphonate method. In the chemical synthesis method, a commercially available automated nucleic acid synthesizer can be used. In addition, amidites are generally used in the chemical synthesis method. The amidites are not particularly limited, and may be prepared by known methods (e.g., methods described in Patent Document 3 and WO 2020 / 166551), or commercially available amidites may be used.

[0073] The oligonucleotide produced by the method of the present invention may be subjected to a step similar to step (III) of the evaluation method of the present invention to actually evaluate central toxicity.

[0074] 3. Oligonucleotides with Reduced Central Toxicity As described above, the present invention allows the design or production of oligonucleotides with reduced central toxicity. Accordingly, in another aspect, there is provided an oligonucleotide having a phosphorothioate bond with reduced central toxicity, wherein the 5' carbon atom of the sugar moiety of at least one nucleoside constituting the oligonucleotide is represented by the following formula I:

[0075]

[0076] (In the formula, R ６ and R ７ is the same as that in formula I in 1 above), or the following formula I'

[0077]

[0078] (In the formula, R ４ and R ５ The definition of R is the same as that in formula I' in 1 above.) In one embodiment, R ６ and R ７ and are both hydrogen atoms. In the ASO of the present invention, from the viewpoint of further reducing central toxicity, it is preferable that at least one internucleoside bond is a phosphodiester bond.

[0079] Therefore, at least one nucleoside constituting the ASO of the present invention can be represented by the following formula III or III'.

[0080]

[0081] (In the formula, R ６ , R ７ , R ８ , R ９ The definitions of Base are the same as those in Formula I and Formula II of 1. In one embodiment, R ６ and R ７ are both hydrogen atoms.)

[0082]

[0083] (In the formula, R ４ , R ５ , R ８ , R ９ The definitions of Base are the same as those in Formula I' and Formula II of 1.

[0084] The ASO of the present invention may be an inhibitory ASO or an activating ASO. In the case of an inhibitory ASO, it is preferably a gapmer-type oligonucleotide, but is not limited thereto. In the case of an activating ASO, it is preferably a mixmer-type oligonucleotide, but is not limited thereto.

[0085] Nucleosides having the structure represented by Formula I or Formula I' above may be present at any position in the ASO of the present invention, but it is preferred that at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleoside is located in the gap region.

[0086] When a phosphodiester bond is present in the internucleoside linkages of an ASO of the present invention, the phosphodiester bond may be present between any nucleosides of the ASO of the present invention, but it is preferred that at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) internucleoside phosphodiester bond is located in a gap region. Furthermore, when a phosphodiester bond is present in the internucleoside linkages of an ASO of the present invention, the phosphodiester bond may or may not be adjacent to the 5' or 3' side of a nucleoside having the structure represented by Formula I or Formula I' above.

[0087] In the ASO of the present invention, at least one nucleoside constituting each of the 3' wing region and the 5' wing region is preferably a bridged nucleoside, such as the same bridged nucleosides described in 1. above.

[0088] The ASO of the present invention can suppress or enhance the function of target mRNA, thereby controlling gene expression. Therefore, the ASO of the present invention can be used as a reagent for controlling gene expression (hereinafter, sometimes referred to as the "reagent of the present invention"). The term "reagent for controlling gene expression" encompasses both the terms "reagent for suppressing gene expression" and "reagent for enhancing gene expression."

[0089] When the reagent of the present invention contains two or more ASOs or other reagents, the reagent can be provided as a reagent kit containing each ASO or reagent separately. In the present invention, unless otherwise specified, the term "gene expression" is used to mean at least "the production of a functional protein encoded by a target mRNA," but also "the production of a target mRNA." Therefore, suppression of gene expression may include not only the reduction in the amount of functional protein encoded by the gene present in cells by administration of the ASO of the present invention, but also the reduction in the amount of mRNA transcribed from the gene present in cells. Similarly, enhancement of gene expression may include not only the increase in the amount of functional protein encoded by the gene present in cells by administration of the ASO of the present invention, but also the increase in the amount of mRNA transcribed from the gene present in cells.

[0090] The reagent of the present invention can be used to administer the ASO alone or together with a pharmacologically acceptable carrier to a subject. Examples of subjects to which the ASO is to be administered include cells, tissues, and organs of mammals, including humans. Therefore, a method for regulating gene expression in a subject, comprising administering the ASO of the present invention to the subject, is also provided.

[0091] To facilitate the delivery of the ASO of the present invention into target cells, the reagent of the present invention may further comprise a nucleic acid delivery reagent, such as calcium chloride, calcium enrichment reagents, atelocollagen, liposomes, nanoparticles, lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or cationic lipids such as poly(ethyleneimine) (PEI).

[0092] As mentioned above, the ASO of the present invention has reduced central nervous system toxicity, making it particularly suitable for use as a pharmaceutical. Therefore, a therapeutic agent for a disease (hereinafter, sometimes referred to as the "therapeutic agent of the present invention") comprising the ASO of the present invention is provided. In the present invention, "treatment" also includes alleviating or ameliorating symptoms, and preventing, delaying, or halting the progression of a disease or symptom, or the manifestation of symptoms. Examples of such diseases include those caused by the overexpression or excessive accumulation of a protein encoded by the target mRNA of the ASO of the present invention, or the deletion or reduction of a protein encoded by the target mRNA of the ASO of the present invention. Examples of such diseases include neurological diseases (e.g., neurodegenerative diseases such as tauopathy).

[0093] As a therapeutic agent of the present invention, an effective amount of the ASO of the present invention may be used alone, or may be formulated as a pharmaceutical composition together with any carrier, such as a pharmaceutically acceptable carrier (the formulated product is also referred to as a therapeutic agent).

[0094] Examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, flavorings such as citric acid and menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolide, dispersing agents such as surfactants, diluents such as water and physiological saline, and base waxes.

[0095] To promote the introduction of the ASO of the present invention into target cells, the therapeutic agent of the present invention may further contain a nucleic acid transfer reagent, which may be the same as those described above.

[0096] The therapeutic agent of the present invention may also be a pharmaceutical composition comprising the ASO of the present invention encapsulated in a liposome. Liposomes are small, closed vesicles with an internal phase surrounded by one or more lipid bilayers, and can typically hold a water-soluble substance in the internal phase and a lipid-soluble substance within the lipid bilayer. As used herein, the term "encapsulated" refers to the ASO of the present invention being held either in the internal phase of the liposome or within the lipid bilayer. The liposomes used in the present invention may be monolayer or multilayer membranes, and the particle size can be appropriately selected, for example, within the range of 10 to 1,000 nm, preferably 50 to 300 nm. In consideration of delivery to target tissues, the particle size is, for example, 200 nm or less, preferably 100 nm or less.

[0097] Methods for encapsulating water-soluble compounds such as oligonucleotides into liposomes include, but are not limited to, the lipid film method (vortex method), reverse phase evaporation, surfactant removal method, freeze-thaw method, and remote loading method, and any known method can be appropriately selected.

[0098] The pharmaceutical composition of the present invention can be administered orally or parenterally to mammals (e.g., humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cattle, dogs, cats, and monkeys), preferably parenterally. Therefore, a method for treating a disease in a mammal is also provided, which comprises administering an effective amount of the ASO of the present invention to the mammal.

[0099] Suitable formulations for parenteral administration (e.g., subcutaneous injection, intramuscular injection, intravenous infusion, local injection (topical application, local application), intraventricular administration, intrathecal administration, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. Such formulations can be packaged in unit-dose or multi-dose containers such as ampoules or vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that only requires dissolution or suspension in an appropriate sterile vehicle immediately before use. Other formulations suitable for parenteral administration include aerosols, etc.

[0100] The content of the ASO of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the total pharmaceutical composition.

[0101] The dosage of the therapeutic agent of the present invention varies depending on the purpose of administration, the administration method, the type and severity of the target disease, and the condition of the patient (sex, age, body weight, etc.), but for example, when administered systemically to an adult, the ASO of the present invention is usually administered at a single dose of 0.01 mg / kg to 1000 mg / kg, and when administered locally, it is preferably administered at a single dose of 0.001 mg / body to 100 mg / body. It is desirable to administer such a dose 1 to 10 times, more preferably 5 to 10 times.

[0102] The therapeutic agents of the present invention can also be used in combination with, for example, therapeutic agents for diseases that are already on the market. These concomitant drugs can be formulated together with the pharmaceutical agent of the present invention and administered as a single formulation, or they can be formulated separately from the pharmaceutical agent of the present invention and administered simultaneously or at different times via the same or a different route as the pharmaceutical agent of the present invention. The dosage of these concomitant drugs may be the amount normally used when the drug is administered alone, or it may be reduced from the amount normally used.

[0103] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0104] Example 1: Design and Synthesis of Antisense Nucleotides To evaluate the central nervous system toxicity of antisense oligonucleotides, the following oligonucleotides were designed and synthesized. LX-A4285 has a phosphorothioate internucleoside linkage and is a sequence known to exhibit central nervous system toxicity according to WO 2016 / 127000 A1. In the table, 5 represents 5-methylcytosine, Ln represents LNA, ^ represents a phosphorothioate linkage, and Cp represents DNA with a cyclopropane structure introduced at the 5' position of the sugar moiety (referred to herein as "5'-cyclopropane DNA").

[0105]

[0106]

[0107] Oligonucleotides related to the present invention were synthesized by the methods described in Tetrahedron Letters 22, 1859-1862 (1981), WO 2011 / 052436, etc.

[0108] As the sugar-modified nucleoside, a locked nucleic acid (LNA) represented by formula (a) or a 5'-cyclopropane DNA (5'-CP-DNA) represented by formula (b) was used.

[0109]

[0110]

[0111] (In the formula, Base is a 5-methylcytosin-1-yl group, a thymin-1-yl group, an adenin-9-yl group, or a guanin-9-yl group.)

[0112] Oligonucleotides containing 5'-cyclopropane DNA (5'-CP-DNA) were synthesized with reference to the method described in WO 2020 / 158910.

[0113] Oligonucleotides containing locked nucleic acid (LNA) or 5'-cyclopropane DNA (5'-CP-DNA) were synthesized using an automated nucleic acid synthesizer (nS-8, Gene Design, Inc.). Chain elongation was performed using a standard phosphoramidite protocol (solid-phase support: CPG, iodine was used for oxidation to form the phosphorodiester (PO) backbone, and DDTT (((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione) was used for sulfuration to form the phosphorothioate (PS) backbone). Oligonucleotides containing locked nucleic acid (LNA) or 5'-cyclopropane DNA (5'-CP) were obtained in which the terminal 5' hydroxyl group was not protected with a 4,4'-dimethoxytrityl (DMTr) group and the 3' position was supported on the solid phase. Subsequently, the target compound was cleaved from the solid support by treatment with a base, and the solvent was then distilled off. The resulting crude product was purified by reverse phase HPLC to obtain the target compound.

[0114] The purity and structure of each of the resulting oligonucleotides were confirmed by LC-MS (Waters).

[0115] Example 2: Development of a method for evaluating the toxicity of antisense nucleotides in the mouse central nervous system BALB / cCrSlc (male, 7 weeks old, Japan SLC Co., Ltd.) was used to evaluate the central toxicity of antisense oligonucleotides in mice. The animals were housed in an open rack under an environment of temperature: 18-28°C, humidity: 30-80%, and light exposure: 12 hours / day, and all animals were allowed free access to food and water.

[0116] Antisense oligonucleotides were administered intracerebroventricularly to mice to assess central nervous system toxicity. Under deep anesthesia with a triple anesthetic mixture (butorphanol tartrate 5 mg / kg, midazolam 4 mg / kg, and medetomidine hydrochloride 0.3 mg / kg) administered intraperitoneally at a volume of 10 mL / kg, mice were fixed in a stereotaxic apparatus. The skull was exposed, and a hole was drilled at the injection site (brain coordinates: −0.2 mm caudal to the bregma, −1.0 mm to the right, −2.5 mm deep) using a dental drill. A single dose of the antisense oligonucleotide was administered intracerebroventricularly using a 30G needle, microsyringe, and polyethylene tubing. The antisense oligonucleotide used was LX-A4285, listed in Table 1.

[0117] Behavioral analysis of mice was performed 1 to 24 hours after a single intracerebroventricular administration of LX-A4285 using a modified Irwin method. Evaluation items included spontaneous movement, rearing, hyperactivity, walking, grooming, ptosis, vocalization, tremors, and convulsions.

[0118] The results are shown in Figure 1. The scores for LX-A4285 were calculated from the deviation from the normal score using the modified Irwin method and are shown as the median for each group. Normal behavior is indicated by a score of approximately 0, whereas LX-A4285, which showed a deviation, was shown to cause central nervous system toxicity. In other words, a method for evaluating the toxicity of antisense nucleotides in the mouse central nervous system has been properly established.

[0119] Example 3: In vitro mRNA expression suppression by antisense nucleotides in human neuroblastoma cells The antisense oligonucleotides used in Example 1 above were used to evaluate the MAPT mRNA expression suppression effect in human neuroblastoma cells. A sample without oligonucleotide addition served as a control. For comparison, a negative control oligonucleotide (also referred to as "Negative Control; NC") was used: 5(Y)^A(Y)^T(Y)^t^t^a^g^a^a^g^t^c^5(Y)^T(Y)^c (LX-A0070; SEQ ID NO: 6). In this sequence, 5 represents 5-methylcytosine, and Y represents AmNA.

[0120] SH-SY5Y cells (ECACC, EC94030304-F0) were used as human neuroblastoma cells. Each antisense oligonucleotide (LX-A0070, LX-A4285, LX-A5106, LX-A5108, and LX-A5113) was transfected into SH-SY5Y cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine 3000). mRNA expression levels were measured by qRT-PCR, and knockdown activity (mRNA expression suppression) was examined. The procedure is as follows: 3.0 x 10 SH-SY5Y cells in the logarithmic growth phase were transfected into SH-SY5Y cells. 4 The cells were seeded at 1000 cells / well in a 24-well plate containing an equal ratio mixture of Ham's F12 and Eagle's minimum essential medium (E-MEM) containing 10% fetal bovine serum (FBS). After 24 hours, each antisense oligonucleotide was added to the wells (antisense oligonucleotide concentration in the medium: 1, 3, 10, 30, or 100 nM) and incubated for 24 hours or longer.

[0121] After incubation, cells were harvested and total RNA was extracted using an RNA extraction reagent (Macherey-Nagel, Nucleo ZOL). Using the total RNA as a template, reverse transcription and PCR amplification were performed using a nucleic acid amplification reagent (Qiagen, QuantiFast Probe RT-PCR kit). Nucleic acid amplification was performed using a temperature cycling protocol of 50°C for 10 minutes, 95°C for 5 minutes, and then 40 cycles of 95°C for 10 seconds, followed by 60°C for 30 seconds. In real-time PCR, the mRNA level of the housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also quantified, and the relative mRNA level of MAPT was evaluated. The mRNA levels of each antisense oligonucleotide are shown relative to the mRNA level of cells without oligonucleotides, which is set to 1.

[0122] The primer sets used are as follows: (Primer set for MAPT detection) TaqMan Gene Expression Assay Hs00902194_m1_4331182 (ThermoFisher Scientific) (Primer set for GAPDH detection) TaqMan Gene Expression Assay Hs02786624_g1_4331182 (ThermoFisher Scientific)

[0123] The results are shown in Figure 2. Compared to cells without oligonucleotides ("control") and cells with negative control, the antisense oligonucleotides demonstrated dose-dependent knockdown activity (mRNA expression suppression). Therefore, it was concluded that antisense nucleotides with 5'-modified nucleosides maintained their mRNA expression suppression activity.

[0124] Example 4: Acute toxicity of antisense nucleotides in the mouse central nervous system Two representative antisense nucleotides (LX-A5108 and LX-A5113) were selected from the antisense nucleotides listed in Tables 1 and 2 that maintained knockdown activity while exhibiting low cytotoxicity (data not shown), and the acute toxicity of these nucleotides in the mouse central nervous system was evaluated. The evaluation method followed the method described in Example 2. The results are shown in Figure 3. Figure 3 confirms that, compared to antisense nucleotides containing only phosphorothioate bonds, sequences containing 5'-CP and an increased proportion of phosphodiester bonds exhibit reduced central toxicity.

[0125] From the results of Examples 3 and 4 above, it was confirmed that antisense nucleotides having a 5'-modified nucleoside retain the activity of suppressing mRNA expression while reducing central nervous system toxicity.

[0126] The present invention enables the design of oligonucleotides with reduced central nervous system toxicity. Oligonucleotides synthesized based on the design results can be administered to cells or individuals to control gene expression while suppressing side effects, and can therefore be used as therapeutic agents for diseases.

[0127] This application is based on Japanese Patent Application No. 2021-078507 (filing date: May 6, 2021) and Japanese Patent Application No. 2021-151543 (filing date: September 16, 2021), the contents of which are incorporated in their entirety herein.

Claims

1. A method for designing an oligonucleotide having reduced central nervous system toxicity, comprising: (1) The 5' carbon atom of the sugar moiety of at least one nucleoside constituting an oligonucleotide having a phosphorothioate modification is replaced with a carbon atom of the following formula I: 【Chemistry 1】 (In the formula, R ６ and R ７ are each independently a hydrogen atom, a halogen atom, or a methyl group. Or the following formula I' 【Chemistry 2】 (In the formula, R ４ and R ５ are each independently a hydrogen atom, a methyl group, or an ethyl group (provided that R ４ and R ５ (Except when both are hydrogen atoms) and optionally replacing the structure represented by (2) replacing at least one phosphorothioate bond in the oligonucleotide with a phosphodiester bond; A method comprising:

2. The method of claim 1 , wherein the oligonucleotide is a gapmer-type oligonucleotide.

3. 3. The method of claim 2, wherein at least one of the nucleosides substituted in step (1) is located in the gap region.

4. The method of claim 2, wherein at least one of the phosphorothioate bonds replaced in step (2) is located in a gap region.

5. The method of claim 2, wherein at least one nucleoside constituting the 3' wing region and the 5' wing region is a bridged nucleoside.

6. (3) The method described in claim 1, comprising a step of confirming a reduction in the central neurotoxicity of the designed oligonucleotide, or a step of evaluating the degree of reduction in the central neurotoxicity of the designed oligonucleotide.

7. The method according to claim 1, wherein the number of nucleosides substituted in step (1) is 1 to 4.

8. The method according to claim 1, wherein the number of nucleosides substituted in step (1) is 1.

9. The method according to claim 1, wherein the number of nucleosides substituted in step (1) is two.

10. The method according to claim 1, wherein the number of nucleosides substituted in step (1) is three.

11. The method according to claim 1, wherein the number of nucleosides substituted in step (1) is four.

12. The method of claim 1, wherein at least one of the phosphorothioate bonds substituted in step (2) is adjacent to the 5' end of the nucleoside substituted in step (1).

13. The method according to claim 2, wherein at least one of the phosphorothioate bonds substituted in step (2) is located between the 5' wing region and the gap region.

14. The method of claim 2, wherein the number of nucleosides substituted in step (1) is 1 to 4, at least one of the phosphorothioate bonds substituted in step (2) is adjacent to the 5' end of the nucleoside substituted in step (1), and at least one of the phosphorothioate bonds substituted in step (2) is located between the 5' wing region and the gap region.

15. A method for evaluating the degree of reduction in central neurotoxicity of an oligonucleotide having a phosphorothioate modification, comprising the following steps (1) to (3): (1) providing an oligonucleotide having a phosphorothioate modification; (2) preparing an oligonucleotide designed by the method according to any one of claims 1 to 14; and (3) comparing the central neurotoxicity of the oligonucleotide prepared in step (2) with the central neurotoxicity of the oligonucleotide prepared in step (1); A method comprising:

16. An oligonucleotide having a phosphorothioate bond with reduced central nervous toxicity, wherein the 5' carbon atom of the sugar moiety of at least one nucleoside constituting the oligonucleotide is represented by the following formula I: 【Chemistry 3】 (In the formula, R ６ and R ７ are each independently a hydrogen atom, a halogen atom, or a methyl group. Or the following formula I' 【Chemistry 4】 (In the formula, R ４ and R ５ are each independently a hydrogen atom, a methyl group, or an ethyl group (provided that R ４ and R ５ (Except when both are hydrogen atoms) and optionally at least one internucleoside bond is a phosphodiester bond.

17. The oligonucleotide of claim 16 which is a gapmer-type oligonucleotide.

18. The oligonucleotide of claim 17, wherein at least one nucleoside having the structure represented by formula I or I' is located in the gap region.

19. 18. The oligonucleotide of claim 17, wherein at least one of the phosphodiester bonds is located in a gap region.

20. 18. The oligonucleotide of claim 17, wherein at least one nucleoside constituting the 3' wing region and the 5' wing region is a bridged nucleoside.

21. A reagent for controlling gene expression, comprising the oligonucleotide according to any one of claims 16 to 20.

22. A therapeutic agent for a disease, comprising the oligonucleotide according to any one of claims 16 to 20.

23. (1) designing an oligonucleotide by the method according to any one of claims 1 to 14; and (2) synthesizing the oligonucleotide designed in step (1) A method for producing an oligonucleotide with low central nervous toxicity, comprising:

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