JP2023130528A - Compound, method and pharmaceutical composition for modulating plp1 expression - Google Patents

Abstract

To provide compounds, methods, and pharmaceutical compositions for inhibiting the expression of Proteolipid protein 1 (PLP1) and/or for treating, preventing, delaying, or ameliorating PLP1-related diseases.SOLUTION: A modified oligonucleotide for inhibiting PLP1 expression consists of 12 to 30 residues, and contains at least 8 consecutive nucleobase sequences that are complementary to an equal-length portion of any one of positions 10014 to 10152, positions 10180 to 10252, or positions 14033 to 14113 from the 5' end of the nucleobase sequence of a specific sequence, and the full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to an equal length portion of the nucleobase sequence of the specific sequence.SELECTED DRAWING: None

Description

The present invention provides a compound for reducing at least one of the pre-mRNA level, mRNA level, and protein level of Proteolipid protein 1 (PLP1) in animals, a method of using the compound, and a pharmaceutical composition containing the compound. Regarding. The method of the invention treats a PLP1-related disease, such as Pelizaeus-Merzbacher Disease (PMD),
Useful for prevention or delay of progression.

Pelizaus-Merzbacher disease (PMD) is the most common congenital cerebral leukodysplasia, with an estimated incidence of 1 in 200,000 to 500,000 people. Many patients develop nystagmus and head tremors, which then progress to convulsions and spastic paralysis.

Mutations in the propiolipid protein gene (PLP1 gene) have been reported as a cause of the onset of PMD (Non-Patent Documents 1 to 4). PMD is classified into fetal PMD and classical PMD depending on the type of mutation in the PLP1 gene. Fetal PMD is caused by amino acid substitutions and splicing abnormalities due to point mutations in the PLP1 gene, and accounts for approximately 15 to 25% of all patients with PLP1 gene mutations. It is a severe disease that develops at birth or during the neonatal period, resulting in death in infancy. Classic PMD is caused by duplication of the PLP1 gene and accounts for 50-75% of cases. Onset begins in early infancy and death occurs in early childhood.

To date, multiple therapeutic methods have been attempted for the treatment of PMD. Yamauchi et al. attempted a treatment method using a substance that inhibits MAPK pathway activation (Patent Document 1), but kinase inhibitors generally have safety issues due to low target molecule specificity. It is believed that there is. In addition, gene therapy using a vector containing miRNA that suppresses the expression of PLP1 by Inoue et al. and genome editing by gene therapy by TESAR et al. have also been attempted (Patent Documents 2 and 3). Furthermore, treatment methods using antisense nucleic acids have been attempted, and Elit et al. have found that suppressing the expression of PLP1 using an antisense nucleic acid complementary to the base sequence of the intron region of the PLP1 gene can treat fetal PMD. It has been confirmed that it exhibits therapeutic effects in disease model mice that develop similar symptoms (Non-Patent Document 5). However, since the above-mentioned antisense nucleic acid was designed based on the base sequence of the mouse PLP1 gene, it is difficult to apply it clinically as an actual method for treating PMD patients.

JP5391401B WO2019/156115 WO2018/106782

Willard et al., Science 1985 230, 940-942 Gencic et al., Am. J. Hum. Genet. 1989 45, 435-442 Hudson et al., Proc. Natl. Acad. Sci. USA 1989 86, 8128-8131 Trofatter et al., Proc. Natl. Acad. Sci USA 1989 86, 9427-9430 Elitt et al., biRxiv 2018 preprint doi:doi. org/10.1101/508192

At present, there is no fundamental treatment for PMD, and medications and therapy are used as symptomatic treatments for conditions such as epilepsy, dystonia, and mental retardation, which are insufficiently effective and place a heavy burden on patients. It is therefore an object of the present invention to provide compounds, compositions and methods for treating, preventing, delaying or ameliorating PLP1-related diseases such as PMD.

It is also an object of the present invention to provide compounds, methods, and pharmaceutical compositions for suppressing PLP1 expression.

The present inventors conducted extensive studies, discovered a modified oligonucleotide that strongly inhibits PLP1 expression, discovered that the modified oligonucleotide is effective in treating and preventing PLP1-related diseases such as PMD, and completed the present invention. I ended up letting it happen.

The gist of the invention is as follows.
[1] A modified oligonucleotide consisting of 12 to 30 residues, and any one of positions 10014 to 10152, 10180 to 10252, or 14033 to 14113 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. at least 8 consecutive nucleobase sequences complementary to equal-length portions, and the full-length nucleobase sequence of the modified oligonucleotide is at least 85% complementary to the equal-length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. A modified oligonucleotide that suppresses the expression of Proteolipid protein 1 (PLP1).
[2] A modified oligonucleotide consisting of 12 to 30 residues, and positions 10036 to 10055, 10064 to 10089, 10101 to 10130, and 10202 to 10230 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. The modified oligonucleotide contains at least 8 consecutive nucleobase sequences complementary to the isometric portion of position 1 or 14055 to 14091, and the full-length nucleobase sequence of the modified oligonucleotide is the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. A modified oligonucleotide that suppresses the expression of PLP1, having at least 85% complementarity to an isometric portion of.
[3] A modified oligonucleotide consisting of 12 to 30 residues, and any one of positions 10101 to 10130, 10202 to 10230, or 14055 to 14091 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. at least 8 consecutive nucleobase sequences complementary to equal-length portions, and the full-length nucleobase sequence of the modified oligonucleotide is at least 85% complementary to the equal-length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. A modified oligonucleotide that suppresses the expression of PLP1.
[4] Positions 10104-10119, 10109-10124, 10114-10129, 10202-10217, 10203-10218, 14056-14071, 14057- from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing A modified oligonucleotide that suppresses the expression of PLP1, comprising a nucleobase sequence complementary to any one of positions 14072, 14074 to 14089, or 14075 to 14090.
[5] The modified oligonucleotide according to any one of [1] to [4], which is single-stranded.
[6] The modified oligonucleotide according to any one of [1] to [5], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified sugar.
[7] The modified sugar according to [6] is selected from the group consisting of a bicyclic sugar, a sugar modified with 2'-O-methoxyethyl, and a sugar modified with 2'-O-methyl. Modified oligonucleotides.
[8] the bicyclic sugar is selected from the sugar moiety of LNA, ENA, cEt, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz], The modified oligonucleotide described in [7].
[9] The modified oligonucleotide according to any one of [1] to [8], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified nucleobase.
[10] The modified oligonucleotide according to [9], wherein the modified nucleobase is 5-methylcytosine.
[11] The modified oligonucleotide according to any one of [1] to [10], wherein at least one internucleoside bond constituting the modified oligonucleotide is a modified internucleoside bond.
[12] The modified internucleoside bond is a phosphorothioate internucleoside bond, [1
1].
[13] The modified oligonucleotide is
1) gap segment;
2) 5' wing segment;
3) a 3' wing segment;
the gap segment is positioned between the 5′ wing segment and the 3′ wing segment;
The modified oligonucleotide according to any one of [1] to [12], wherein the nucleosides constituting the 5' wing segment and 3' wing segment contain a modified sugar.
[14] A pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [13], and a pharmaceutically acceptable carrier.
[15] The pharmaceutical composition according to [14] for treating, preventing, or delaying the progression of a PLP1-related disease.
[16] The pharmaceutical composition according to [15], wherein the PLP1-related disease is Pelizaeus-Merzbacher disease.
[17] The pharmaceutical composition according to [16], wherein the Pelizaus-Merzbacher disease is fetal Pelizaus-Merzbacher disease or classical Pelizaus-Merzbacher disease.
[18] Treatment, prevention, or prevention of PLP1-related diseases in a subject, characterized by administering a therapeutically effective amount of the modified oligonucleotide according to any one of [1] to [13] to a subject in need thereof. Methods for slowing down its progress.
[19] Use of the modified oligonucleotide according to any one of [1] to [13] in the manufacture of a medicament for treating, preventing, or delaying the progression of a PLP1-related disease.
[20] The modified oligonucleotide according to any one of [1] to [13] for treating, preventing, or delaying the progression of a PLP1-related disease.

According to the present invention, it is possible to efficiently suppress PLP1 expression, improve the symptoms of a PLP1-related disease, for example, Pelizaus-Merzbacher disease, and provide a medicament that is effective for the prevention and treatment of the disease. .

It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and are not intended to limit the invention as claimed. In this specification, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Additionally, unless otherwise stated, terms such as "element" encompass elements containing one unit and elements containing more than one subunit.

The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, and papers, with respect to any portion of the documents discussed herein, and in their entirety. expressly incorporated herein by reference.

(definition)
Unless specific definitions are given, the nomenclature utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and medicinal chemistry, and the procedures and techniques thereof described herein, are those of ordinary skill in the art. It is well known and commonly used. Standard techniques can be used for the chemical synthesis and analysis used herein. Where permissible, all patents, applications, published applications and other publications referenced throughout this disclosure, including GenBank accession numbers and numbers available through databases such as the National Center for Biotechnology Information (NCBI); Related sequence information and other data are incorporated by reference with respect to portions of the documents discussed herein, as well as in their entirety.
Further, although this specification is filed with a sequence listing in electronic format, the information of the sequence listing described in the electronic format is incorporated herein by reference in its entirety.

Unless otherwise specified, the following terms have the following meanings:

"Nucleobase" means a heterocyclic moiety that is capable of pairing with a base of another nucleic acid.

"Nucleobase sequence" means the sequential order of nucleobases that make up the oligonucleotide of the invention.

"Nucleoside" means a molecule in which a sugar and a nucleobase are linked. In certain embodiments, the nucleoside is linked to a phosphate group.

"Nucleotide" refers to a molecule in which a phosphate group is attached to the sugar moiety of a nucleoside. The sugar moiety of naturally occurring nucleotides is ribose or deoxyribose, which are covalently bonded via a phosphate group by a phosphodiester bond.

"Oligomeric compound" or "oligomer" means a polymer of linked monomer subunits that can hybridize to at least one region of a nucleic acid molecule.

"Oligonucleotide" means a polymer of nucleosides in which each nucleoside and each internucleoside linkage are independently linked to each other.

"Internucleoside linkage" refers to a chemical bond between nucleosides.

"Naturally occurring internucleoside linkage" means a 3'-5' phosphodiester linkage.

"Modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage). Examples include, but are not limited to, phosphorothioate internucleoside linkages.

"Phosphorothioate internucleoside linkage" means an internucleoside linkage in which the phosphodiester linkage is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is one example of such a modified internucleoside linkage.

"Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil. Examples include, but are not limited to, 5-methylcytosine. "Unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

"Modified oligonucleotide" means an oligonucleotide comprising at least one such modified nucleoside and/or such modified internucleoside linkage.

"Salt" is a general term for compounds in which one or more dissociable hydrogen ions contained in an acid are replaced with a cation such as a metal ion or ammonium ion. Salts of modified oligonucleotides include phosphorothioate, or salts (e.g., sodium salts, magnesium salts) formed with inorganic ions (e.g., sodium ions, magnesium ions) on phosphodiesters or on functional groups (e.g., amino groups) in modified nucleobases. However, it is not limited to these.

"Sugar" or "sugar moiety" refers to a natural sugar moiety or a modified sugar moiety.

"Modified sugar" refers to a substitution or change from a naturally occurring sugar. Modified sugars include, for example, substituted sugar moieties and bicyclic sugars.

"Substituted sugar moiety" means a furanosyl other than the naturally occurring sugar of RNA or DNA.

"Bicyclic sugar" means a furanosyl ring modified by a bridge of two different carbon atoms on the same ring.

"Single-stranded oligonucleotide" means an oligonucleotide that has not hybridized to a complementary strand.

"PLP1" means a nucleic acid or protein called Proteolipid protein 1. PLP1 may include, for example, sequence variants such as various splicing variants and single nucleotide substitutions (SNPs) transcribed from the PLP1 gene.

"Complementary" refers to the capacity for pairing between the nucleobases of a first nucleic acid and a second nucleic acid. In certain embodiments, adenine is complementary to thymidine or uracil. In certain embodiments, cytosine is complementary to guanine. In certain embodiments, 5-methylcytosine is complementary to guanine.

"Completely complementary (also referred to as complementarity)" or "100% complementary (also referred to as complementarity)" means that the nucleobase sequence of a first nucleic acid is completely complementary to the nucleobase sequence of a second nucleic acid. It means something. In certain embodiments, the first nucleic acid is a modified oligonucleotide and the target nucleic acid is a second nucleic acid.

A "mismatch" or "non-complementary nucleobase" refers to when a nucleobase of a first nucleic acid is unable to pair with a corresponding nucleobase of a second or target nucleic acid.

"Target nucleic acid," "target RNA," and "target RNA transcript" all refer to a nucleic acid that can be targeted by a modified oligonucleotide. In certain embodiments, the target nucleic acid comprises a region of PLP1 mRNA or PLP1 pre-mRNA.

"Motif" refers to a combination of chemically heterogeneous regions in a modified oligonucleotide.

"Pharmaceutically acceptable salts" are physiologically and pharmaceutically acceptable salts of the modified oligonucleotides of the present invention, i.e., those that retain the desired biological activity of the modified oligonucleotide and do not impart undesirable toxic effects to it. Means no salt.

"Administering" means giving the drug to an animal, including, but not limited to, administration by a medical professional and self-administration.

"Amelioration" refers to reducing at least one indicator, sign or symptom of an associated disease, disorder or condition. The severity of an indicator can be determined by subjective or objective measures known to those skilled in the art.

"Animal" means humans or non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and chimpanzees. Point.

"Effective amount" means an amount of a modified oligonucleotide of the invention that is sufficient to achieve the desired physiological outcome in an individual in need of the drug. Effective amounts will vary among individuals depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors. obtain.

"Individual" means a human or non-human animal selected for treatment or therapy.

"Prevent" means delaying the onset or occurrence of a disease, disorder, or undesirable health condition, or one or more symptoms associated with the disease, disorder, or undesirable health condition, for a period of minutes to an indefinite period; or It means to prevent something from happening. Preventing also means reducing the risk of developing a disease, disorder or undesirable health condition.

"Treat" means to reduce or eliminate a disease, disorder, or undesirable health condition, or one or more symptoms associated with the disease, disorder, or undesirable health condition; or to partially eliminate or eradicate one or more causes of the undesirable health condition itself.

(Specific implementation)
Certain specific embodiments set forth below provide, but are not limited to, compounds for inhibiting the expression of PLP1, methods of using the compounds, and pharmaceutical compositions containing the compounds.

(1) Modified oligonucleotide The modified oligonucleotide of the present invention (hereinafter sometimes referred to as "compound of the present invention" or "modified oligonucleotide of the present invention") is an antisense oligonucleotide of PLP1 having the activity of suppressing PLP1 expression. It is. Suppression of PLP1 expression (level) in the present invention refers to the pre-mRNA level transcribed from the PLP1 gene, the mRNA level spliced from the pre-mRNA, and the PLP1 protein level translated from the mRNA (these are collectively referred to as "PLP1 (sometimes referred to as "expression level"). PLP1 mRNA is not particularly limited and may include sequence variations, but for example, GenBank accession number NG_0088
63.2:4996-21109 (SEQ ID NO: 1 in the sequence listing).

The degree of suppression of PLP1 expression by the compound of the present invention is that at least one of the pre-mRNA level, mRNA level, and protein level of PLP1 is reduced compared to when the compound is not administered, and as a result, PLP1-related diseases are reduced. Any degree may be used as long as prevention and/or improvement of accompanying symptoms is observed, but specifically, for example, in the in vitro PLP1 expression measurement method described below, after contacting cells with the compound of the present invention, The PLP1 expression level is at least 70% or less, preferably 50% or less, more preferably 40% or less, even more preferably 30% or less, particularly preferably 20% or less, compared to the non-contact state or the negative control substance contact state. is used. Furthermore, in the in vitro PLP1 expression measurement method described below, those having an IC ₅₀ of preferably 100 nM or less, more preferably 50 nM or less, and even more preferably 20 nM or less are used.

As a method for verifying the activity of the compound of the present invention, any method may be used as long as it can verify that the compound of the present invention suppresses PLP1 expression, and the method described in "Method for evaluating the compound of the present invention" below is exemplified. be done.

The compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues that has the activity of suppressing PLP1 expression, and is located at positions 10014 to 10152, 10180 to 10180 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. Any sequence containing at least 8 consecutive nucleobase sequences (hereinafter referred to as "PLP1 complementary nucleobase sequence") that are complementary to the isometric portion of either position 10252 or 14033 to 14113. good. Further, the PLP1 complementary nucleobase sequence is preferably a sequence of 8 to 25 consecutive nucleobases, more preferably a sequence of 12 to 20 consecutive nucleobases, and still more preferably 16, 17 consecutive nucleobases. , 18, 19 or 20 consecutive nucleobase sequences.

The compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues and has the activity of suppressing PLP1 expression, and is located at positions 10036 to 10055, 10064 to 10055 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. At least 8 consecutive nucleobase sequences complementary to the equal length portion of any of positions 10089, 10101 to 10130, 10202 to 10230, or 14055 to 14091 (hereinafter referred to as "PLP1 complementary nucleobase sequence") Any type of material may be used as long as it contains the following. Further, the PLP1 complementary nucleobase sequence is preferably a sequence of 8 to 25 consecutive nucleobases, more preferably a sequence of 12 to 20 consecutive nucleobases, and still more preferably 16, 17 consecutive nucleobases. , 18, 19 or 20 consecutive nucleobase sequences.

The compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues that has the activity of suppressing PLP1 expression, and is located at positions 10101 to 10130, 10202 to 10202 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. Any sequence containing at least eight consecutive nucleobase sequences (hereinafter referred to as "PLP1 complementary nucleobase sequences") that are complementary to the isometric portion of either position 10230 or 14055 to 14091. good. Further, the PLP1 complementary nucleobase sequence is preferably a sequence of 8 to 25 consecutive nucleobases, more preferably a sequence of 12 to 20 consecutive nucleobases, and still more preferably 16, 17 consecutive nucleobases. , 18, 19 or 20 consecutive nucleobase sequences.

PLP1 complementary nucleobase sequences include positions 10036 to 10051, 10037 to 10052, 10038 to 10053, 10039 to 10054, 10040 to 10055, and 10064 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. ～10079th place, 10065th to 10080th place, 10066th to 10081st place, 10067th to 10082nd place, 1
0068-10083rd, 10069-10084, 10070-10085, 10071-10086, 10072-10087, 10073-10088, 10074-10089, 10101-10116, 10102-10117, 10103 ～10118th place, 10104-10119, 10105-10120, 10106-10121, 10107-10122, 10108-10123, 10109-10124, 10110-10125, 10111-10126, 10112-10127, 1011 3rd to 10128th place, 10114-10129, 10115-10130, 10202-10217, 10203-10218, 10204-10219, 10205-10220, 10206-10221, 10207-10222, 10208-10223, 1020 9th to 10224th place, 10210-10225, 10211-10226, 10212-10227, 10213-10228, 10214-10229, 10215-10230, 14055-14070, 14056-14071, 14057-14072, 1406 2~14077th place, It may be a nucleobase sequence complementary to any of positions 14063-14078, 14074-14089, 14075-14090, and 14076-14091.

Here, the modified oligonucleotide of the present invention has a total length of 12 to 30 residues in addition to the above-mentioned PLP1 complementary nucleobase sequence, and the entire length of the nucleobase sequence is the nucleobase of SEQ ID NO: 1 in the sequence listing. Additional sequences may be present at the 5' end and/or 3' end as long as they have at least 85% complementarity to the equal length portion of the sequence. Furthermore, any additional sequence may be used as long as the modified oligonucleotide of the present invention has the activity of suppressing PLP1 expression.

The full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to the equal length portion of SEQ ID NO: 1 in the sequence listing, and the complementarity is preferably 90%, more preferably 95%, More preferably, it is 100%. Here, the term "equal-length portion" refers to a portion that has homology when the nucleobase sequence of the modified oligonucleotide of the present invention and the nucleobase sequence of PLP1 pre-mRNA or mRNA are aligned using software such as BLAST. It means a part that is detected as a part. In other words, it is desirable that the nucleobase sequence of the oligonucleotide of the present invention be completely complementary to an equal length portion of the nucleobase sequence of PLP1 pre-mRNA or mRNA, but may contain one or more mismatched nucleobases. Complementarity of 85% or more, 90% or more, preferably 95% or more is used.

The above-mentioned mismatched nucleobases may be continuous, or PLP1 complementary nucleobase sequences may be sandwiched between them. The percent complementarity of the antisense oligonucleotides of the invention with PLP1 pre-mRNA or mRNA was determined using the BLAST program (basic local alignment search tools) and PowerB, which are known in the art.
can be routinely determined using the LAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). . Percent homology, sequence identity, or complementarity can be measured using, for example, the GAP program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Re
search Park, Madison Wis. ), using default settings using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489). For example, when 18 of the 20 nucleobases of the modified oligonucleotide are complementary to and hybridize to an equal length portion of PLP1 pre-mRNA, the modified oligonucleotide has 90 percent complementarity.

As an example of a specific nucleobase sequence of the modified oligonucleotide of the present invention,
TACCGTTGCGCTCAGG (complementary sequence of positions 10104 to 10119 of SEQ ID NO: 1) (SEQ ID NO: 2 in the sequence listing)
CCTGTTACCGTTGCGC (complementary sequence of positions 10109 to 10124 of SEQ ID NO: 1) (SEQ ID NO: 3 in the sequence listing)
GGCCCCCTGTTACCGT (complementary sequence of positions 10114 to 10129 of SEQ ID NO: 1) (SEQ ID NO: 4 in the sequence listing)
TCGGGATGTCCTAGCC (complementary sequence of positions 10202 to 10217 of SEQ ID NO: 1) (SEQ ID NO: 5 in the sequence listing)
GTCGGGATGTCCTAGC (complementary sequence of positions 10203 to 10218 of SEQ ID NO: 1) (SEQ ID NO: 6 in the sequence listing)
ATGAGTTTAAGGACGG (complementary sequence of positions 14056 to 14071 of SEQ ID NO: 1) (SEQ ID NO: 7 in the sequence listing)
CATGAGTTTAAGGACG (complementary sequence of positions 14057 to 14072 of SEQ ID NO: 1) (SEQ ID NO: 8 in the sequence listing)
AACTTGGTGCCTCGGC (complementary sequence of positions 14074 to 14089 of SEQ ID NO: 1) (SEQ ID NO: 9 in the sequence listing), or
GAACTTGGTGCCTCGG (complementary sequence of positions 14075 to 14090 of SEQ ID NO: 1) (SEQ ID NO: 10 in the sequence listing)
Examples include the nucleobase sequences described in .

Although the modified oligonucleotide of the present invention may be a double-stranded modified oligonucleotide, a single-stranded modified oligonucleotide is preferably used.

(Modified sugar)
The modified oligonucleotide of the present invention is preferably used in which at least one nucleoside constituting the oligonucleotide contains a modified sugar. In the present invention, a modified sugar refers to one in which the sugar moiety has been modified, and a modified oligonucleotide containing one or more of the modified sugars has advantageous characteristics such as enhanced nuclease stability and increased binding affinity. Preferably, at least one of the modified sugars is selected from the group consisting of bicyclic sugars, 2'-MOE-modified sugars, and 2'-OMe-modified sugars. Examples of bicyclic sugars include sugars of LNA, ENA, cEt, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz], as shown below. The sugar moiety of ALNA[Ms] is preferably used.

Examples of substituted sugar moieties include, but are not limited to, 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'- _OCH (2'-OMe). , 2'-OCH ₂ CH ₃ , 2'-OCH ₂ CH ₂ F and 2'-O(CH ₂ ) ₂ OCH ₃ (2'-MOE) substituents. Substituents at the 2' position include allyl, amino, azide, thio, O-allyl, O-C ₁ -C ₁₀ alkyl, OCF ₃ , OCH ₂ F, O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ -O-N(R _m )(R _n ), O-CH ₂ -C(=O)-N(R _m )(R _n ) and O-CH ₂ -C(=O)-N(R _l )-(CH ₂ ) ₂ -N(R _m )(R _n ) (wherein each R _l , R _m and R _n is independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl) You can choose from.

Examples of nucleosides with bicyclic sugars include, but are not limited to, nucleosides that include a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the oligonucleotides provided herein include a nucleoside with one or more bicyclic sugars, where the bridge comprises one of the following formulas: 4'-( _CH2 )-O-2'(LNA);4'
-(CH ₂ )-S-2';4'-(CH ₂ ) ₂ -O-2'(ENA);4'-CH (CH ₃ )-O-2' (cEt) and 4'-CH ( CH ₂ OCH ₃ )-O-2' (and analogs thereof; see US Pat. No. 7,399,845); 4'-C(CH ₃ )(CH ₃ )-O-2' (and analogs thereof, see WO 2009/006478); 4'-CH ₂ -N(OCH ₃ )-2' (and analogs thereof, see WO 2008/150729); 4'-CH ₂ -O-N(CH ₃ )-2' (and analogs thereof, see US 2004-0171570); 4'-CH ₂ -N(R)-O-2' (wherein R is , H, C1-C12 alkyl or protecting group) (see US Pat. No. 7,427,672); 4'-CH ₂ -C(H)(CH ₃ )-2' (and similar (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'- _CH2 -C(= _CH2 )-2' (and analogs thereof). Please refer to WO2008/154401).

Nucleosides with additional bicyclic sugars have been reported in the published literature (eg: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561
; Braasch et al. , Chem. Biol. , 2001, 8, 1-7; Orum et al. , Curr. Opinion Mol. Ther. ,2001,3,23
9-243; Wahlestedt et al. , Proc. Natl. Acad. Sci. U. S. A. , 2000, 97, 5633-5638; Singh et al. , Chem. Commun. , 1998, 4, 455-456; Koshkin et al. , Tetrahedron, 1998, 54, 3607-3630; Kumar et al. , Bioorg. Med. Chem. Lett. , 1998, 8, 2219-2222; Singh et al. , ; United States Patent No. 7,399,845; No. 6,770,748; No. 6,525,191; No. 6,268,490; United States Patent No. 2008-0039618; No. 0012281; WO2010/036698; WO 2009/067647; WO2
009/067647; WO2007/134181; WO2005/021570; WO2004/106356; WO94/14226; WO 2009/0064
78; WO2008/154401; and WO2008/150729). Each of the foregoing bicyclic sugar-bearing nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose.

GuNA, a nucleoside with a bicyclic sugar, has been reported as an artificial nucleoside with a guanidine bridge (see WO2014/046212, WO2017/047816). Bicyclic nucleosides ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Trz] and ALNA [Oxz] have been reported as cross-linked artificial nucleic acid amino LNA (ALNA) (see WO2020/100826). Please refer).

In certain embodiments, nucleosides having bicyclic sugars include a bridge between the 4' and 2' carbon atoms of the pentofuranosyl sugar moiety, including, but not limited to, -[C(R _a )(R _b )] _n -, -C(R _a )=C(R _b )-, -C(R _a )=N-, -C(=NR _a )-, -C(=O)-, One or one to four independently selected from -C(=S)-, -N(R _a )-, -O-, -Si(R _a ) ₂ - and -S(=O) _x - where X is 0, 1 or 2; n is 1, 2, 3 or 4; each R _a and R _b is independently H, a protecting group , hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, aromatic ring group, substituted aromatic ring group, heterocyclic group, substituted heterocyclic group, C ₅ - C ₇ alicyclic group, substituted C ₅ - C ₇ alicyclic group, halogen, OJ ₁ , NJ ₁ J ₂ , SJ ₁ , N ₃ , COOJ ₁ is acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) ₂ -J ₁ ) or sulfoxyl (S(=O)-J ₁ ),
Each J ₁ and J ₂ is independently H, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, Substituted C ₂ -C ₁₂ alkynyl, aromatic ring group, substituted aromatic ring group, acyl (C(=O)-H), substituted acyl, heterocyclic group, substituted heterocyclic group, C ₁ -C ₁₂ aminoalkyl, substituted C ₁ to C ₁₂ aminoalkyl or a protecting group.

In certain embodiments, the bridge of the bicyclic sugar moiety is -[C(R _a )(R _b )] _n -, -[C(R _a )(R _b )] _n -O-, -C (R _a R _b )-N(R)-O- or C(R _a R _b )-O-N(R)-. In certain embodiments, the bridges include 4'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) ₃ -2', 4'-CH ₂ -O -2' (the nucleoside having a bicyclic sugar in this case is also referred to as LNA), 4'-(CH ₂ ) ₂ -O-2' (the nucleoside having a bicyclic sugar in this case is also referred to as ENA), 4'-CH(CH ₃ )-O-2' (the nucleoside having a bicyclic sugar in this case is also referred to as cEt), 4'-CH ₂
-O-N(R)-2' and 4'-CH ₂ -N(R)-O-2'-, where each R is independently H, a protecting group, or a C ₁ -C ₁₂ alkyl It is.

In certain embodiments, the bridge of the bicyclic sugar moiety is 4'- _CH2 -O-2'-(LNA) or _CH2 -N(R)-, where each R is independently -SO ₂ -CH ₃ (ALNA[Ms]), -CO-NH-CH ₃ (ALNA[mU]), 1,5-dimethyl-1,2,4-triazol-3-yl (ALNA[Trz]) , -CO-NH-CH(CH ₃ ) ₂ (ALNA
[ipU]), 5-methyl-1,2,4-oxadiazol-3-yl (ALNA[Ox
z]) (WO2020/100826).

In certain embodiments, nucleosides having bicyclic sugars are further defined by isomeric configuration. For example, a nucleoside containing a 4'-(CH ₂ )-O-2' bridge may exist in the α-L-configuration or in the β-D-configuration.

In certain embodiments, nucleosides with bicyclic sugars include those with 4'-2' bridges, including, but not limited to, α-L-4'-(CH ₂ ). -O-2', β-D-4'-CH ₂ -O-2', 4'-(CH ₂ ) ₂ -O-2', 4'-CH ₂ -O-N(R)-2' , 4'-CH ₂ -N(R)-O-2', 4'-CH(CH ₃ )-O-2', 4'-CH ₂ -S-2', 4'-CH ₂ -CH( CH ₃ )-2' and 4'-(CH ₂ ) ₃ -2' (wherein R is optionally substituted with H, a protecting group, C ₁ -C ₁₂ alkyl, or C ₁ -C ₁₂ alkyl) urea or guanidine).

式中、
Ｂｘは複素環塩基部分であり；
Ｔ_ａ及びＴ_ｂはそれぞれ独立に、水素原子、水酸基の保護基、置換されてもよいリン酸基、リン部分又は支持体への共有結合等であり；
Ｚ_ａは、Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル、置換Ｃ_１～Ｃ_６アルキル、置換Ｃ_２～Ｃ_６アルケニル、置換Ｃ_２～Ｃ_６アルキニル、アシル、置換アシル、置換アミド、チオール又は置換チオールである。 In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

During the ceremony,
Bx is a heterocyclic base moiety;
T _a and T _b each independently represent a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety, or a covalent bond to a support;
Z _a is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, acyl , substituted acyl, substituted amide, thiol or substituted thiol.

In certain embodiments, each of the substituents is independently halogen, oxo, hydroxyl, OJ _c , NJ _c J _d , SJ _c , N ₃ , OC(=X) J _c and NJ _e C(=X) from NJ _c J _d (wherein each J _c , J _d and J _e is independently H, C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl, and X is O or NJ _c ) Mono- or polysubstituted with independently selected substituents.

式中、
Ｂｘは複素環塩基部分であり；
Ｔ_ａ及びＴ_ｂはそれぞれ独立に、水素原子、水酸基の保護基、置換されてもよいリン酸基、リン部分又は支持体への共有結合等であり；
Ｚ_ｂは、Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル、置換Ｃ_１～Ｃ_６アルキル、置換Ｃ_２～Ｃ_６アルケニル、置換Ｃ_２～Ｃ_６アルキニル又は置換アシル（Ｃ（＝Ｏ）－）である。 In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

During the ceremony,
Bx is a heterocyclic base moiety;
T _a and T _b each independently represent a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety, or a covalent bond to a support;
Z _b is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C 6 alkynyl, substituted C ₁ -C ₆ alkyl, substituted C _{2 -C 6 alkenyl} , substituted C ₂ -C ₆ alkynyl or substituted Acyl (C(=O)-).

式中、
Ｂｘは複素環塩基部分であり；
Ｔ_ａ及びＴ_ｂはそれぞれ独立に、水素原子、水酸基の保護基、置換されてもよいリン酸基、リン部分又は支持体への共有結合等であり；
Ｒ_ｄは、Ｃ_１～Ｃ_６アルキル、置換Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、置換Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル又は置換Ｃ_２～Ｃ_６アルキニルであり；
各ｑ_ａ、ｑ_ｂ、ｑ_ｃ及びｑ_ｄは独立に、Ｈ、ハロゲン、Ｃ_１～Ｃ_６アルキル、置換Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、置換Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル又は置換Ｃ_２～Ｃ_６アルキニル、Ｃ_１～Ｃ_６アルコキシル、置換Ｃ_１～Ｃ_６アルコキシル、アシル、置換アシル、Ｃ_１～Ｃ_６アミノアルキル又は置換Ｃ_１～Ｃ_６アミノアルキ
ルである。 In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

During the ceremony,
Bx is a heterocyclic base moiety;
T _a and T _b each independently represent a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety, or a covalent bond to a support;
R _d is C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl; ;
Each q _a , q _b , q _c and q _d independently represents H, halogen, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxyl, substituted C ₁ -C ₆ alkoxyl, acyl, substituted acyl, C ₁ -C ₆ aminoalkyl or substituted C ₁ -C ₆ amino It is an alkyl.

式中、
Ｂｘは複素環塩基部分であり；
Ｔ_ａ及びＴ_ｂはそれぞれ独立に、水素原子、水酸基の保護基、置換されてもよいリン酸基、リン部分又は支持体への共有結合等であり；
ｑ_ａ、ｑ_ｂ、ｑ_ｅ及びｑ_ｆはそれぞれ独立に、水素、ハロゲン、Ｃ_１～Ｃ_１２アルキル、置換Ｃ_１～Ｃ_１２アルキル、Ｃ_２～Ｃ_１２アルケニル、置換Ｃ_２～Ｃ_１２アルケニル、Ｃ_２～Ｃ_１２アルキニル、置換Ｃ_２～Ｃ_１２アルキニル、Ｃ_１～Ｃ_１２アルコキシ、置換Ｃ_１～Ｃ_１２アルコキシ、ＯＪ_ｊ、ＳＪ_ｊ、ＳＯＪ_ｊ、ＳＯ_２Ｊ_ｊ、ＮＪ_ｊＪ_ｋ、Ｎ_３、ＣＮ、Ｃ（＝Ｏ）ＯＪｊ、Ｃ（＝Ｏ）ＮＪ_ｊＪ_ｋ、Ｃ（＝Ｏ）Ｊ_ｊ、Ｏ－Ｃ（＝Ｏ）ＮＪ_ｊＪ_ｋ、Ｎ（Ｈ）Ｃ（＝ＮＨ）ＮＪ_ｊＪ_ｋ、Ｎ（Ｈ）Ｃ（＝Ｏ）-ＮＪ_ｊＪ_ｋもしくはＮ
（Ｈ）Ｃ（＝Ｓ）ＮＪ_ｊＪ_ｋであり；
又は、ｑ_ｅ及びｑ_ｆは共に＝Ｃ（ｑ_ｇ）（ｑ_ｈ）であり；
ｑ_ｇ及びｑ_ｈはそれぞれ独立に、Ｈ、ハロゲン、Ｃ_１～Ｃ_１２アルキルもしくは置換Ｃ_１～Ｃ_１２アルキルである。 In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

During the ceremony,
Bx is a heterocyclic base moiety;
T _a and T _b each independently represent a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety, or a covalent bond to a support;
q _a , q _b , q _e and q _f each independently represent hydrogen, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxy, substituted C ₁ -C ₁₂ alkoxy, OJ _j , SJ _j , SOJ _j , SO ₂ J _j , NJ _j J _k , N ₃ , CN, C(=O)OJj, C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H)C(=NH) NJ _j J _k , N(H)C(=O)-NJ _j J _k or N
(H)C(=S)NJ _j J _k ;
Or, q _e and q _f are both =C(q _g )(q _h );
q _g and q _h are each independently H, halogen, C ₁ -C ₁₂ alkyl or substituted C ₁ -C ₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides (also referred to as LNA) with 4'-CH2- _O -2' bridges are important for their oligomerization and nucleic acid It has been described along with its recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of nucleosides with bicyclic sugars is also described in WO98/39352 and WO99/14226.

Various bicyclics with a 4' _- 2' bridging group, such as 4'-CH 2 -O-2' (the bicyclic nucleoside in this case is also referred to as LNA) and 4'-CH ₂ -S-2'. Nucleoside analogs have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of oligodeoxyribonucleotide duplexes containing bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Additionally, the synthesis of 2'-amino-LNA (bicyclic nucleosides in this case also referred to as ALNA), conformationally restricted high affinity oligonucleotide analogs, has been described in the art ( Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Additionally, 2'-amino- and 2'-methylamino-LNAs have been prepared and their duplex thermal stability with complementary RNA and DNA strands has been previously reported.

式中、
Ｂｘは複素環塩基部分であり；
Ｔ_ａ及びＴ_ｂはそれぞれ独立に、水素原子、水酸基の保護基、置換されてもよいリン酸基、リン部分又は支持体への共有結合等であり；
各ｑ_ｉ、ｑ_ｊ、ｑ_ｋ及びｑ_ｌは独立に、Ｈ、ハロゲン、Ｃ_１～Ｃ_１２アルキル、置換Ｃ_１～Ｃ_１２アルキル、Ｃ_２～Ｃ_１２アルケニル、置換Ｃ_２～Ｃ_１２アルケニル、Ｃ_２～Ｃ_１２アルキニル、置換Ｃ_２～Ｃ_１２アルキニル、Ｃ_１～Ｃ_１２アルコキシル、置換Ｃ_１～Ｃ_１２アルコキシル、ＯＪ_ｊ、ＳＪ_ｊ、ＳＯＪ_ｊ、ＳＯ_２Ｊ_ｊ、ＮＪ_ｊＪ_ｋ、Ｎ_３、ＣＮ、Ｃ（＝Ｏ）ＯＪ_ｊ、Ｃ（＝Ｏ）ＮＪ_ｊＪ_ｋ、Ｃ（＝Ｏ）Ｊ_ｊ、Ｏ－Ｃ（＝Ｏ）ＮＪ_ｊＪ_ｋ、Ｎ（Ｈ）Ｃ（＝ＮＨ）ＮＪ_ｊＪ_ｋ、Ｎ（Ｈ）Ｃ（＝Ｏ）ＮＪ_ｊＪ_ｋ又はＮ（Ｈ）Ｃ（＝Ｓ）ＮＪ_ｊＪ_ｋであり；
ｑ_ｉ及びｑ_ｊ又はｑ_ｌ及びｑ_ｋは共に＝Ｃ（ｑ_ｇ）（ｑ_ｈ）であり、式中、ｑ_ｇ及びｑ_ｈはそれぞれ独立に、Ｈ、ハロゲン、Ｃ_１～Ｃ_１２アルキル又は置換Ｃ_１～Ｃ_１２アルキルである。 In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

During the ceremony,
Bx is a heterocyclic base moiety;
T _a and T _b each independently represent a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety, or a covalent bond to a support;
Each q _i , q _j , q _k and q _l independently represents H, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxyl, substituted C ₁ -C ₁₂ alkoxyl, OJ _j , SJ _j , SOJ _j , SO ₂ J _j , NJ _j J _k , N ₃ , CN, C(=O)OJ _j , C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H)C(=NH )NJ _j J _k , N(H)C(=O)NJ _j J _k or N(H)C(=S)NJ _j J _k ;
q _i and q _j or q _l and q _k are both =C(q _g )(q _h ), where q _g and q _h each independently represent H, halogen, C ₁ -C ₁₂ alkyl, or Substituted C ₁ -C ₁₂ alkyl.

One carbocyclic bicyclic nucleoside has been described with a 4'-(CH ₂ )3-2' bridge and an alkenyl analog bridge 4'-CH=CH-CH ₂ -2' (Frier et al. , Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362- 8379).

In certain embodiments, nucleosides with bicyclic sugars include, but are not limited to, compounds such as those shown below.

式中、Ｂｘは塩基部分であり、Ｒは独立に、保護基、Ｃ_１～Ｃ_６アルキル又はＣ_１～Ｃ_６アルコキシである。

where Bx is a base moiety and R is independently a protecting group, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy.

In certain embodiments (LNA), the nucleoside having a bicyclic sugar has the following general formula:
[In the formula,
B is a nucleobase;
X and Y can each independently represent a nucleoside represented by a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety, a covalent bond to a support, etc. (WO98/ 39352). A typical example is the following formula:
Examples include nucleotides represented by

In certain embodiments (GuNA), the bicyclic-containing nucleoside has the following general formula:
[In the formula, B is a nucleobase, and R ₃ , R ₄ , R ₅ , and R ₆ are each independently a hydrogen atom or a C _1-6 alkyl group which may be substituted with one or more substituents. R ₇ and R ₈ each independently represent a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety or a covalent bond to a support, and R ₉ , R ₁₀ , R Each of ₁₁ is independently a hydrogen atom, a C _1-6 alkyl group which may be substituted with one or more substituents, or a protecting group for an amino group. ]
(For example, see WO2014/046212 and WO2017/047816).

In certain embodiments (ALNA [mU]), the bicyclic sugar-containing nucleoside has the following general formula (I):
[In the formula,
B is a nucleobase;
R ₁ , R ₂ , R ₃ and R ₄ are each independently a hydrogen atom or a C _1-6 alkyl group optionally substituted with one or more substituents;
R ₅ and R ₆ are each independently a hydrogen atom, a hydroxyl group protecting group, an optionally substituted phosphoric acid group, a phosphorus moiety, a covalent bond to a support, etc.;
m is 1 or 2;
X is the following formula (II-1):
is a group represented by;
Symbols described in formula (II-1):
indicates the bonding point with the 2'-amino group;
One of R ₇ and R ₈ is a hydrogen atom, and the other is a methyl group which may be substituted with one or more substituents. ]
(For example, see WO2020/100826). A typical example is a nucleoside in which one of R ₇ and R ₈ is a hydrogen atom and the other is an unsubstituted methyl group.

In certain embodiments (ALNA[ipU]), the bicyclic sugar-containing nucleoside is a nucleoside having the general formula (I) as defined in ALNA[mU] above, where:
X is the following formula (II-1):
is a group represented by;
One of R ₇ and R ₈ is a hydrogen atom, and the other is an isopropyl group that may be substituted with one or more substituents (see, for example, WO2020/100826). A typical example is a nucleoside in which one of R ₇ and R ₈ is a hydrogen atom and the other is an unsubstituted isopropyl group.

In certain embodiments (ALNA[Trz]), the bicyclic-containing nucleoside is a nucleoside having the general formula (I) above, where X is the following formula (II-2):
is a group represented by;
A is a triazolyl group optionally substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). A typical example of ALNA[Trz] is a triazolyl group in which A may have one or more methyl groups, more specifically, 1,5-dimethyl-1,2,4- It is a nucleoside that is a triazol-3-yl group.

In certain embodiments (ALNA[Oxz]), a nucleoside having the general formula (I) as defined in ALNA[mU] above, wherein:
X is the following formula (II-2):
is a group represented by;
A is an oxadiazolyl group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). A typical example is an oxadiazolyl group in which A may have one or more methyl groups, more specifically a 5-methyl-1,2,4-oxadiazol-3-yl group. is a nucleoside or nucleotide.

In certain embodiments (ALNA[Ms]), the bicyclic-containing nucleoside is a nucleoside having the general formula (I) above, where X is the following general formula (II-3):
is a group represented by;
M is a sulfonyl group substituted with a methyl group optionally substituted with one or more substituents (see, for example, WO2020/100826). A typical example of ALNA[Ms] is a nucleoside in which M is a sulfonyl group substituted with an unsubstituted methyl group.

In certain embodiments, the nucleoside is modified by replacing the ribosyl ring with a sugar substitute. Such modifications include, but are not limited to, substitute ring systems (sometimes referred to as DNA analogs), such as morpholino, cyclohexenyl, cyclohexyl or tetrahydropyranyl rings, such as those of the formula Mention may be made of the replacement of the ribosyl ring with one having:

式中、
Ｂｘは複素環塩基部分であり；
Ｔ_３及びＴ_４はそれぞれ独立に、テトラヒドロピランヌクレオシド類似物をオリゴマー化合物に連結するヌクレオシド間連結基、又はＴ_３及びＴ_４の一方は、テトラヒドロピランヌクレオシド類似物をオリゴマー化合物もしくはオリゴヌクレオチドに連結するヌクレオシド間連結基であり、Ｔ_３及びＴ_４の他方は、Ｈ、ヒドロキシル保護基、連結共役基又は５’もしくは３’－末端基であり；
ｑ_１、ｑ_２、ｑ_３、ｑ_４、ｑ_５、ｑ_６及びｑ_７はそれぞれ独立に、Ｈ、Ｃ_１～Ｃ_６アルキル、置換Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、置換Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル又は置換Ｃ_２～Ｃ_６アルキニルであり；
Ｒ_１及びＲ_２の一方は水素であり、他方はハロゲン、置換又は無置換のアルコキシ、ＮＪ_１Ｊ_２、ＳＪ_１、Ｎ_３、ＯＣ（＝Ｘ）Ｊ_１、ＯＣ（＝Ｘ）ＮＪ_１Ｊ_２、ＮＪ_３Ｃ（＝Ｘ）ＮＪ_１Ｊ_２及びＣＮ（式中、ＸはＯ、Ｓ又はＮＪ_１であり、各Ｊ_１、Ｊ_２及びＪ_３は独立に、Ｈ又はＣ_１～Ｃ_６アルキルである）から選択される。 In certain embodiments, a sugar substitute having the following formula is selected:

During the ceremony,
Bx is a heterocyclic base moiety;
T ₃ and T ₄ are each independently an internucleoside linking group that links the tetrahydropyran nucleoside analog to the oligomeric compound, or one of T ₃ and T ₄ connects the tetrahydropyran nucleoside analog to the oligomeric compound or oligonucleotide. an internucleoside linking group, the other of T ₃ and T ₄ being H, a hydroxyl protecting group, a linking conjugate group, or a 5' or 3'-terminal group;
q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are each independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl;
One of R ₁ and R ₂ is hydrogen, and the other is halogen, substituted or unsubstituted alkoxy, NJ ₁ J ₂ , SJ ₁ , N ₃ , OC(=X)J ₁ , OC(=X)NJ ₁ J _{2 , NJ 3 C ( =} X)NJ ₁ J ₂ and CN ₍ wherein, alkyl).

In certain embodiments, q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are each H. In certain embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ is other than H. In certain embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ is methyl. In certain embodiments, THP nucleosides are provided where one of R ₁ and R ₂ is F. In certain embodiments, R ₁ is fluoro and R ₂ is H; R ₁ is methoxy and R ₂ is H, and R ₁ is methoxyethoxy, and R ₂ is It is H.

Such sugar substitutes include, but are not limited to, those referred to in the art as hexitol nucleic acid (HNA), altritol nucleic acid (ANA), and mannitol nucleic acid (MNA) (Leumann, C.J.). , Bioorg. & Med.Chem
．． , 2002, 10, 841-854).

In certain embodiments, the sugar substitute includes a ring with more than 5 atoms and more than 1 heteroatom. For example, its use in nucleoside and oligomeric compounds containing morpholino sugar moieties has been reported (e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and US Pat. No. 5,698,685; 5,166 , 315; 5,185,444; and 5,034,506).

As used herein, the term "morpholino" refers to a sugar substitute having the following structure:

In certain embodiments, morpholinos can be modified, for example, by adding or changing various substituents from the morpholino structures described above. Such sugar substitutes are referred to herein as "modified morpholinos."

In certain embodiments, the oligonucleotide comprises one or more modified cyclohexenyl nucleosides, which are nucleosides that have a 6-membered cyclohexenyl in place of the naturally occurring nucleoside pentofuranosyl residue. Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (e.g., co-owned WO 2010/036696, published April 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130 (6), 1979-1984; Horvath et al., Tetrahedron Letters
, 2007, 48, 3621-3623; Nauwelaerts et al. , J. Am. Chem. Soc. , 2007, 129(30), 9340-9348; Gu et al. , Nucleosides, Nucleotides & Nucleic Aci
ds, 2005, 24(5-7), 993-998; Nauwelaerts et al. , Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al. , Acta Crystallographic
a, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-
586; Gu et al. , Tetrahedron, 2004, 60(9), 2111-2123; Gu et al. , Oligonucleotides, 2003, 13(6), 479-489; Wang et al. , J. Org. Chem. , 2003, 68, 4499-4505; Verbeure et al. , Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al.
．． , J. Org. Chem. , 2001, 66, 8478-82; Wang et al. , Nucleosides, Nucleotides & Nucleic Acids,
2001, 20(4-7), 785-788; Wang et al. , J. Am. Chem. , 2000, 122, 8595-8602; WO 06/047842; and WO 01/049687; each text of which is incorporated herein by reference in its entirety).

式中、
Ｂｘは複素環塩基部分であり；
Ｔ_３及びＴ_４はそれぞれ独立に、シクロヘキセニルヌクレオシド類似物をオリゴヌクレオチド化合物に連結するヌクレオシド間連結基であり、又はＴ_３及びＴ_４の一方は、テトラヒドロピランヌクレオシド類似物をオリゴヌクレオチド化合物に連結するヌクレオシド間連結基であり、Ｔ_３及びＴ_４の他方は、Ｈ、ヒドロキシル保護基、連結共役基もしくは５’－もしくは３’－末端基であり；
ｑ_１、ｑ_２、ｑ_３、ｑ_４、ｑ_５、ｑ_６、ｑ_７、ｑ_８及びｑ_９はそれぞれ独立に、Ｈ、Ｃ_１～Ｃ_６アルキル、置換Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、置換Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル、置換Ｃ_２～Ｃ_６アルキニル又は他の糖置換基である。 Certain modified cyclohexenyl nucleosides have the formula:

During the ceremony,
Bx is a heterocyclic base moiety;
T ₃ and T ₄ are each independently an internucleoside linking group that links the cyclohexenyl nucleoside analog to the oligonucleotide compound, or one of T ₃ and T ₄ connects the tetrahydropyran nucleoside analog to the oligonucleotide compound. an internucleoside linking group, and the other of T ₃ and T ₄ is H, a hydroxyl protecting group, a linking conjugate group, or a 5'- or 3'-terminal group;
q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , q ₇ , q ₈ and q ₉ each independently represent H, C ₁ to C ₆ alkyl, substituted C ₁ to C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₂ -C ₆ alkynyl or other sugar substituents.

Many other bicyclic and tricyclic sugar surrogate ring systems are known in the art that can be used to modify nucleosides for incorporation into oligonucleotides (e.g., review: Leumann, Christian J., Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo a variety of further substitutions to enhance activity.

Methods for preparing modified sugars are well known to those skilled in the art. Several representative U.S. publications teach the preparation of such modified sugars. S. Patents include, but are not limited to, U. S. :4,981,957;5,118,800;5,319,080;5,359,044;5,393,878;5,446,137;5,466,786;5,514,785;5 ,519,134;5,567,811;5,576,427;5,591,722;5,597,909;5,610,300;5,627,053;5,639,873;5,646 , 265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and WO2005/121371, each of which is incorporated herein by reference in its entirety. It will be done.

For nucleotides with modified sugar moieties, the nucleobase moiety (natural, modified or a combination thereof) is maintained during hybridization with an appropriate nucleic acid target.

Preferred modified sugars are the sugar moieties of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz], among which the sugar moiety of ALNA[Ms] is more preferred. That is, in certain embodiments, the modified oligonucleotide of the present invention is a modified oligonucleotide consisting of 12 to 25 residues, preferably 16, 17, 18, 19 or 20 bases, which has the activity of suppressing PLP1 expression. and the nucleobase sequence of the modified oligonucleotide has at least 85%, at least 90%, or at least 95% complementarity to an equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, and At least one of the constituent nucleosides is a modified oligonucleotide having a modified sugar selected from the sugar moiety of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz]. .

(modified nucleobase)
Modifications or substitutions of nucleobases (or bases) are structurally distinct from, and functionally compatible with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases can participate in hydrogen bonding. Such nucleobase modifications can confer nuclease stability, binding affinity or some other beneficial biological property to the oligonucleotide compound. The modified oligonucleotide of the present invention, in which at least one nucleoside constituting the oligonucleotide contains a modified nucleobase, is preferably used. Examples of modified nucleobases include 5-methylcytosine (5-me-C). 5-Methylcytosine means a cytosine modified with a methyl group attached to the 5-position. Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful in increasing the binding affinity of oligonucleotides. For example, 5-methylcytosine substitutions have been shown to increase the duplex stability of nucleic acids by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Further modified nucleobases include 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2 -thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ₃ )uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5 - uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5 - trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- Examples include deazaadenine, 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties can also include those in which purine or pyrimidine bases are replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Nucleobases particularly useful for increasing the binding affinity of modified oligonucleotides include 5-substituted pyrimidines, 6-azapyrimidine and N-2, N-6 and O-6 substituted purines (2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine).

(Modified internucleoside bond)
The naturally occurring internucleoside linkages in RNA and DNA are 3'-5' phosphodiester linkages. Oligonucleotides with one or more modified, i.e., non-naturally occurring, internucleoside linkages may have, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases. are often preferred over oligonucleotides with naturally occurring internucleoside linkages because of their properties.

Oligonucleotides with modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, one or more of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.

The internucleoside bonds of the modified oligonucleotide of the present invention may be of any type as long as the modified oligonucleotide has the activity of suppressing PLP1 expression, but at least one
One in which one of the internucleoside bonds comprises a phosphorothioate internucleoside bond is preferably used; for example, all the internucleoside bonds may be phosphorothioate internucleoside bonds.

(modified oligonucleotide motif)
In certain embodiments, the modified oligonucleotides of the invention are modified to acquire increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for a target nucleic acid, and/or increased PLP1 expression suppression activity. can have a gapmer motif.

"Gapmer" refers to a modified oligonucleotide in which an internal region with multiple nucleosides that supports cleavage by RNase H is located between an external region with one or more nucleosides. The inner region can be referred to as a "gap segment" and the outer region can be referred to as a "wing segment." A wing segment existing on the 5' side of the gap segment can be referred to as a "5' wing segment", and a wing segment existing on the 3' side of the gap segment can be referred to as a "3' wing segment". In gapmers, the sugar moiety of each wing nucleoside that approaches the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) is It is different from the sugar part. For example, the sugar moieties of the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing are modified sugars, and the sugar moieties of the adjacent gap nucleosides are the sugar moieties of the natural DNA. It is.

In certain embodiments, the modified oligonucleotide of the present invention comprises: 1) a gap segment, 2) a 5' wing segment, and 3) a 3' wing segment, wherein the gap segment has the 5' wing segment and the 3' wing segment. The modified oligonucleotide is positioned between the wing segment and the nucleosides of the 5' wing segment and the 3' wing segment include nucleosides having modified sugars. The nucleosides of the gap segment may have only natural DNA sugars or may have one or more modified sugars. The modified sugar is preferably selected from the group consisting of bicyclic sugars, 2'-MOE-modified sugars, and 2'-OMe-modified sugars, and the bicyclic sugars include, for example, Examples include at least one sugar moiety of LNA, ENA, cEt, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz] and ALNA [Trz].

The number of nucleosides contained in the gap segment, 5' wing segment, and 3' wing segment may be any number as long as it inhibits PLP1 expression.

In certain embodiments, a cap structure is included at one or both ends of the modified oligonucleotide to enhance properties such as nuclease stability. Suitable cap structures include 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol. Nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, phosphorodithioate bonds, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, Acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-reverse nucleotide moiety, 3'-3'-reverse abasic moiety, 3'-2'-reverse nucleotide moiety, 3'-2' - inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, bridged methylphosphonate moiety and Non-crosslinked methylphosphonate moiety, 5'-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 5 '-5'-reverse nucleotide moiety, 5'-5'-reverse abasic moiety, 5'-phosphoramidate, 5'-phosphorothioate, 5'-amino, bridged and/or non-bridged 5'-phospho ruamidate, phosphorothioate, as well as 5'-mercapto moieties.

(Method for evaluating compounds of the present invention)
The compound of the present invention may be evaluated using any method as long as it can verify that the compound of the present invention suppresses the expression level of PLP1 in cells, but specifically, for example, the following in vitro and in vivo A method for measuring PLP1 expression is used.

The in vitro PLP1 expression measurement method for evaluating the inhibition of intracellular PLP1 expression by the compound of the present invention can be carried out in any cell in which PLP1 is expressed (hereinafter sometimes referred to as "PLP1-expressing cells"). For example, A375 cells (human malignant melanoma cells, eg, ATCC CRL-1619) can be used.

There are no particular limitations on the method of contacting the compound of the present invention with PLP1-expressing cells, but examples include methods generally used to introduce nucleic acids into cells. Specifically, for example, lipofection method, electroporation method, gymnosis method, etc. are used. In the lipofection method, the compound of the invention can be treated, for example, at a final concentration of 3, 10, 30 or 100 nM.

Intracellular mRNA levels of PLP1 can be assayed by various methods known in the art. Specific examples include Northern blot analysis, competitive polymerase chain reaction (PCR), quantitative real-time PCR, and the like.

Intracellular protein levels of PLP1 can be assayed by various methods known in the art. Specifically, for example, immunoprecipitation, Western blot analysis (immunoblot), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry. , immunocytochemistry, or fluorescence-activated cell sorting (FACS).

In vivo for evaluating the suppression of PLP1 expression in cells of the compound of the present invention
Examples of methods for measuring PLP1 expression include a method in which the compound of the present invention is administered to an animal that expresses PLP1, and the above-mentioned PLP1 expression level is analyzed in the cells.

The compound of the present invention can be synthesized by the phosphoramidite method using commercially available amidites for DNA/RNA synthesis (including LNA). Oligomers of the artificial nucleic acids ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], and ALNA [Trz] can be synthesized by the method described in WO2020/100826.

(Treatment of PLP1-related diseases using the compound of the present invention)
The compounds of the present invention can treat PLP1-related diseases by suppressing PLP1 expression. PLP1-related diseases are not particularly limited as long as they are caused by abnormalities in the PLP1 gene, and congenital cerebral leukodystrophy is exemplified.

An example of congenital cerebral white matter hypoplasia caused by an abnormality in the PLP1 gene is Pellizaeus-Merzbacher disease, which includes fetal Pellizaus-Merzbacher disease and classical Pellizaeus-Merzbacher disease.

Therefore, the present invention provides modified oligonucleotides for use in treating, preventing or delaying the progression of PLP1-related diseases; pharmaceutical compositions for use in treating, preventing or delaying the progression of PLP1-related diseases; Use of a modified oligonucleotide for the treatment, prevention or delay of progression; Use of a modified oligonucleotide in the manufacture of a medicament for the treatment, prevention or delay of progression of a PLP1-related disease; Use of a modified oligonucleotide for the treatment, prevention or delay of progression of a PLP1-related disease A modified oligonucleotide for use in the manufacture of a medicament; a method for treating, preventing or delaying the progression of a PLP1-related disease, which comprises administering an effective amount of the modified oligonucleotide to a subject in need thereof;

(2) Pharmaceutical composition containing modified oligonucleotide The compound of the present invention, or a pharmaceutically acceptable salt thereof, and the compound of the present invention containing a pharmaceutically acceptable carrier can be used as a pharmaceutical composition. For the preparation of pharmaceutical compositions, modified oligonucleotides can be mixed with one or more pharmaceutically acceptable active or inert substances. Compositions and methods for formulating pharmaceutical compositions can be selected according to a number of criteria, including route of administration, severity of disease, or dose administered.
For example, an injection can be used as a composition for parenteral administration. Such injections are prepared according to methods known per se, for example, by dissolving, suspending, or emulsifying the above-mentioned modified oligonucleotide in a sterile aqueous or oily liquid commonly used for injections. Aqueous solutions for injection include, for example, phosphate buffered saline, physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents, such as alcohol (e.g., ethanol). , polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mo1) adduct of hydrogenated castor oil), etc. may be used in combination. It may also contain buffering agents, pH adjusting agents, tonicity agents, soothing agents, preservatives, stabilizing agents, and the like. Such compositions are manufactured by known methods.

Examples of parenteral administration include subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intracranial administration, intrathecal administration, and intraventricular administration. Administration may be continuous or chronic, short term or intermittent.

Compositions for oral administration include solid or liquid dosage forms, in particular tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions, etc. Such compositions are manufactured by known methods and contain carriers, diluents or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate, and examples of diluents include physiological saline.

Furthermore, the pharmaceutical composition of the present invention can contain a reagent for introducing nucleic acid. As the reagent for introducing the nucleic acid, cationic lipids such as liposome, lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly(ethyleneimine) (PEI) are used. Can be used.

The modified oligonucleotides included in the pharmaceutical compositions of the present invention may also include cholesterol, carbohydrates, phospholipids, biotin, phenazine, vitamins, peptides, folate, phenanthridine, anthraquinone, acridine, etc. at one or more locations. , fluorescein, rhodamine, coumarin and dyes conjugated with conjugate groups are preferably used. The conjugated modified oligonucleotides described above can be produced by known methods and selected to enhance their activity, cellular distribution, or cellular uptake.

The conjugate group may be directly attached to the modified oligonucleotide, or the conjugate group may be an amino, hydroxyl, carboxylic acid, thiol, unsaturated moiety (e.g., double or triple bond), 8-amino-3 , 6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted The modified oligonucleotide is attached by a linking moiety selected from substituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. Here, the substituents are selected from amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

The administration form of the pharmaceutical composition of the present invention may be systemic administration such as oral administration, intravenous administration, or intraarterial administration, or local administration such as intracranial administration, intrathecal administration, or intraventricular administration. There may be. The dosage of the pharmaceutical composition of the present invention can be changed as appropriate depending on the purpose of use, severity of disease, patient's age, weight, sex, etc., but usually the amount of modified oligonucleotide is 0.1 ng to 100 mg/kg. /day, preferably from the range of 1 ng to 10 mg/kg/day.

NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE While certain compounds, compositions and methods described herein are specifically described in accordance with certain embodiments, the following examples are incorporated herein by reference. It serves only to exemplify and is not intended to be limiting. Each of the references mentioned in this application is herein incorporated by reference in its entirety.

Example 1
Synthesis and purification of modified oligonucleotide compounds for in vitro evaluation
Modified oligonucleotide compounds (SEQ ID NOs: 2 to 54) having the base sequences listed in Table 1 were synthesized using a general oligonucleotide analog solid-phase synthesis method and ALNA[Ms]amidite (described in WO2020/100826). It was synthesized and purified by Nippon Gene Co., Ltd. using the following method.

Table 1 shows the target positions of the synthesized modified oligonucleotide compounds. The target position is indicated from the target start position to the target end position, and the target start position is the PLP1 pre-mRNA 5' target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the sequence listing, which corresponds to the 3' end of the modified oligonucleotide). and the target end position indicates the PLP1 pre-mRNA 3' target site of the modified oligonucleotide (position of SEQ ID NO: 1 in the sequence listing, which corresponds to the 5' end of the modified oligonucleotide).
The internucleoside linkages of each modified oligonucleotide are phosphorothioate internucleoside linkages, and each modified oligonucleotide contains a sugar moiety of 5-methylcytosine and/or ALNA [Ms].

Example 2
In vitro PLP1 knockdown activity test
A375 cells were seeded at 3×10 ³ cells/well on a transfection reagent containing a mixture of the synthesized modified oligonucleotide and Lipofectamine RNAi Reagent, and cultured in a CO ₂ incubator for about 24 hours. Thereafter, using SuperPrep II Lysis & RT Kit for qPCR (TOYOBO), RNA preparation and reverse transcription reaction were performed to synthesize cDNA. The mRNA expression level was measured by quantitative real-time PCR using the synthesized cDNA. For quantitative real-time PCR, probes for PLP1 gene (Hs.PT.58.39005119, INTEGRATED DNA TECHNOLOGIES) and HPRT1 gene (Hs.PT.58v.45621572, INTEGRATED DNA TECHNOLOGIES) were used, and ΔΔ HPRT1 mRNA expression level by Ct method The relative expression level of PLP1 mRNA was calculated. The inhibition rate of the modified oligonucleotide was calculated as a percentage from the rate of decrease in the expression level of PLP1 mRNA when the modified oligonucleotide was added, and the IC ₅₀ value was calculated from the two concentrations that sandwiched the 50% inhibition rate. The results are shown in Table 1. As a result, it was found that the modified oligonucleotide of the present invention has an excellent effect of suppressing PLP1 expression.

Claims (20)

A modified oligonucleotide consisting of 12 to 30 residues, and an isometric portion of any one of positions 10014 to 10152, positions 10180 to 10252, or positions 14033 to 14113 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. the full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to an equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing; A modified oligonucleotide that suppresses the expression of Proteolipid protein 1 (PLP1). A modified oligonucleotide consisting of 12 to 30 residues, and positions 10036 to 10055, 10064 to 10089, 10101 to 10130, 10202 to 10230, or 14055 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. The modified oligonucleotide contains at least 8 consecutive nucleobase sequences complementary to any equal-length portion of positions 14091, and the full-length nucleobase sequence of the modified oligonucleotide is of equal length to the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. A modified oligonucleotide that suppresses the expression of PLP1, having at least 85% complementarity in portions. A modified oligonucleotide consisting of 12 to 30 residues, and an isometric portion of any one of positions 10101 to 10130, 10202 to 10230, or 14055 to 14091 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. the full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to an equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing; A modified oligonucleotide that suppresses the expression of PLP1. Positions 10104-10119, 10109-10124, 10114-10129, 10202-10217, 10203-10218, 14056-14071, 14057-14072 from the 5' end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, A modified oligonucleotide that suppresses the expression of PLP1, consisting of a nucleobase sequence complementary to either position 14074-14089 or position 14075-14090. The modified oligonucleotide according to any one of claims 1 to 4, which is single-stranded. The modified oligonucleotide according to any one of claims 1 to 5, wherein at least one nucleoside constituting the modified oligonucleotide contains a modified sugar. 7. The modified oligonucleotide of claim 6, wherein the modified sugar is selected from the group consisting of bicyclic sugars, 2'-O-methoxyethyl-modified sugars, and 2'-O-methyl-modified sugars. . 7. The bicyclic sugar is selected from the sugar moieties of LNA, ENA, cEt, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz]. The modified oligonucleotide described in . The modified oligonucleotide according to any one of claims 1 to 8, wherein at least one nucleoside constituting the modified oligonucleotide contains a modified nucleobase. 10. The modified oligonucleotide according to claim 9, wherein the modified nucleobase is 5-methylcytosine. The modified oligonucleotide according to any one of claims 1 to 10, wherein at least one internucleoside bond constituting the modified oligonucleotide is a modified internucleoside bond. 12. The modified oligonucleotide of claim 11, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage. The modified oligonucleotide is
1) gap segment;
2) 5' wing segment;
3) a 3' wing segment;
the gap segment is positioned between the 5′ wing segment and the 3′ wing segment;
The modified oligonucleotide according to any one of claims 1 to 12, wherein the nucleosides constituting the 5' wing segment and the 3' wing segment contain a modified sugar. A pharmaceutical composition comprising the modified oligonucleotide according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 14, for treating, preventing, or delaying the progression of a PLP1-related disease. 16. The pharmaceutical composition according to claim 15, wherein the PLP1-related disease is Pellizaeus-Merzbacher disease. 17. The pharmaceutical composition according to claim 16, wherein the Pellizaeus-Merzbacher disease is fetal Pellizaeus-Merzbacher disease or classical Pellizaeus-Merzbacher disease. Treatment, prevention, or progression of PLP1-related diseases in a subject, characterized by administering a therapeutically effective amount of the modified oligonucleotide according to any one of claims 1 to 13 to a subject in need thereof. Methods for delaying. Use of the modified oligonucleotide according to any one of claims 1 to 13 in the manufacture of a medicament for treating, preventing or delaying the progression of a PLP1-related disease. The modified oligonucleotide according to any one of claims 1 to 13, for treating, preventing, or delaying the progression of a PLP1-related disease.