WO2021261538A1

WO2021261538A1 - Compound, method, and pharmaceutical composition for regulating plp1 expression

Info

Publication number: WO2021261538A1
Application number: PCT/JP2021/023883
Authority: WO
Inventors: 新司熊谷; 峻幸金川; 浩昭澤本
Original assignee: 田辺三菱製薬株式会社
Priority date: 2020-06-24
Filing date: 2021-06-24
Publication date: 2021-12-30
Also published as: JP2023130528A

Abstract

The present invention pertains to: a modified oligonucleotide that is formed of 12-30 residues, that has at least eight consecutive nucleic acid base sequences complementary to an equal length portion at, from the 5'-end in the nucleic acid base sequence indicated in SEQ ID NO: 1 of the sequence listing, positions 9964-10286, positions 140007-14152, positions 4439-4470, positions 4569-4616, positions 9061-9164, positions 11926-12015, positions 12817-12874, positions 14839-14920, or positions 15088-15114, that has a total length nucleic acid base sequence being at least 85% complementation to an equal length portion of the nucleic acid base sequence indicated in SEQ ID NO: 1 of the sequence listing, and that inhibits expression of proteolipid protein 1 (PLP1); and a medicinal drug that contains the modified oligonucleotide and that is for treating, preventing, delaying, or improving PLP1-related diseases.

Description

Compounds, methods and pharmaceutical compositions for regulating PLP1 expression

The present invention relates to a compound for reducing at least one of the pre-mRNA level, mRNA level and protein level of Proteinipid protein 1 (PLP1) in an animal, a method using the compound, and a pharmaceutical composition containing the compound. Regarding. The method of the present invention is useful for treating, preventing, or delaying progression of PLP1-related diseases such as Pelizaeus-Merzbacher Disease (PMD).

Pelizaeus-Merzbacher's disease (PMD) is the most common congenital cerebral white matter imperfecta that occurs in about 1 in 200,000 to 500,000 people. Many patients develop nystagmus and head tremor, which subsequently develop into convulsions and spastic paralysis.

Mutations in the proteolipid protein 1 gene (PLP1 gene) have been reported as the cause of the onset of PMD (Non-Patent Documents 1 to 4). PMDs are classified into congenital PMDs and classical PMDs according to the type of mutation in the PLP1 gene. Congenital PMD is caused by amino acid substitutions and splicing abnormalities due to point mutations in the PLP1 gene, and accounts for about 15-25% of all patients with PLP1 gene mutations. It is severe, develops from birth or newborn, and dies in infancy. Classic PMD is caused by duplication of the PLP1 gene and accounts for 50-75% of the same. It develops from early infancy and dies in early childhood.

So far, multiple treatment methods have been tried for the treatment of PMD. Yamauchi et al. Tried a therapeutic method using a substance that inhibits MAPK pathway activation (Patent Document 1), but in general, kinase inhibitors have problems in terms of safety 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, a therapeutic method using an antisense nucleic acid has also been attempted, and Elitt et al. Suppressed the expression of PLP1 by using an antisense nucleic acid complementary to the base sequence of the intron region of the PLP1 gene, thereby congenital PMD. It has been confirmed that it shows a therapeutic effect in disease model mice that develop similar symptoms (Non-Patent Document 5). However, since the above-mentioned antisense nucleic acid is a method designed based on the base sequence of the mouse PLP1 gene, it is difficult to clinically apply it as a therapeutic method for an actual PMD patient.

JP5391401B WO2019 / 156115 WO2018 / 106782

Currently, there is no fundamental treatment as a PMD treatment method, such as medication and medical treatment as symptomatic treatment for epilepsy, dystonia, mental retardation, etc., and the effect is insufficient and the burden on the patient is heavy. Therefore, an object of the present invention is to provide compounds, compositions and methods for treating, preventing, delaying, or ameliorating PLP1-related diseases such as PMD.

An object of the present invention is also to provide a compound, a method, and a pharmaceutical composition for suppressing PLP1 expression.

The present inventors have conducted diligent studies, found a modified oligonucleotide that strongly suppresses PLP1-expression, and found that the modified oligonucleotide is effective for the treatment and prevention of PLP1-related diseases such as PMD, and completed the present invention. I came to let you.

The gist of the present invention is as follows.
[1] A modified oligonucleotide consisting of 12 to 30 residues, and 9964 to 10286 positions, 140007 to 14152 positions, 4439 to 4470 positions, 4569 to 4616 positions from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. Containing at least eight contiguous nucleic acid sequences complementary to any of the equilength moieties of positions 9061-9164, 11926-12015, 12817-12874, 14389-14920 or 15088-15114, said. A modified oligonucleotide that suppresses the expression of Proteolipid protein 1 (PLP1), wherein the full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to the equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing.
[2] A modified oligonucleotide consisting of 12 to 30 residues, and either at positions 10014 to 10152 or positions 14033 to 14113 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. The full-length nucleobase sequence of the modified oligonucleotide contains at least eight contiguous nucleic acid sequences complementary to the equilength portion and is at least 85% complementary to the equilongate 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).
[3] A modified oligonucleotide consisting of 12 to 30 residues, and from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, positions 10036 to 10059, positions 10064 to 10089, positions 10101 to 10130, and 10202 to 10230. The full-length nucleobase sequence of the modified oligonucleotide contains at least eight contiguous nucleobase sequences complementary to the equilength portion of the position or position 14055 to 14091, and the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. A modified oligonucleotide that suppresses the expression of PLP1 with at least 85% complementarity to the equal length portion of.
[4] A modified oligonucleotide consisting of 12 to 30 residues, and either at positions 10101 to 10130 or 14055 to 14091 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. The full-length nucleobase sequence of the modified oligonucleotide contains at least eight contiguous nucleic acid sequences complementary to the equilength portion and is at least 85% complementary to the equilongate portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. A modified oligonucleotide that suppresses the expression of PLP1.
[5] From the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, 10036 to 10051, 10037 to 10025, 10038 to 10053, 10039 to 10045, 10040 to 10055, 10064 to 10024, 10065 to 10065 to 10065. 10080th, 10066-10081th, 10067-10082th, 10062-10083th, 10069-10084th, 10070-10085th, 10071-10086th, 10072-10087th, 10073-10088th, 10064-10089th, 10101- 10116th, 10102-10117th, 10103-10118th, 10104-10119th, 10105-10120th, 10106-10121th, 10107-10122th, 10108-10123th, 10109-10124th, 10110-10125th, 10111- 10126th, 10112-10127th, 10113-10128th, 10114-10129th, 10115-10130th, 10202-10217th, 10203-10218th, 10204-10219th, 10205-10220th, 10206-10221th, 10207- 10222th, 10208-10223th, 10209-10224th, 10210-10225th, 10211-10226th, 10212-10227th, 10213-10228th, 10214-10229th, 10215-10230th, 14055-14070th, 14056- Expression of PLP1 consisting of a nucleobase sequence complementary to any of positions 14071, 14057 to 14072, 14062 to 14077, 14063 to 14078, 14074 to 14089, 14075 to 14090, or 14076 to 14091. A modified oligonucleotide that suppresses.
[6] A modified oligonucleotide consisting of 12 to 30 residues, and 4439 to 4470, 4569 to 4616, 961 to 9164, 9964 to 10055 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. , 10056 to 10286, 11926 to 12015, 12817 to 12874, 14007 to 14095, 14112 to 14152, 14039 to 14920, or 15088 to 15114, at least eight complementary to any of the equal length portions. Of Proteolipid protein 1 (PLP1), which comprises a contiguous sequence of nucleobases, wherein the full length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to the equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. A modified oligonucleotide that suppresses expression.
[7] From the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, positions 4450 to 4467, 4571 to 4589, 9072 to 9089, 9119 to 9136, 10035 to 10025, 10037 to 10045, 10072 to 10089th, 10088-10105th, 10101-10118th, 10102-10119th, 10103-10120th, 10104-10121th, 10105-10122th, 10108-10125th, 10109-10126th, 10112-10129th, 10113- 10130, 11959 to 11976, 11989 to 12006, 12830 to 12847, 14020 to 14037, 14025 to 14042, 14055 to 14072, 14056 to 14073, 14060 to 14077, 14065 to 14082, 14075 to Expression of PLP1 consisting of a nucleobase sequence complementary to any of positions 14092, 14130-14147, 14847-14864, 14852-14869, 14857-14874, 14866-14883, or 15091-15108. A modified oligonucleotide that suppresses.
[8] The modified oligonucleotide according to any one of [1] to [7], which is a single strand.
[9] The modified oligonucleotide according to any one of [1] to [8], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified sugar.
[10] The above-mentioned [9], wherein the modified sugar 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 oligonucleotide.
[11] Bicyclic sugars are selected from the sugar moieties of LNA, ENA, cEt, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz]. [10] The modified oligonucleotide according to [10].
[12] The modified oligonucleotide according to any one of [1] to [11], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified nucleobase.
[13] The modified oligonucleotide according to [12], wherein the modified nucleobase is 5-methylcytosine.
[14] The modified oligonucleotide according to any one of [1] to [13], wherein the at least one nucleoside link constituting the modified oligonucleotide is a modified nucleoside bond.
[15] The modified oligonucleotide according to [14], wherein the modified nucleoside linkage is a phosphorothioate nucleoside linkage.
[16] The modified oligonucleotide is
1) Gap segment and
2) 5'wing segment and
3) Including 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 [13], wherein the nucleoside constituting the 5'wing segment and the 3'wing segment contains a modified sugar.
[17] A pharmaceutical composition comprising the modified oligonucleotide according to any one of [1] to [16] or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
[18] The pharmaceutical composition according to [17] for treating, preventing, or delaying the progression of PLP1-related diseases.
[19] The pharmaceutical composition according to [18], wherein the PLP1-related disease is Pelizaeus-Merzbacher's disease.
[20] The pharmaceutical composition according to [19], wherein the Pelizaeus-Merzbacher disease is congenital Pelizaeus-Merzbacher disease or classical Pelizaeus-Merzbacher disease.
[21] Treatment, prophylaxis or prevention of PLP1-related diseases in subjects, characterized in that a therapeutically effective amount of the modified oligonucleotide according to any one of [1] to [16] is administered to a subject in need thereof. A method for delaying its progress.
[22] Use of the modified oligonucleotide according to any one of [1] to [16] in the manufacture of a pharmaceutical for the treatment, prevention or delay of its progression of PLP1-related diseases.
[23] The modified oligonucleotide according to any one of [1] to [16], for treating, preventing or delaying the progression of PLP1-related diseases.

According to the present invention, PLP1 expression can be efficiently suppressed, the symptoms of PLP1-related diseases such as Pelizaeus-Merzbacher's disease can be improved, and an effective drug for the prevention and treatment of the diseases is provided. ..

It should be understood that both the above outline and the following detailed description are merely exemplary and descriptive and do not limit the claimed invention. Unless otherwise stated herein, the use of the singular includes the plural. As used herein, the use of the term "included" and other forms, such as "includes" and "included", is not limiting. Further, unless otherwise stated, terms such as "element" include an element comprising one unit and an element comprising more than one subunit.

The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described. All documents or parts of documents cited in this application, including but not limited to patents, patent applications, articles, books and articles, may, in part, with respect to, in whole, in part of the documents discussed herein. It is expressly incorporated herein by reference.

(Definition)
Unless a specific definition is given, the nomenclatures used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and medicinal chemistry described herein, and their procedures and techniques, are in the art. It is well known and commonly used in. Standard techniques can be used for chemical synthesis and analysis as used herein. Where permitted, all patents, applications, published applications and other publications referred to throughout the disclosure of this specification, GenBank accession numbers and GenBank accession numbers available through databases such as the National Center for Biotechnology Information (NCBI). Relevant sequence information and other data are incorporated by reference with respect to and in whole the documents discussed herein.
In addition, although this specification is filed together with an electronic format sequence listing, the information in the sequence listing described in the electronic format is incorporated herein by reference in its entirety.

Unless otherwise instructed, the following terms have the following meanings.

"Nucleobase" means a heterocyclic moiety that can be paired with the base of another nucleic acid.

"Nucleobase sequence" means a continuous sequence of nucleobases constituting the oligonucleotide of the present 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" means a molecule in which a phosphate group is bonded to the sugar portion of a nucleoside. The naturally occurring nucleotide has a sugar moiety of ribose or deoxyribose, which is covalently bonded by a phosphodiester bond via a phosphate group.

"Oligomer 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 the bonds between each nucleoside are linked independently of each other.

"Nucleoside bond" refers to a chemical bond between nucleosides.

"Naturally occurring nucleoside bond" means a 3'-5'phosphodiester bond.

"Modified nucleoside bond" refers to a substitution or arbitrary change from a naturally occurring nucleoside bond (ie, a phosphodiester nucleoside bond). For example, there are, but are not limited to, phosphorothioate nucleoside linkages.

"Phosphodiester bond between nucleosides" means a bond between nucleosides in which the phosphodiester bond is modified by replacing one of the non-crosslinked oxygen atoms with a sulfur atom. The phosphorothioate bond is an example of the modified nucleoside-linked bond.

"Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil. For example, there is, but is not limited to, 5-methylcytosine. "Unmodified nucleobase" means the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

"Modified oligonucleotide" means an oligonucleotide containing at least one of the modified nucleosides and / or the linkage between the modified nucleosides.

"Salt" is a general term for compounds in which one or more dissociable hydrogen ions contained in an acid are replaced with cations such as metal ions and ammonium ions, and the salt of the modified oligonucleotide is phosphorothioate. Alternatively, a salt (eg, sodium salt, magnesium salt) formed with an inorganic ion (eg, sodium ion, magnesium ion) on a phosphodiester or a functional group (eg, amino group) in a modified nucleic acid base can be mentioned. However, it is not limited to these.

"Sugar" or "sugar moiety" means a natural sugar moiety or a modified sugar moiety.

"Modified sugar" refers to a substitution or change from a natural sugar. Examples of the modified sugar include a substituted sugar moiety and a bicyclic sugar.

"Substituted sugar moiety" means furanosyl other than natural sugar in RNA or DNA.

"Bicyclic sugar" means a furanosyl ring modified by cross-linking two different carbon atoms present on the same ring.

"Single-stranded oligonucleotide" means an oligonucleotide that does not hybridize with the complementary strand.

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

"Complementary" means the ability of the first nucleic acid to form a pair between the nucleobases of the 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 the first nucleic acid is completely complementary to the nucleobase sequence of the second nucleic acid. It means that there is. In certain embodiments, the first nucleic acid is a modified oligonucleotide and the target nucleic acid is a second nucleic acid.

"Mismatch" or "non-complementary nucleobase" refers to the case where the nucleobase of the first nucleic acid cannot be paired with the corresponding nucleobase of the second or target nucleobase.

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

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

A "pharmaceutically acceptable salt" is a physiologically and pharmaceutically acceptable salt of a modified oligonucleotide of the invention, i.e., which retains the desired biological activity of the modified oligonucleotide and imparts an undesired toxic effect to it. Means no salt.

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

"Improvement" refers to reducing at least one indicator, sign or symptom of a related disease, disorder or condition. The severity of the indicator can be determined by subjective or objective measures known to those of skill in the art.

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

"Effective amount" means the amount of the modified oligonucleotide of the invention that is sufficient to achieve the desired physiological outcome in an individual in need of the drug. Effective amounts vary among individuals depending on the health and physical condition of the individual being treated, the taxon 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.

"Preventing" delays or delays the onset or onset of a disease, disorder or unfavorable health condition, or one or more symptoms associated with the disease, disorder or unfavorable health condition over a period of minutes to an indefinite period of time. It means to prevent it. Prevention also means reducing the risk of developing a disease, disorder or unfavorable health condition.

"Treatment" reduces or eliminates or eliminates a disease, disorder or unfavorable health condition, or one or more symptoms associated with the disease, disorder or unfavorable health condition, or the disease, disorder. , Or means to partially eliminate or eradicate one or more causes of the unfavorable health condition itself.

(Specific embodiment)
Certain specific embodiments shown below are not limited to these, but provide a compound for suppressing the expression of PLP1, a method using the compound, and a pharmaceutical composition containing the compound.

(1) Modified oligonucleotide The modified oligonucleotide of the present invention (hereinafter, may be referred to as "the compound of the present invention" or "the modified oligonucleotide of the present invention") is an antisense oligonucleotide of PLP1 having an activity of suppressing PLP1 expression. Is. Suppression of PLP1 expression (level) in the present invention refers to the pre-mRNA level transcribed from the PLP1 gene, the pre-mRNA spliced mRNA level, and the PLP1 protein level translated from the mRNA (collectively, "PLP1". It means suppressing at least one of (sometimes referred to as "expression level"). The PLP1 mRNA is not particularly limited and may include sequence variations. For example, the PLP1 mRNA represented by the sequence (SEQ ID NO: 1 in the sequence listing) described in GenBank accession number NG_00863.2: 4996-21109 is used. Can be mentioned. In the RNA sequence, T is replaced with U in SEQ ID NO: 1.

The degree of suppression of PLP1 expression possessed by the compound of the present invention is lower than that when at least one of the pre-mRNA level, mRNA level and protein level of PLP1 is not administered with the compound, and as a result, PLP1-related diseases occur. Any degree may be used as long as the prevention and / or improvement of the accompanying symptoms is observed. Specifically, for example, in the method for measuring in vitro PLP1 expression described later, after contacting the compound of the present invention with cells, the cells are contacted with each other. The PLP1 expression level is at least 70% or less, preferably 50% or less, more preferably 40% or less, still more preferably 30% or less, and particularly preferably 20% or less, as compared with the case of non-contact or contact with a negative control substance. Is used. Further, in the in vitro PLP1 expression measuring method described later, an IC ₅₀ having an IC 50 of preferably 100 nM or less, more preferably 50 nM or less, and more preferably 20 nM or less is used.

The method for verifying the activity of the compound of the present invention may be any method as long as it can be verified that the compound of the present invention suppresses PLP1 expression, and the method described in "Method for evaluating the compound of the present invention" described later is exemplified. Will be done.

In one embodiment, the compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues, preferably 16 to 18 residues, having an activity of suppressing PLP1 expression, and the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. 9964 to 10286, 140007 to 14152, 4439 to 4470, 4569 to 4616, 9061 to 9164, 11926 to 12015, 12817 to 12874, 14039 to 14920 or 15088 to 15114 from the 5'end of the Any one may be used as long as it contains at least eight consecutive nucleobase sequences (hereinafter referred to as "PLP1 complementary nucleobase sequence") complementary to any of the equal length portions. The PLP1 complementary nucleobase sequence is preferably 8 to 25 consecutive nucleobase sequences, more preferably 12 to 20 consecutive nucleobase sequences, and even more preferably 16,17. , 18, 19 or 20 consecutive nucleobase sequences.

In one embodiment, the compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues, preferably 16 to 18 residues, having an activity of suppressing PLP1 expression, and the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. Any one containing at least 8 consecutive nucleobase sequences complementary to any of the equilength portions of positions 10014 to 10152, 10180 to 10252, or 14033 to 14113 from the 5'end of the above. .. The PLP1 complementary nucleobase sequence is preferably 8 to 25 consecutive nucleobase sequences, more preferably 12 to 20 consecutive nucleobase sequences, and even more preferably 16,17. , 18, 19 or 20 consecutive nucleobase sequences.

In one embodiment, the compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues, preferably 16 to 18 residues, having an activity of suppressing PLP1 expression, and the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. At least 8 consecutive nucleobase sequences complementary to any of the equilongate moieties at positions 10036 to 10055, 10064 to 10089, 10101 to 10130, 10202 to 10230, or 14055 to 14091 from the 5'end of Anything containing (hereinafter referred to as "PLP1 complementary nucleic acid base sequence") may be used. The PLP1 complementary nucleobase sequence is preferably 8 to 25 consecutive nucleobase sequences, more preferably 12 to 20 consecutive nucleobase sequences, and even more preferably 16,17. , 18, 19 or 20 consecutive nucleobase sequences.

In one embodiment, the compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues, preferably 16 to 18 residues, having an activity of suppressing PLP1 expression, and the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. At least eight consecutive nucleobase sequences complementary to any of the equal length portions of positions 10101 to 10130 or 14055 to 14091 from the 5'end of the above (hereinafter referred to as "PLP1 complementary nucleobase sequence"). Anything may be used as long as it contains (referred to as). The PLP1 complementary nucleobase sequence is preferably 8 to 25 consecutive nucleobase sequences, more preferably 12 to 20 consecutive nucleobase sequences, and even more preferably 16,17. , 18, 19 or 20 consecutive nucleobase sequences.

More specifically, as the PLP1 complementary nucleobase sequence, from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, positions 10036 to 10051, positions 10037 to 10025, positions 10028 to 10053, positions 10039 to 10045 to 10040 to 10055, 10064 to 10094, 10065 to 10080, 10066 to 10081, 10067 to 10082, 10068 to 10083, 10069 to 10084, 10070 to 10085, 10071 to 10086, 10072 to 10087, 10073 to 10087. 10088th, 10074-10089th, 10101-10116th, 10102-10117th, 10103-10118th, 10104-10119th, 10105-10120th, 10106-10121th, 10107-10122th, 10108-10123th, 10109- 10124th, 10110-10125th, 10111-10126th, 10112-10127th, 10113-10128th, 10114-10129th, 10115-10130th, 10202-10217th, 10203-10218th, 10204-10219th, 10205- 10220, 10206 to 10221, 10207 to 10222, 10208 to 10223, 10209 to 10224, 10210 to 10225, 10211 to 10226, 10212 to 10227, 10213 to 10228, 10214 to 10229, 10215 to Nucleic acid complementary to any of positions 10230, 14055 to 14070, 14056 to 14071, 14057 to 14072, 14062 to 14077, 14063 to 14078, 14074 to 14089, 14075 to 14090, 14076 to 14091. It may be a base sequence.

In one embodiment, the compound of the present invention is a modified oligonucleotide consisting of 12 to 30 residues, preferably 16 to 18 residues, having an activity of suppressing PLP1 expression, and the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. From the 5'end, 4439-4470, 4569-4616, 9061-9164, 9964-10055, 10056-10286, 11926-12015, 12817-12874, 14007-14895, 14112-14152, Any one containing at least eight consecutive nucleobase sequences complementary to any of the equilength portions of positions 14839 to 14920 or 15088 to 15114 may be used. The PLP1 complementary nucleobase sequence is preferably 8 to 25 consecutive nucleobase sequences, more preferably 12 to 20 consecutive nucleobase sequences, and even more preferably 16,17. , 18, 19 or 20 consecutive nucleobase sequences.

More specifically, as the PLP1 complementary nucleobase sequence, positions 4450 to 4467, 4572 to 4589, 9072 to 9089, 9119 to 9136, and 10035 to the 5'end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. 10027, 10037 to 10049, 10072 to 10089, 10088 to 10105, 10101 to 10118, 10102 to 10119, 10103 to 10120, 10104 to 10121, 10105 to 10122, 10108 to 10125, 10109 to 10126th, 10112-10129th, 10113-10130th, 11959-11976th, 11989-12006th, 12830-12847th, 14020-14037th, 14025-14042th, 14055-14072th, 14056-14073th, 14060- Complementary to any of 14077, 14065 to 14082, 14075 to 14092, 14130 to 14147, 14847 to 14864, 14852 to 14869, 14857 to 14874, 14866 to 14883, or 15091 to 15108. It may be a nucleic acid base sequence.

Here, the modified oligonucleotide of the present invention has a total length of 12 to 30 residues in addition to the PLP1 complementary nucleic acid base sequence, and the full length nucleic acid base sequence is the nucleic acid base of SEQ ID NO: 1 in the sequence listing. It may have an additional sequence on its 5'end and / or 3'end, as long as it has at least 85% complementarity to the equal length portion of the sequence. Further, the addition sequence may be any as long as the modified oligonucleotide of the present invention has an activity of suppressing PLP1 expression.

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

The mismatched nucleobase may be continuous or may be sandwiched between PLP1 complementary nucleobase sequences. The percentage of complementarity of the antisense oligonucleotide of the present invention with PLP1 pre-mRNA or mRNA is described in BLAST programs (basic local alignment sequences) and PowerBLAST programs (Altschul et al., J. Mol. Biol) known in the art. , 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649, 656). Percent of homology, sequence identity or complementarity can be determined, for example, by the Gap Program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group, Universals Computer Group, University ResearchAp. It can be determined using the default settings that use the algorithms of., 1981, 2, 482, 489). For example, when 18 of the 20 nucleobases of a modified oligonucleotide are complementary to and hybridize to the equivalescent portion of PLP1 pre-mRNA, the modified oligonucleotide has 90% complementarity.

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

As an example of a specific nucleic acid base sequence of the modified oligonucleotide of the present invention,
Complementary sequence of positions 10037 to 10025 of SEQ ID NO: 1,
Complementary sequence at positions 10028 to 10053 of SEQ ID NO: 1,
Complementary sequence at positions 10039 to 10045 of SEQ ID NO: 1,
Complementary sequence at positions 10040 to 10055 of SEQ ID NO: 1,
Complementary sequence at positions 10065 to 10080 of SEQ ID NO: 1,
Complementary sequence at positions 10068 to 10083 of SEQ ID NO: 1,
Complementary sequence at positions 10072 to 10087 of SEQ ID NO: 1,
Complementary sequence at positions 10102 to 10117 of SEQ ID NO: 1,
Complementary sequence at positions 10103 to 10118 of SEQ ID NO: 1,
Complementary sequence at positions 10106 to 10121 of SEQ ID NO: 1,
Complementary sequence at positions 10110 to 10125 of SEQ ID NO: 1,
Complementary sequence at positions 10115 to 10130 of SEQ ID NO: 1,
Complementary sequence at positions 10206 to 10221 of SEQ ID NO: 1,
Complementary sequence at positions 10207 to 10222 of SEQ ID NO: 1,
Complementary sequence at positions 10209 to 10224 of SEQ ID NO: 1,
Complementary sequence at positions 10211 to 10226 of SEQ ID NO: 1,
Complementary sequence at positions 10212 to 10227 of SEQ ID NO: 1,
Complementary sequence at positions 10213 to 10228 of SEQ ID NO: 1,
Complementary sequence at positions 10214 to 10229 of SEQ ID NO: 1,
Complementary sequence at positions 14055 to 14070 of SEQ ID NO: 1,
Complementary sequence at positions 14062 to 14077 of SEQ ID NO: 1,
Complementary sequence at positions 14063 to 14078 of SEQ ID NO: 1, or complementary sequence at positions 14076 to 14091 of SEQ ID NO: 1,
The nucleic acid base sequence described in is mentioned.

As an example of a specific nucleic acid base sequence of the modified oligonucleotide of the present invention,
Complementary sequence at positions 4450 to 4467 of SEQ ID NO: 1,
Complementary sequence at positions 14020 to 14037 of SEQ ID NO: 1,
Complementary sequence at positions 14025 to 14042 of SEQ ID NO: 1,
Complementary sequence at positions 14055 to 14072 of SEQ ID NO: 1, or complementary sequence at positions 14060 to 14077 of SEQ ID NO: 1,
The nucleic acid base sequence described in is mentioned.

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

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

Examples of substituted sugar moieties are, but are not limited to, 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ₃ (2'-OMe). , 2'-OCH ₂ CH ₃ , 2'-OCH ₂ CH ₂ F and 2'-O (CH ₂ ) ₂ OCH ₃ (2'-MOE) nucleosides containing substituents. Substituents at the 2'position are allyl, amino, azide, thio, O-allyl _{, OC 1} to C ₁₀ alkyl, OCF ₃ , OCH ₂ F, O (CH ₂ ) ₂ SCH ₃ , O (CH ₂ ). _2- ON (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 ) (In the formula, each R _l , R _m and R _n are independently H or substituted or unsubstituted C ₁ to C ₁₀ alkyl). You can choose from.

Examples of nucleosides with bicyclic sugars include, but are not limited to, nucleosides that include crosslinks between the 4'and 2'ribosyl ring atoms. In certain embodiments, the oligonucleotides provided herein comprise a nucleoside having one or more bicyclic sugars whose cross-linking comprises one of the following formulas: 4'-(CH _2). ) -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 their analogs, see US Pat. No. 7,399,845); 4'-C (CH _3). _{) (CH 3) -O-2} '( and see like No. .WO2009 / 006478 _{thereof); 4'-CH 2 -N (} OCH 3) -2' ( and analogs thereof .WO2008 / See 150729); 4'-CH ₂ -ON (CH ₃ ) -2'(and their analogs, see US2004-0171570); 4'-CH _2- N (R). ) -O-2 '(wherein, R is H, C1 ~ C12 alkyl or a protecting group) (see U.S. Pat. No. _{7,427,672); 4'-CH 2 -C} (H) (CH ₃ ) -2'(and their analogs; see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH _2- C (= CH). ₂ ) -2'(and their analogs, see WO 2008/154401).

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; Eladi et al., Curr. Opinion Events. Drugs, 2001, 2, 558-561; Braach et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Walestedt et al., Proc. Natl. Acad. Sci. USA, 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. ,; US Pat. No. 7,399,845; 6,770,748; 6,525,191; 6,268,490; US US2008-0039618; US2007-0287831; US2004-0171570; US2009-01281; WO2010 / 036698; WO2009 / 067647; WO2009 / 067647; WO2007 / 134181; WO2005 / 021570; WO2004 / 106356; WO94 / 14226; WO2009 / 006478 See WO2008 / 154401; and WO2008 / 150729). Each of the aforementioned 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 of nucleosides with bicyclic sugars has been reported as artificial nucleosides with guanidine crosslinks (see WO2014 / 046212, WO2017 / 047816). The bicyclic nucleosides ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Trz] and ALNA [Oxz] have been reported as crosslinked artificial nucleic acid amino LNA (ALNA) (WO2020 / 100826). Please refer to).

In certain embodiments, nucleosides with bicyclic sugars include, but are not limited to, bridges between the 4'and 2'carbon atoms of the pentoflanosyl sugar moiety, but are not limited to-[C ( _Ra). ) (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- A bridge containing two linking groups is included, in which X is 0, 1 or 2; n is 1, 2, 3 or 4; each _Ra and R _b are independently H, a protective group. , Hydroxyl, C ₁ to C ₁₂ alkyl, substituted C ₁ to C ₁₂ alkyl, C ₂ to C ₁₂ alkenyl, substituted C ₂ to C ₁₂ alkenyl, C ₂ to C ₁₂ alkynyl, substituted C ₂ to C ₁₂ alkynyl, aromatic ring. group, a substituted aromatic group, a heterocyclic group, substituted heterocyclic _group, C 5 ~ _{C 7} alicyclic, substituted _C 5 ~ _{C 7} alicyclic, _{_{_{_{halogen, OJ 1, NJ 1 J 2}}}} , SJ 1, N 3 , COOJ ₁ , 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 are independently H, C ₁ to C ₁₂ alkyl, substituted C ₁ to C ₁₂ alkyl, C ₂ to C ₁₂ alkenyl, substituted C ₂ to C ₁₂ alkenyl, C ₂ to C ₁₂ alkynyl, respectively. Substituted C ₂ to C ₁₂ alkynyl, aromatic ring group, substituted aromatic ring group, acyl (C (= O) -H), substituted acyl, heterocyclic group, substituted heterocyclic group, C ₁ to C ₁₂ aminoalkyl, substituted C ₁ to C ₁₂ aminoalkyl or protective group.

In certain embodiments, the cross-linking 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 ) -ON (R)-. In certain embodiments, the crosslinks are 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'(in this case, the nucleoside having a bicyclic sugar is also referred to as cEt), 4'-CH ₂ -ON (R) -2'and 4'-CH. ₂ -N (R) -O-2'- wherein the average each R is independently, H, a protecting group or _C 1 _{~ C 12} alkyl.

In certain embodiments, the cross-linking of the bicyclic sugar moiety is 4'-CH _2- O-2'-(LNA) or CH _2- N (R)-, where each R is independent. -SO _2- CH ₃ (ALNA [Ms]), -CO-NH-CH ₃ (ALNA [mU]), 1,5-dimethyl-1,2,4-triazole-3-yl (ALNA [Trz]) , -CO-NH-CH (CH ₃ ) ₂ (ALNA [ipU]), 5-Methyl-1,2,4-oxadiazole-3-yl (ALNA [Oxz]) (WO2020 / 100926). ..

In certain embodiments, nucleosides with bicyclic sugars are further defined by isomer configuration. For example, a nucleoside containing a 4'-(CH ₂ ) -O-2'crosslink may be present in an α-L-configuration or a β-D-configuration.

In certain embodiments, nucleosides with bicyclic sugars include, but are 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 -ON (R) -2' 4, 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, H, a protecting _group, C 1 _{~ C 12} alkyl, or _C 1 in _{~ C 12} alkyl which may be substituted Urea or guanidine).

In certain embodiments, nucleosides with bicyclic sugars have the following formula:

During the ceremony
Bx is the heterocyclic base portion;
The T _a and T _b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
Z _a is C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, C ₂ to C ₆ alkynyl, substituted C ₁ to C ₆ alkyl, substituted C ₂ to C ₆ alkenyl, substituted C ₂ to 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 (in the formula, each J _c , J _d and J _e are independently H, C ₁ to C ₆ alkyl or substituted C ₁ to C ₆ alkyl, and X is O or NJ _c ). It is mono- or poly-substituted with independently selected substituents.

During the ceremony
Bx is the heterocyclic base portion;
The T _a and T _b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
Z _b is C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, C ₂ to C ₆ alkynyl, substituted C ₁ to C ₆ alkyl, substituted C ₂ to C ₆ alkenyl, substituted C ₂ to C ₆ alkynyl or substituted. It is an acyl (C (= O)-).

During the ceremony
Bx is the heterocyclic base portion;
The T _a and T _b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
R _d is C ₁ to C ₆ alkyl, substituted C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, substituted C ₂ to C ₆ alkynyl, C ₂ to C ₆ alkynyl or substituted C ₂ to C ₆ alkynyl. ;
Each q _a , q _b , q _c and q _d are independently H, halogen, C ₁ to C ₆ alkyl, substituted C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, substituted C ₂ to C ₆ alkenyl, C ₂ to C ₆ alkynyl or substituted C ₂ to C ₆ alkynyl, C ₁ to C ₆ alkoxyl, substituted C ₁ to C ₆ alkoxyl, acyl, substituted acyl, C ₁ to C ₆ aminoalkyl or substituted C ₁ to C ₆ amino It is alkyl.

During the ceremony
Bx is the heterocyclic base portion;
The T _a and T _b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
q _a , q _b , q _e and q _f are independently hydrogen, halogen, C ₁ to C ₁₂ alkyl, substituted C ₁ to C ₁₂ alkyl, C ₂ to C ₁₂ alkoxy, substituted C ₂ to C ₁₂ alkoxy, respectively. C ₂ to C ₁₂ alkynyl, substituted C ₂ to C ₁₂ alkynyl, C ₁ to C ₁₂ alkoxy, substituted C ₁ to 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 ;
Alternatively, q _e and q _f are both = C (q _g ) (q _h );
q _g and q _h are independently H, halogen, C ₁ to C ₁₂ alkyl or substituted C ₁ to C ₁₂ alkyl, respectively.

The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides (also referred to as LNAs) with 4'-CH _{2-O-2'bridges are their oligomerizations and nucleic acids.} Described with recognition characteristics (Koshkin etal., Tetrahedron, 1998, 54, 3607-3630). The synthesis of nucleosides with bicyclic sugars is also described in WO98 / 39352 and WO99 / 14226.

Various bicyclics with 4'-2'crosslinking groups 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). Preparation of a bicyclic nucleoside-containing oligodeoxyribonucleotide double strand for use as a substrate for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Further described in the art are the synthesis of 2'-amino-LNA (bicyclic nucleosides in this case also referred to as ALNA), ie, conformationally restricted high affinity oligonucleotide analogs (in the art). Singh et al., J. Org. Chem., 1998, 63, 1003-10039). In addition, 2'-amino- and 2'-methylamino-LNA have been prepared and double-stranded thermal stability with complementary RNA and DNA strands has been previously reported.

During the ceremony
Bx is the heterocyclic base portion;
The T _a and T _b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
Each q _i , q _j , q _k and q _l are independently H, halogen, C ₁ to C ₁₂ alkyl, substituted C ₁ to C ₁₂ alkyl, C ₂ to C ₁₂ alkoxy, substituted C ₂ to C ₁₂ alkoxy, respectively. C ₂ to C ₁₂ alkynyl, substituted C ₂ to C ₁₂ alkynyl, C ₁ to C ₁₂ alkoxyl, substituted C ₁ to 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 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 ), and in the equation, q _g and q _h are independently H, halogen, C ₁ to C ₁₂ alkyl or Substituted C ₁ to C ₁₂ alkyl.

_{4 '- (CH 2) 3-2} ' bridge and alkenyl analogs crosslinked _{4'-CH = CH-CH 2} 1 single carbocyclic bicyclic nucleosides having 2 'have been described (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 has also been described along with their oligomerization and biochemical studies (Srivastava et al., J. Am. Chem. Soc. 2007, 129 (26), 8362-. 8379).

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

Wherein, Bx is the base moiety, R represents independently a protecting _group, C 1 ~ _{C 6} alkyl or _C 1 ~ _{C 6} alkoxy.

In certain embodiments (LNA), nucleosides having bicyclic sugars have the following general formula:

[During the ceremony,
B is a nucleobase;
Each of X and Y can independently include a nucleoside represented by a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.] (WO98 / See 39352). A typical specific example is the following formula:

The nucleotides indicated by can be mentioned.

In certain embodiments (GuNA), nucleosides, including bicyclics, have the following general formula:

[In the formula, B is a nucleic acid base, 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. Yes, R ₇ and R ₈ are independently hydrogen atoms, hydroxyl group protective groups, optionally substituted phosphate groups, covalent bonds to phosphorus moieties or supports, etc., and R ₉ , R ₁₀ , R ₁₁ _{is a C 1-6} alkyl group or amino group protective group, each of which may be independently substituted with a hydrogen atom or one or more substituents. ]
It is a nucleoside represented by (see, for example, WO2014 / 046212, WO2017 / 047816).

In certain embodiments (ALNA [mU]), nucleosides comprising bicyclic sugars are represented by the following general formula (I):

[During the ceremony,
B is a nucleobase;
R ₁ , R ₂ , R ₃ and R ₄ _{are C 1-6} alkyl groups, each independently of which may be substituted with a hydrogen atom or one or more substituents;
R ₅ and R ₆ are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, and the like;
m is 1 or 2;
X is the following formula (II-1):

It is a group indicated by;
Symbols described in formula (II-1):

Indicates the binding 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. ]
It is a nucleoside represented by (see, for example, WO2020 / 100826). A typical embodiment _{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]), a nucleoside comprising a bicyclic sugar is a nucleoside having the general formula (I) as defined in ALNA [mU] above, wherein the nucleoside comprises the above formula.
X is the following formula (II-1):

It is a group indicated by;
One of R ₇ and R ₈ is a hydrogen atom and the other is an isopropyl group which may be substituted with one or more substituents (see, eg, WO2020 / 100826). A typical embodiment _{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 nucleoside comprising the bicyclic formula is a nucleoside having the above general formula (I), wherein X is the following formula (II-2) :.

It is a group indicated by;
A is a triazolyl group which may be substituted with one or more substituents (see, eg, Japanese Patent Application No. 2018-212424). A typical embodiment 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, which is a triazole-3-yl group.

In certain embodiments (ALNA [Oxz]), a nucleoside having the general formula (I) as defined in ALNA [mU] above, wherein the nucleoside has the same formula.
X is the following formula (II-2):

It is a group indicated by;
A is an oxadiazolyl group that may be substituted with one or more substituents (see, eg, Japanese Patent Application No. 2018-212424). A typical embodiment is an oxadiazolyl group in which A may have one or more methyl groups, more specifically a 5-methyl-1,2,4-oxadiazole-3-yl group. Is a nucleoside or nucleotide.

In certain embodiments (ALNA [Ms]), the nucleoside comprising the bicyclic formula is a nucleoside having the above general formula (I), wherein X is the following general formula (II-3) :.

It is a group indicated by;
M is a sulfonyl group substituted with a methyl group optionally substituted with one or more substituents (see, eg, WO2020 / 100826). A typical embodiment of ALNA [Ms] is a nucleoside, which is a sulfonyl group in which M is 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 rings, cyclohexenyl rings, cyclohexyl rings or tetrahydropyranyl rings, such as: The replacement of the ribosyl ring with one having one is mentioned:

In certain embodiments, sugar substitutes having the following formula are selected:

During the ceremony
Bx is the heterocyclic base portion;
T ₃ and T ₄ independently link the tetrahydropyran nucleoside analog to the oligomer compound, or one of T ₃ and T ₄ links the tetrahydropyran nucleoside analog to the oligomer compound or oligonucleotide. A nucleoside interlinking group, _{the other of T 3} and T ₄ being an H, hydroxyl protecting group, linking conjugate group or 5'or 3'-terminal group;
q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are independently H, C ₁ to C ₆ alkyl, substituted C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, substituted, respectively. 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, 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 (in the formula, X is O, S or NJ ₁ , and each J ₁ , J ₂ and J ₃ are independently H or C ₁ to C ₆ It is selected from (is alkyl).

In certain embodiments, q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are H, respectively. 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, a THP nucleoside in which one of _{R 1} and R _{2 is F is provided.} 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), altritor nucleic acid (ANA) and mannitol nucleic acid (MNA) (Leumann, C.J. , Bioorg. & Med. Chem., 2002, 10, 841-854).

In certain embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, its use in nucleosides and oligomeric compounds containing morpholino sugar moieties has been reported (eg, Braach 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" means a sugar substitute having the following structure:

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

In certain embodiments, the oligonucleotide comprises one or more modified cyclohexenyl nucleosides, which are nucleosides having a 6-membered cyclohexenyl in place of the pentoflanosyl residue of the naturally occurring nucleoside. Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (eg, WO2010 / 036696, published April 10, 2010, Robeyns et al., Concerning sharing. J. Am. Chem. Soc., 2008, 130 (6), 1979-1984; Harbor et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nucleoside et al., J. Am. Chem. Soc. 2007, 129 (30), 9340-9348; Guetal., Nucleosides, Nucleosides & Nucleic Acids, 2005, 24 (5-7), 993-998; , 2452-2463; Robeyns et al., Acta Crystallogica, Section F: Structural Biology and Crystallation Communications, 2005, F61 (6), 585-586; Guet2, Gu et al., Oligonucleosides, 2003, 13 (6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29 (24), 4941-4497; Wang etal., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleosides & 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 is by reference in its entirety herein. Will be incorporated).

Certain modified cyclohexenyl nucleosides have the following formula:

During the ceremony
Bx is the heterocyclic base portion;
T ₃ and T ₄ are each independently nucleoside interlinking groups that link the cyclohexenyl nucleoside analog to the oligonucleotide compound, or one of T ₃ and T ₄ ligates the tetrahydropyran nucleoside analog to the oligonucleotide compound. The other of T ₃ and T ₄ is an H, hydroxyl protecting group, linking conjugate group or 5'-or 3'-terminal group;
q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , q ₇ , q ₈ and q ₉ are independently H, C ₁ to C ₆ alkyl, substituted C ₁ to C ₆ alkyl, C _{2 respectively.} ~ C ₆ alkenyl, substituted C ₂ to C ₆ alkynyl, C ₂ to C ₆ alkynyl, substituted C ₂ to C ₆ alkynyl or other sugar substituents.

Many other bicyclic and tricyclic sugar-substituting ring systems that can be used to modify nucleosides for incorporation into oligonucleotides are known in the art (eg, Review: Leumann, Christian). See J., Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various further substitutions to enhance their activity.

The method for preparing the modified sugar is well known to those skilled in the art. Some representative U.S. teaches the preparation of such modified sugars. S. Patents are, but are not limited to, U.S.A. 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. Is done.

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

Preferred modified sugars are the sugar moieties of ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz], of 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 an activity of suppressing PLP1 expression. The nucleic acid base sequence of the modified oligonucleotide has at least 85%, at least 90%, or at least 95% complementarity to the equal length portion of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, and the oligonucleotide is used. At least one of the constituent nucleic acids 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]. ..

Another preferred modified sugar is a 2'-MOE modified sugar. 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 an activity of suppressing PLP1 expression. The nucleic acid base sequence of the modified oligonucleotide has at least 85%, at least 90%, or at least 95% complementarity to the equal length portion of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, and the oligonucleotide is used. At least one of the constituent nucleic acid is a modified oligonucleotide having a sugar modified with 2'-MOE.

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

Further modified nucleic acid bases include 5-hydroxymethylcytosine, xanthin, hypoxanthin, 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-thiocitosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ₃ ) uracil and citocin and other alkynyl derivatives of pyrimidine base, 6-azouracil, cytosine and chimin, 5 -Uracil (Pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halos, especially 5-bromo, 5 -Trifluoromethyl and other 5-substituted uracils and citocins, 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.

The heterocyclic base moiety can also include those in which the purine or pyrimidine base is replaced with another heterocycle, for example, 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-azapyrimidines and N-2, N-6 and O-6 substituted purines (2-aminopropyladenine, Includes 5-propynyluracil and 5-propynylcytosine).

(Binding between modified nucleosides)
The naturally occurring nucleoside bond between RNA and DNA is a 3'-5'phosphodiester bond. One or more modified, i.e., non-naturally occurring, oligonucleotides with nucleoside linkages include, for example, enhanced cell uptake, enhanced affinity for target nucleic acids and increased stability in the presence of nucleases. Often preferred over naturally occurring oligonucleotides with internucleoside linkages because of their properties.

Oligonucleotides with modified nucleoside bonds include nucleoside bonds that retain a phosphorus atom and nucleoside bonds that do not have a phosphorus atom. Representative phosphorus-containing nucleoside bonds include, but are not limited to, one or more of phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known.

The nucleoside linkage of the modified oligonucleotide of the present invention may be any as long as the modified oligonucleotide has an activity of suppressing PLP1 expression, but one in which at least one nucleoside linkage contains a phosphorothioate nucleoside linkage is preferably used. For example, all nucleoside linkages may be phosphorothioate nucleoside linkages.

(Modified oligonucleotide motif)
In certain embodiments, the modified oligonucleotides of the invention obtain increased resistance to degradation by nucleases, increased cell uptake, increased binding affinity for target nucleic acids and / or increased PLP1 expression inhibitory activity. Can have a gapmer motif.

"Gapmer" means a modified oligonucleotide in which an internal region with multiple nucleosides that assists cleavage by RNase H is located between external regions 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 called a "5'wing segment", and a wing segment existing on the 3'side of the gap segment can be called a "3'wing segment". In Gapmar, the sugar portion of each wing's nucleoside approaching the gap (the 5'-the most 3'side nucleoside of the wing and the 3'-the most 5'side of the wing) is the sugar portion of the adjacent gap nucleoside. It is different from the sugar part. For example, the sugar moiety of the nucleoside on the most 3'side of the 5'-wing and the nucleoside on the most 5'side of the 3'-wing are modified sugars, and the sugar moiety of the adjacent gap nucleoside is the sugar moiety of natural DNA. Is.

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

The number of nucleosides contained in the gap segment, the 5'wing segment and the 3'wing segment may be any number as long as it suppresses the inhibition of PLP1 expression.
As the gap mar, the length is preferably 3-10-3 or 5-8-5 in the order of 5'wing segment-gap segment-3'wing segment, and ALNA [Ms] or 2 for both wing segments. '-Preferably those containing MOE.

In certain embodiments, cap structures are included at the ends of one or both of the modified oligonucleotides 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-pentoflanosyl nucleotides, acyclic 3', 4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, Acyclic 3,5-dihydroxypentylnucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted debasement moiety, 3'-2'-inverted nucleotide moiety, 3'-2' -Inverted debasement, 1,4-butanediol phosphate, 3'-phosphoruamidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phospholothioate, phosphorodithioate, crosslinked methylphosphonate moiety and Non-bridged 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'-Inverted nucleotide moiety, 5'-5'-Inverted debasement moiety, 5'-phosphoruamidate, 5'-phosphorothioate, 5'-amino, crosslinked and / or non-crosslinked 5'-phospho There are lumidates, phosphorothioates, and 5'-mercapto moieties.

(Evaluation Method for Compound of the Present Invention)
The method for evaluating the compound of the present invention may be any method as long as the compound of the present invention can verify the suppression of the intracellular expression level of PLP1, but specifically, for example, in vitro and in vivo shown below. A method for measuring PLP1 expression is used.

The in vitro PLP1 expression measuring method for evaluating the suppression of PLP1 expression in cells of the present invention can be any cell expressing PLP1 (hereinafter, may be referred to as "PLP1-expressing cell"). Can also be used, and examples thereof include A375 cells (human malignant melanoma cells, for example, ATCC CRL-1619).

The method of contacting the compound of the present invention with PLP1-expressing cells is also not particularly limited, but a method generally used for introducing nucleic acid into cells can be mentioned. Specifically, for example, a lipofection method, an electroporation method, a Gymnosis method, or the like. In the lipofection method, the compound of the present 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 a variety of 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 a variety of methods known in the art. Specifically, for example, immunoprecipitation, Western blotting (immun blot), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (eg, caspase activity assay), immunohistochemistry. , Immunocytochemistry or fluorescence activated cell sorting (FACS) and the like.

The in vivo PLP1 expression measuring method for evaluating the suppression of PLP1 expression in cells of the present invention is, for example, to administer the compound of the present invention to an animal expressing PLP1 and to express the above-mentioned PLP1 in the cells. There is a method of performing level analysis.

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

(Treatment of PLP1-related diseases with the compound of the present invention)
The compound of the present invention can treat PLP1-related diseases by suppressing PLP1-expression. The PLP1-related disease is not particularly limited as long as it is a disease caused by an abnormality in the PLP1 gene, and examples thereof include congenital cerebral white matter dysplasia.

Examples of congenital cerebral white matter dysplasia caused by an abnormality in the PLP1 gene include Pelizaeus-Merzbacher's disease, and Pelizaeus-Merzbacher's disease includes congenital Pelizaeus-Merzbacher's disease and classical Pelizaeus-Merzbacher's disease.

Accordingly, the present invention comprises modified oligonucleotides for use in the treatment, prevention or delay of progression of PLP1-related diseases; pharmaceutical compositions for use in the treatment, prevention or delay of progression of PLP1-related diseases; Use of modified oligonucleotides to treat, prevent or delay progression; use of modified oligonucleotides in the manufacture of drugs for the treatment, prevention or delay of progression of PLP1-related diseases; for treatment, prevention or delay of progression of PLP1-related diseases Provided are modified oligonucleotides for use in the manufacture of a pharmaceutical; a method for treating, preventing or delaying progression of a PLP1-related disease, comprising administering an effective amount of the modified oligonucleotide to a subject in need thereof.

(2) Pharmaceutical composition containing a modified oligonucleotide The compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof 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. The composition and method for formulating the pharmaceutical composition can be selected according to several criteria including the route of administration, the degree of disease or the dose to be administered.
For example, as a composition for parenteral administration, for example, an injection is used. Such injections are prepared according to methods known per se, for example, by dissolving, suspending or emulsifying the modified oligonucleotide in a sterile aqueous or oily solution usually used for injections. As the aqueous solution for injection, for example, a phosphate buffered saline solution, a physiological saline solution, an artificial cerebrospinal fluid, an isotonic solution containing glucose and other adjuvants, and the like are used, and suitable solubilizing agents such as, for example, are used. Alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysolvate 80, HCO-50 (polyoxyethylene (50mo1) adduct of hydrogenated castor oil)], etc. May be used together. Further, a buffering agent, a pH adjusting agent, an tonicity agent, a soothing agent, a preservative, a stabilizer and the like can be included. Such compositions are produced by known methods.

Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intracranial administration, intrathecal administration, and intracerebroventricular administration. Administration may be continuous or long-term, short-term or intermittent.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspending agents and the like. Such compositions are produced by known methods and contain carriers, diluents or excipients commonly used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, magnesium stearate and the like are used, and as the diluent, for example, physiological saline is used.

Further, the pharmaceutical composition of the present invention can contain a reagent for introducing nucleic acid. Examples of the nucleic acid introduction reagent include liposomes, lipofectin, lipofectamine, DOGS (transferase), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, and cationic lipids such as poly (ethyleneimine) (PEI). Can be used.

In addition, the modified oligonucleotide contained in the pharmaceutical composition of the present invention is biotin, a protein having affinity for in vivo molecules such as fatty acids, cholesterol, sugars, phospholipids, and antibodies at one or a plurality of places. , Phenazine, vitamins, peptides, folates, phenanthridines, anthraquinones, acridines, fluorescein, rhodamine, coumarins and dyes conjugated with conjugate groups are preferably used. The conjugated modified oligonucleotide is produced by a known method, and one that enhances its activity, tissue distribution, cell distribution or cell uptake can be selected.

The conjugate group is directly attached to the modified oligonucleotide, or the conjugate group is amino, hydroxyl, carboxylic acid, thiol, unsaturated moiety (eg, double or triple bond), 8-amino-3. , 6-Dioxaoctanoic acid (ADO), succinimidyl 4- (N-maleimidemethyl) cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), azide, substituted C1-C10 alkyl, substituted Alternatively, it is bound to the modified oligonucleotide by a linking moiety selected from the unsubstituted C2-C10 alkenyl and the substituted or unsubstituted C2-C10 alkynyl. Here, the substituent is selected from amino, alkoxy, carboxy, azide, 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 intracerebral administration. There may be. The dose of the pharmaceutical composition of the present invention may be appropriately changed depending on the purpose of use, the severity of the disease, the age, weight, sex, etc. of the patient, but usually, the amount of the modified oligonucleotide is 0.1 ng to 100 mg / kg. / Day, preferably in the range of 1 ng to 10 mg / kg / day.

Non-Limited Disclosure and Incorporation by Reference Certain compounds, compositions and methods described herein are specifically described according to certain embodiments, while the following examples are described herein. It serves only as an example of the compounds of, and is not intended to limit it. Each of the references described in this application is incorporated herein by reference in its entirety.

Example 1
Synthesis and purification of modified oligonucleotide compounds for in vitro evaluation
Modified oligonucleotide compounds having the nucleotide sequences shown in Table 1 (SEQ ID NOs: 11 to 63) are described in a general solid phase synthesis method for oligonucleotide analogs and ALNA [Ms] amidite (WO2020 / 100926). Synthesized and purified by Nippon Gene Co., Ltd. using the above method).

Table 1 shows the target positions of the synthesized modified oligonucleotide compound (16-residue modified oligonucleotide).
In the notation of the compound, each nucleotide is represented by three letters. However, the nucleotide at the 3'end is represented by two letters because there is no nucleoside bond.
1) The first letter is capitalized to indicate the following nucleobases:
A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,
2) The second letter indicates each sugar part below:
m = ALNA [Ms], d = 2'-deoxyribose,
3) The third letter indicates the following nucleoside bond:
s = phosphorothioate.
The target position is indicated by the target start position to the target end position, and the target start position is the PLP1 pre-mRNA5'target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the sequence table corresponding to the 3'end of the modified oligonucleotide). The target termination position shown indicates the PLP1 pre-mRNA3'target site of the modified oligonucleotide (position of SEQ ID NO: 1 in the sequence listing corresponding to the 5'end of the modified oligonucleotide).

Example 2
An In vitro PLP1 knockdown activity test synthesized modified oligonucleotide and transfection reagent on a mixture of Lipofectamine RNAi Reagent, were seeded such that the 4 × ¹⁰ 3 cells / well of A375 cells, about 24 hours culture in a CO ₂ incubator did. Then, using SuperPrep II Lysis & RT Kit for qPCR (TOYOBO), RNA was prepared and a reverse transcription reaction was performed to synthesize cDNA. The mRNA expression level was measured by quantitative real-time PCR using the synthesized cDNA. For quantitative real-time PCR, the probe of PLP1 gene (Hs.PT.58.390511, INTERGRATED DNA TECHNOLOGIES) and HPRT1 gene (Hs.PT.58v.45621572, INTERGRATED DNA TECHNOLOGIES) was used, and the amount of HPRT1 mRNA expressed by the ΔΔCt method. The relative expression level of PLP1 mRNA corrected in 1 was calculated. Modification inhibition rate oligonucleotides the added during the PLP1 mRNA expression level of a modified oligonucleotide from the reduction rate was calculated in percentage, IC ₅₀ values were calculated from the concentration of 2 points sandwiching 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.

Example 3
Synthesis and purification of modified oligonucleotide compounds for in vitro evaluation
Modified oligonucleotide compounds (SEQ ID NOs: 64-96) having the nucleotide sequences shown in Table 2 were synthesized and purified by Nippon Gene Co., Ltd. using a general solid-phase synthesis method for oligonucleotide analogs.

Table 2 shows the target positions of the synthesized modified oligonucleotide compound (18-residue modified oligonucleotide).
In the notation of the compound, each nucleotide is represented by three letters. However, the nucleotide at the 3'end is represented by two letters because there is no nucleoside bond.
1) The first letter is capitalized to indicate the following nucleobases:
A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,
2) The second letter indicates each sugar part below:
e = 2'-MOE, d = 2'-deoxyribose,
3) The third letter indicates the following nucleoside bond:
s = phosphorothioate.
The target position is indicated by the target start position to the target end position, and the target start position is the PLP1 pre-mRNA5'target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the sequence table corresponding to the 3'end of the modified oligonucleotide). The target termination position shown indicates the PLP1 pre-mRNA3'target site of the modified oligonucleotide (position of SEQ ID NO: 1 in the sequence listing corresponding to the 5'end of the modified oligonucleotide).

Example 4
An In vitro PLP1 knockdown activity test synthesized modified oligonucleotide and transfection reagent on a mixture of Lipofectamine RNAi Reagent, were seeded such that the 4 × ¹⁰ 3 cells / well of A375 cells, about 24 hours culture in a CO ₂ incubator did. Then, using SuperPrep II Lysis & RT Kit for qPCR (TOYOBO), RNA was prepared and a reverse transcription reaction was performed to synthesize cDNA. The mRNA expression level was measured by quantitative real-time PCR using the synthesized cDNA. For quantitative real-time PCR, the probe of PLP1 gene (Hs.PT.58.390511, INTERGRATED DNA TECHNOLOGIES) and HPRT1 gene (Hs.PT.58v.45621572, INTERGRATED DNA TECHNOLOGIES) was used, and the amount of HPRT1 mRNA expressed by the ΔΔCt method. The relative expression level of PLP1 mRNA corrected in 1 was calculated. Modification inhibition rate oligonucleotides the added during the PLP1 mRNA expression level of a modified oligonucleotide from the reduction rate was calculated in percentage, IC ₅₀ values were calculated from the concentration of 2 points sandwiching the 50% inhibition rate. The results are shown in Table 2. As a result, it was found that the modified oligonucleotide of the present invention has an excellent effect of suppressing PLP1 expression.

Claims

It is a modified oligonucleotide consisting of 12 to 30 residues, and is 9964 to 10286, 140007 to 14152, 4439 to 4470, 4569 to 4616, 9061 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. The modified oligonucleotide comprises at least eight contiguous nucleic acid sequences complementary to any of the equilength moieties of positions -9164 to 11926 to 12015, 12817 to 12874, 14839 to 14920, or 15088 to 15114. A modified oligonucleotide that suppresses the expression of Proteolipid protein 1 (PLP1), wherein the full-length nucleobase sequence has at least 85% complementarity to the equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing.
A modified oligonucleotide consisting of 12 to 30 residues, and an equal length portion of either 10014 to 10152 positions, 10180 to 10252 positions, or 14033 to 14113 positions from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. The full-length nucleobase sequence of the modified oligonucleotide contains at least 85% complementarity to the equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, comprising at least eight contiguous nucleic acid sequences complementary to. A modified oligonucleotide that suppresses the expression of Proteolipid protein 1 (PLP1).
It is a modified oligonucleotide consisting of 12 to 30 residues, and is located at positions 10036 to 10089 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, positions 10064 to 10089, positions 10101 to 10130, 10202 to 10230, or 14055. The full-length nucleobase sequence of the modified oligonucleotide contains at least eight consecutive nucleobase sequences complementary to any of the equal-length moieties at positions ~ 14891, and the nucleobase sequence of SEQ ID NO: 1 in the sequence listing is the same length. A modified oligonucleotide that suppresses the expression of PLP1 with at least 85% complementarity to the moiety.
It is a modified oligonucleotide consisting of 12 to 30 residues, and is an equal length portion of either the 10101 to 10130 position, the 1022 to 10230 position, or the 14055 to 14091 position from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. The full-length nucleobase sequence of the modified oligonucleotide contains at least 85% complementarity to the equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, comprising at least eight contiguous nucleic acid sequences complementary to. A modified oligonucleotide that suppresses the expression of PLP1.
From the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, positions 10036 to 10051, 10037 to 10025, 10038 to 10053, 10039 to 10045, 10040 to 10055, 10064 to 10024, 10065 to 10080, 10066-10081, 10067-10082, 10062-10083, 10069-10084, 10070-10085, 10071-10086, 10072-10087, 10073-10088, 10064-10089, 10101-10116, 10102-10117, 10103-10118, 10104-10119, 10105-10120, 10106-10121, 10107-10122, 10108-10123, 10109-10124, 10110-10125, 10111-10126, 10112-10127, 10113-10128, 10114-10129, 10115-10130, 10202-10217, 10203-10218, 10204-10219, 10205-10220, 10206-10221, 10207-10222, 10208 to 10223, 10209 to 10224, 10210 to 10225, 10211 to 10226, 10212 to 10227, 10213 to 10228, 10214 to 10229, 10215 to 10230, 14055 to 14070, 14056 to 14071, Modifications that suppress the expression of PLP1 consisting of a nucleobase sequence complementary to any of positions 14057 to 14072, 14062 to 14077, 14063 to 14078, 14074 to 14089, 14075 to 14090, or 14076 to 14091. Oligonucleotide.
It is a modified oligonucleotide consisting of 12 to 30 residues, and is located at positions 4439 to 4470, 4569 to 4616, 961 to 9164, 9964 to 10055, and 10056 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. At least eight contiguous moieties complementary to any of the equal lengths of positions -10286, 11926 to 12015, 12817 to 12874, 14007 to 14095, 14112 to 14152, 14839 to 14920, or 15088 to 15114. Suppresses the expression of Proteolipid protein 1 (PLP1), which contains a nucleobase sequence and whose full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to the equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. Modified oligonucleotide.
From the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, positions 4450 to 4467, 4571 to 4589, 9072 to 9089, 9119 to 9136, 10035 to 10025, 10037 to 10049, 10072 to 10089, 10088-10105, 10101-10118, 10102-10119, 10103-10120, 10104-10121, 10105-10122, 10108-10125, 10109-10126, 10112-10129, 10113-10130, 11959 to 11976, 11989 to 12006, 12830 to 12847, 14020 to 14037, 14025 to 14042, 14055 to 14072, 14056 to 14073, 14060 to 14077, 14065 to 14082, 14075 to 14092, Modifications that suppress the expression of PLP1 consisting of a nucleobase sequence complementary to any of positions 14130 to 14147, 14847 to 14864, 14852 to 14869, 14857 to 14874, 14866 to 14883, or 15091 to 15108. Oligonucleotide.
The modified oligonucleotide according to any one of claims 1 to 7, which is a single strand.
The modified oligonucleotide according to any one of claims 1 to 8, wherein at least one nucleoside constituting the modified oligonucleotide contains a modified sugar.
The modified oligonucleotide according to claim 9, wherein the modified sugar 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. ..
10. 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 according to.
The modified oligonucleotide according to any one of claims 1 to 11, wherein at least one nucleoside constituting the modified oligonucleotide contains a modified nucleobase.
The modified oligonucleotide according to claim 12, wherein the modified nucleobase is 5-methylcytosine.
The modified oligonucleotide according to any one of claims 1 to 13, wherein at least one nucleoside-linked bond constituting the modified oligonucleotide is a modified nucleoside-modified bond.
The modified oligonucleotide according to claim 14, wherein the modified nucleoside linkage is a phosphorothioate nucleoside linkage.
The modified oligonucleotide is
1) Gap segment and
2) 5'wing segment and
3) Including 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 15, wherein the nucleoside constituting the 5'wing segment and the 3'wing segment contains a modified sugar.
A pharmaceutical composition comprising the modified oligonucleotide according to any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
17. The pharmaceutical composition of claim 17, for treating, preventing, or delaying the progression of PLP1-related diseases.
The pharmaceutical composition according to claim 18, wherein the PLP1-related disease is Pelizaeus-Merzbacher's disease.
The pharmaceutical composition according to claim 19, wherein the Pelizaeus-Merzbacher disease is congenital Pelizaeus-Merzbacher disease or classical Pelizaeus-Merzbacher disease.
The treatment, prevention or progression of a PLP1-related disease in a subject, characterized in that a therapeutically effective amount of the modified oligonucleotide according to any one of claims 1 to 16 is administered to the subject in need thereof. A method for delaying.
Use of the modified oligonucleotide according to any one of claims 1 to 16 in the manufacture of a pharmaceutical for the treatment, prevention or delay of its progression of PLP1-related diseases.
The modified oligonucleotide according to any one of claims 1 to 16, for treating, preventing or delaying the progression of PLP1-related diseases.