Xeroderma pigmentosum group f therapeutic drug

Abstract

To provide therapeutic means of xeroderma pigmentosum group F in which a disease-causing mutation has not been identified.SOLUTION: Provided are an antisense oligonucleotide that has a base sequence which can be hybridized with a part of an intron region of an XPF gene, and also has activity for suppressing abnormal post-transcriptional modification of the XPF gene, or a pharmaceutically acceptable salt thereof, and a therapeutic drug or a therapeutic composition of xeroderma pigmentosum group F which contains it as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for xeroderma pigmentosum group F, an antisense oligonucleotide as an active ingredient thereof, or a pharmaceutically acceptable salt thereof. [Background technology]

[0002] Xeroderma pigmentosum (XP) is caused by congenital abnormalities in genes involved in nucleotide excision repair (NER), which removes photodamage from the genome caused by ultraviolet light in sunlight, and translocation of DNA to the nucleoside transporter (TLS). XP patients suffer from photosensitivity, pigmentation abnormalities in sun-exposed areas, a high incidence of cancer, and neurological symptoms. The prevalence of XP in the Japanese population is estimated to be approximately 1 in 25,000 births. Disease-causing mutations have been reported in the XPA, XPG, and POLH genes, which encode TLS polymerase. XP cases with these mutations are classified into complementation groups: XP-A, XP-G, and XP-V (variant). The proportion of each complementation group among XP cases varies by race, but in the Japanese population, XP-A, followed by XP-V, are most prevalent.

[0003] All XP complementation groups, except for variants with TLS deficiency, exhibit NER deficiency. NER is divided into whole-genome repair (GG-NER) and transcription-coupled repair (TC-NER) depending on the mode of DNA damage recognition. XP-C / E groups exhibit GG-NER deficiency alone, while XP-A / B / D / F / G groups exhibit both GG-NER and TC-NER deficiency. In XP cases, these NER deficiencies lead to the accumulation of DNA damage in the genome, resulting in genomic instability and a predisposition to skin cancer at a young age. The onset of neurological symptoms varies depending on the complementation group and disease-causing mutation, but in particular, cases with the Japanese group A founder mutation (IVS3-1G>C) lose all NER function due to loss of XPA protein expression, resulting in severe neurological symptoms.

[0004] XP is a systemic hereditary disease, and no effective treatment has yet been developed. Patients are often diagnosed with XP when they visit a dermatologist after experiencing severe sunburn during infancy. Genetic testing can also provide a rough prognosis. While it is possible to prevent skin cancer by avoiding sun exposure, there are no effective measures to alleviate the progression of neurological symptoms, which have a significant impact on prognosis and quality of life.

[0005] For the definitive diagnosis of XP, DNA repair activity assays using fibroblasts established from patient skin patches are known. These include the unscheduled DNA synthesis (UDS) assay, which measures GG-NER activity by detecting repair DNA synthesis after removal of photodamaged DNA sites, and the TC-NER assay, which assesses RNA synthesis recovery (RRS) in transcriptionally active regions after DNA damage (Non-Patent Documents 1 and 2). By assessing UDS / RRS in patient-derived cells and investigating which known XP genes restore these activities, it is possible to identify complementation groups (Non-Patent Documents 3 and 4).

[0006] Regarding the genetic mutations carried by XP-F group patients, a mutation in the exon region of the XPF gene (ERCC4) has been reported as an individual mutation in a patient in Non-Patent Document 5. However, no mutation in the intron region of the XPF gene has been disclosed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Nakazawa, Y. et al. DNA Repair 2010, 37(5):714-27 [Non-patent document 2] Limsirichaikul, S. et al. Nucleic Acids Res. 2009, 37(4), e31 [Non-patent document 3] Jia N. et al. Nature Protoc. 2015, 10(1), 12-24 [Non-patent document 4] Nakazawa, Y. et al. Nature Genetics 2012, 44(5), 586-92 [Non-patent document 5] Kashiyama, Y. et al. The American Journal of Human Genetics 2013, 83 92:807-819 Summary of the Invention [Problem to be solved by the invention]

[0008] In the XP-F group, there are cases in which the disease-causing mutation has not been identified, and treatment has not been possible until now. The object of the present invention is to provide a means for treating such XP-F group. [Means for solving the problem]

[0009] The present inventors have identified two novel intronic mutations responsible for the disease in XP-F patients. The first is a novel Japanese founder mutation located deep within the intron (approximately 100 bp downstream from the exon 1-intron 1 boundary) that was shown to generate a new U1 small nuclear ribonucleoprotein (U1 snRNP) binding site, resulting in reduced gene expression through aberrant alternative splicing. The second is a single-base substitution mutation located deep within the intron (several hundred bp downstream from the exon 8-intron 8 boundary). This mutation was shown to result in the formation of a pathological poly(A) addition sequence (cleavage and polyadenylation factor binding site), leading to incomplete transcription termination and mRNA destabilization, resulting in the onset of the disease. Further research led to the completion of this invention.

[0010] That is, the present invention includes the following inventions. [1] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which has a base sequence capable of hybridizing with a portion of the intron region of the XPF gene and has the activity of suppressing abnormal post-transcriptional modification of the XPF gene, and which may be chemically modified at its 5' end and / or 3' end. [2] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], which suppresses abnormal splicing using the 5' splice site on the 5' side of the 193rd guanine and the 3' splice site on the 3' side of the 1658th guanine in the base sequence represented by SEQ ID NO: 63 in post-transcriptional modification of an XPF gene having a mutant intron 1 sequence represented by SEQ ID NO: 63. [3] The antisense oligonucleotide according to [1] or [2], or a pharmaceutically acceptable salt thereof, capable of hybridizing with a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from the 155th to the 217th base in the base sequence represented by SEQ ID NO: 63. [4] An antisense oligonucleotide according to any one of [1] to [3], or a pharmaceutically acceptable salt thereof, which contains a sequence that is 90% or more, preferably 95% or more, and optimally completely complementary to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from the 155th to the 217th base in the base sequence represented by SEQ ID NO: 63. [5] An antisense oligonucleotide according to any one of [1] to [4], or a pharmaceutically acceptable salt thereof, comprising a base sequence represented by any one of SEQ ID NOs: 65 to 82 and 101 to 108 (in the sequence, u may be replaced with t). [6] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [5], which contains one or more sugar-modified nucleosides. [7] Sugar-modified nucleosides are 4'-(CH2) n The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [6], which is a nucleoside containing an -O-2' bridge (wherein n is 1 or 2) or 2'-O-methylation. [8] The antisense oligonucleotide according to any one of [1] to [7], or a pharmaceutically acceptable salt thereof, which contains one or more modified internucleoside bonds. [9] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [8], wherein the modified internucleoside bond is a phosphorothioate bond.

[10] The antisense oligonucleotide according to any one of [1] to [9], which consists of a base sequence represented by any one of SEQ ID NOs: 1 to 18, 37 to 60, and 112 to 123, or a pharmaceutically acceptable salt thereof.

[11] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], which suppresses abnormal polyadenylation using the poly A addition sequence at positions 324 to 329 in the base sequence represented by SEQ ID NO: 64 in the post-transcriptional modification of the XPF gene having a mutant intron 8 sequence represented by SEQ ID NO: 64.

[12] An antisense oligonucleotide according to [1] or

[11] , or a pharmaceutically acceptable salt thereof, capable of hybridizing with a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from the 309th to the 343rd base in the base sequence represented by SEQ ID NO: 64.

[13] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1],

[11] and

[12] , which contains a sequence that is 90% or more, preferably 95% or more, and optimally completely complementary to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from the 309th to the 343rd base in the base sequence represented by SEQ ID NO: 64.

[14] An antisense oligonucleotide according to any one of [1] and

[11] to

[13] , which contains a base sequence represented by any one of SEQ ID NOs: 83 to 100 (in the sequence, u may be replaced with t), or a pharmaceutically acceptable salt thereof.

[15] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] and

[11] to

[14] , which contains one or more sugar-modified nucleosides.

[16] Sugar-modified nucleosides are 4'-(CH2) nThe antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to

[15] , which is a nucleoside comprising an -O-2' bridge (wherein n is 1 or 2) and 2'-O-methylation.

[17] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] and

[11] to

[16] , which contains one or more modified internucleoside bonds.

[18] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to

[17] , wherein the modified internucleoside bond is a phosphorothioate bond.

[19] The antisense oligonucleotide according to any one of [1] and

[11] to

[18] , which consists of a base sequence represented by any one of SEQ ID NOs: 19 to 36, or a pharmaceutically acceptable salt thereof.

[20] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[19] , wherein the chemical modification is the addition of a molecular structure suitable for transport of the oligonucleotide.

[21] A pharmaceutical composition for treating xeroderma pigmentosum group F, comprising the antisense oligonucleotide according to any one of [1] to

[20] or a pharmaceutically acceptable salt thereof.

[22] A method for treating xeroderma pigmentosum group F, comprising administering to a patient an effective amount of the antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[20] .

[23] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[20] for use in the treatment of xeroderma pigmentosum group F.

[24] Use of the antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[20] for the manufacture of a pharmaceutical composition for treating xeroderma pigmentosum group F.

[0011] The present invention also provides the following [A1] to [A20]. [A1] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which has a base sequence capable of hybridizing with a part of the intron region of the XPF gene and has activity of suppressing abnormal post-transcriptional modification of the XPF gene. [A2] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [A1], which suppresses abnormal splicing using the 5' splice site on the 5' side of the 193rd guanine and the 3' splice site on the 3' side of the 1658th guanine in the base sequence represented by SEQ ID NO: 63 in post-transcriptional modification of an XPF gene having a mutant intron 1 sequence represented by SEQ ID NO: 63. [A3] An antisense oligonucleotide according to [A1] or [A2], or a pharmaceutically acceptable salt thereof, capable of hybridizing with a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from the 175th to the 217th in the base sequence represented by SEQ ID NO: 63. [A4] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [A1] to [A3], which contains a sequence that is 90% or more, preferably 95% or more, and optimally completely complementary to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from positions 175 to 217 in the base sequence represented by SEQ ID NO: 63. [A5] An antisense oligonucleotide according to any one of [A1] to [A4], or a pharmaceutically acceptable salt thereof, comprising a base sequence represented by any one of SEQ ID NOs: 65 to 82 and 101 to 108 (in the sequence, u may be replaced with t). [A6] The antisense oligonucleotide according to any one of [A1] to [A5], or a pharmaceutically acceptable salt thereof, which contains one or more sugar-modified nucleosides. [A7] Sugar-modified nucleoside is 4'-(CH2) n The antisense oligonucleotide according to [A6], or a pharmaceutically acceptable salt thereof, which is a nucleoside containing an -O-2' bridge (wherein n is 1 or 2) or 2'-O-methylation. [A8] The antisense oligonucleotide according to any one of [A1] to [A7], or a pharmaceutically acceptable salt thereof, which contains one or more modified internucleoside bonds. [A9] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [A8], wherein the modified internucleoside bond is a phosphorothioate bond. [A10] The antisense oligonucleotide according to any one of [A1] to [A9], which consists of a base sequence represented by any one of SEQ ID NOs: 1 to 18 and 37 to 60, or a pharmaceutically acceptable salt thereof. [A11] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [A1], which suppresses abnormal polyadenylation using the poly A addition sequence at positions 324 to 329 in the base sequence represented by SEQ ID NO: 64 in post-transcriptional modification of an XPF gene having a mutant intron 8 sequence represented by SEQ ID NO: 64. [A12] An antisense oligonucleotide according to [A1] or [A11], or a pharmaceutically acceptable salt thereof, capable of hybridizing with a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from 309 to 343 in the base sequence represented by SEQ ID NO: 64. [A13] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [A1], [A11] and [A12], which contains a sequence that is 90% or more, preferably 95% or more, and optimally completely complementary to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from positions 309 to 343 in the base sequence represented by SEQ ID NO: 64. [A14] An antisense oligonucleotide according to any one of [A1] and [A11] to [A13], or a pharmaceutically acceptable salt thereof, comprising a base sequence represented by any one of SEQ ID NOs: 83 to 100 (in the sequence, u may be replaced with t). [A15] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [A1] and [A11] to [A14], which contains one or more sugar-modified nucleosides. [A16] Sugar-modified nucleoside is 4'-(CH2) nThe antisense oligonucleotide according to [A15], or a pharmaceutically acceptable salt thereof, which is a nucleoside comprising an -O-2' bridge (wherein n is 1 or 2) and 2'-O-methylation. [A17] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [A1] and [A11] to [A16], which contains one or more modified internucleoside bonds. [A18] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to [A17], wherein the modified internucleoside bond is a phosphorothioate bond. [A19] The antisense oligonucleotide according to any one of [A1] and [A11] to [A18], which consists of a base sequence represented by any one of SEQ ID NOs: 19 to 36, or a pharmaceutically acceptable salt thereof. [A20] A pharmaceutical composition for treating xeroderma pigmentosum group F, comprising the antisense oligonucleotide according to any one of [A1] to [A19] or a pharmaceutically acceptable salt thereof. [Effects of the Invention]

[0012] According to the present invention, it is possible to treat a specific XP-F group for which no therapeutic means existed previously. [Brief explanation of the drawings]

[0013] [Figure 1A] Figure 1A shows that the amount of XPF mRNA splicing product is increased in XPF patient-derived cells transfected with the compound of Example 61. Black bars represent the amount of correct splicing product, and open bars represent the amount of aberrant splicing product. [Figure 1B] FIG. 1B shows that XPF protein expression levels are increased in XPF patient-derived cells transfected with the compound of Example 61. [Figure 1C] FIG. 1C shows that DNA repair activity after UV irradiation is increased in XPF patient-derived cells transfected with the compound of Example 61. [Figure 2A]FIG. 2A shows that DNA repair activity after UV irradiation is increased in XPF patient-derived cells transfected with the compounds of Examples 1 to 18. [Figure 2B] FIG. 2B shows that the expression level of XPF protein increases in cells derived from XPF patients transfected with the compounds of Examples 1 to 18. [Figure 3A] Figure 3A shows that DNA repair activity after UV irradiation is increased in XPF patient-derived cells transfected with the compounds of Examples 11 to 14 and 37 to 60. The black bars represent the amount of correctly spliced products, and the open bars represent the amount of aberrantly spliced products. [Figure 3B] Figure 3B shows that XPF protein expression levels are increased in XPF patient-derived cells transfected with the compounds of Examples 11, 13, 37, 38, 44 to 46, and 53 to 60. The upper panel shows the expression levels of XPF protein, and the lower panel shows the expression levels of β-actin. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1.Definition As used herein, "xeroderma pigmentosum group F" (also referred to as XP-F group) is one of the complementation groups of xeroderma pigmentosum, and shows both GG-NER and TC-NER deficiency. The gene whose causative mutation has been reported is the XPF gene.

[0015] As used herein, the term "XPF gene" refers to a human gene encoding an endonuclease involved in the nucleotide excision repair mechanism. The XPF gene is also referred to as the ERCC4 gene, ERCC11 gene, FANCQ gene, RAD1 gene, or XFEPS gene. The sequence of the XPF gene is known, for example, as Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4), RefSeqGene (LRG_463) on chromosome 16 (NCBI-GenBank accession no. NG_011442.1).

[0016] As used herein, the term "mutated intron 1 sequence" refers to the base sequence of intron 1 of the wild-type XPF gene (the base sequence from bases 5217 to 6874 in the aforementioned NG_011442.1), in which T at the 196th position from the 5' end of intron 1 (base number 5412 in NG_011442.1) has been mutated to A, and is the base sequence represented by SEQ ID NO: 63. The mutation site corresponds to base number 196 in the base sequence represented by SEQ ID NO: 63.

[0017] As used herein, the term "mutated intron 8 sequence" refers to the base sequence of intron 8 of the wild-type XPF gene (the base sequence from bases 20588 to 22609 in the aforementioned NG_011442.1), in which C at the 326th position from the 5' end of intron 8 (base number 20913 in NG_011442.1) has been mutated to T, and is the base sequence represented by SEQ ID NO: 64. The mutation site corresponds to base number 326 in the base sequence represented by SEQ ID NO: 64.

[0018] As used herein, the term "post-transcriptional modification" refers to modification that occurs during the process in which a pre-mRNA (pre-mRNA) becomes a mature mRNA, and includes capping at the 5' end, polyadenylation at the 3' end, and splicing.

[0019] As used herein, the term "aberrant post-transcriptional modification" refers to a post-transcriptional modification that is not normally seen in the post-transcriptional modification of a wild-type gene, and includes, for example, aberrant splicing, aberrant polyadenylation, and the like.

[0020] As used herein, "aberrant splicing" refers to splicing not normally observed in post-transcriptional modification of wild-type genes. mRNA splicing occurs when the 5' splice site, branch site, and 3' splice site in the intron are recognized by the splicing factor small nuclear ribonucleoprotein (snRNP). In post-transcriptional modification of the XPF gene having a mutant intron 1 sequence, a mutation at position 196 in the base sequence represented by SEQ ID NO: 63 creates a new recognition sequence (GTATGTAA) for the splicing factor U1 snRNP, making the 5' side of guanine 193 the 5' splice site. As a result, aberrant splicing occurs using the 5' splice site 5' of guanine 193 and the 3' splice site 3' of guanine 1658 in the base sequence represented by SEQ ID NO: 63.

[0021] As used herein, "aberrant polyadenylation" refers to polyadenylation that is not normally observed in post-transcriptional modification of wild-type genes. Polyadenylation of mRNA occurs depending on a poly A addition sequence (also known as a cleavage / polyadenylation factor binding sequence or polyadenylation signal). In humans, the poly A addition sequence typically contains AATAAA. In post-transcriptional modification of the XPF gene with a mutant intron 8 sequence, a mutation at position 326 in the base sequence represented by SEQ ID NO: 64 generates a new poly A addition sequence (AATAAA). As a result, aberrant polyadenylation occurs using the poly A addition sequence at positions 324 to 329 in the base sequence represented by SEQ ID NO: 64.

[0022] As used herein, an "antisense oligonucleotide" refers to a single-stranded oligonucleotide capable of hybridizing to a nucleotide containing a target base sequence. "Able to hybridize" means that it can form a double strand with the target nucleotide through interactions between bases (AG (adenine-guanine) and CT / U (cytosine-thymine / uracil)). An antisense oligonucleotide need only have a sequence complementary to the target sequence sufficient to allow hybridization, and does not necessarily have a completely complementary sequence. Antisense oligonucleotides may also be DNA, RNA, and DNA / RNA chimeras. Antisense oligonucleotides may also contain modifications such as modified nucleosides and modified internucleoside linkages.

[0023] "Hybridizing" includes hybridizing under low stringency conditions, medium stringency conditions, and high stringency conditions. "Low stringency conditions" can be, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, at 32°C, or equivalent conditions. "Medium stringency conditions" can be, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, at 42°C, or 5x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide, at 42°C, or equivalent conditions. "Highly stringent conditions" can be, for example, conditions of 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, 50°C, or 0.2xSSC, 0.1% SDS, 65°C, or equivalent conditions. Factors that affect the stringency of hybridization include temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and those skilled in the art can achieve similar stringency by appropriately selecting these factors. The above conditions can also be achieved using commercially available hybridization reagents. For example, "highly stringent conditions" can be achieved using commercially available hybridization solution ExpressHyb TM This can be achieved by hybridizing in a hybridization solution (Clontech) at 68°C, or by hybridizing a DNA-immobilized filter at 68°C in the presence of 0.7-1.0 M NaCl, followed by washing at 68°C with 0.1-2x SSC solution (1x SSC consists of 150 mM NaCl and 15 mM sodium citrate).

[0024] As used herein, the term "nucleoside" includes both natural and modified nucleosides. "Natural nucleosides" refers to 2'-deoxynucleosides such as 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, thymidine, and 2'-deoxyuridine, as well as ribonucleosides such as adenosine, guanosine, cytidine, 5-methylcytidine, and uridine. Among nucleobases, uracil (U) or (u) and thymine (T) or (t) are interchangeable, and either uracil (U) or (u) or thymine (T) or (t) can be used to form a base pair with adenine (A) or (a) in a complementary strand.

[0025] In this specification, 2'-deoxyadenosine is referred to as A t , 2'-deoxyguanosine to G t , 2'-deoxycytidine C t , 2'-deoxy-5-methylcytidine 5meC t , thymidine to T t , 2'-deoxyuridine to U t In addition, as the corresponding nucleotide, 2'-deoxyadenosine nucleotide is referred to as A p , 2'-deoxyguanosine nucleotide G p , 2'-deoxycytidine nucleotide C p , 2'-deoxy-5-methylcytidine nucleotide 5meC p , thymidine nucleotide T p , 2'-deoxyuridine nucleotide U p It can also be expressed as:

[0026] As used herein, the term "sugar-modified nucleoside" refers to a nucleoside in which the sugar moiety of the nucleoside has been modified. Sugar-modified nucleosides include all types of sugar modifications known in the technical field to which the present invention pertains. Sugar-modified nucleosides include, for example, 2'-modified nucleosides, 4'-thio-modified nucleosides, 4'-thio-2'-modified nucleosides, and bicyclic sugar-modified nucleosides.

[0027] Examples of 2'-modified nucleotides include halo, allyl, amino, azido, O-allyl, and O-C1-C 10 Alkyl, OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ), or O-CH2-C(=O)-N(R m )(R n ) are listed, and each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10The alkyl groups are alkyl. Commercially available reagents can be used for 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methyluridine. 2'-O-Aminoethylguanosine, 2'-O-aminoethyladenosine, 2'-O-aminoethylcytidine, and 2'-O-aminoethyluridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725). 2'-O-Propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine, and 2'-O-propyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838). Commercially available reagents can be used for 2'-O-allylguanosine, 2'-O-allyl adenosine, 2'-O-allyl cytidine, and 2'-O-allyl uridine. 2'-O-Methoxyethylguanosine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethylcytidine, and 2'-O-methoxyethyluridine can be synthesized according to the patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504). 2'-O-Butylguanosine, 2'-O-butyladenosine, 2'-O-butylcytidine, and 2'-O-butyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838). 2'-O-Pentylguanosine, 2'-O-pentyladenosine, 2'-O-pentylcytidine, and 2'-O-pentyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838). 32, 7832-7838. Commercially available reagents can be used for 2'-O-propargylguanosine, 2'-O-propargyladenosine, 2'-O-propargylcytidine, and 2'-O-propargyluridine.

[0028] Examples of 4'-thio modified nucleosides include β-D-ribonucleosides in which the 4'-oxygen atom is replaced with a sulfur atom (Hoshika, S. et al. FEBS Lett. 579, pp. 3115-3118, (2005); Dande, P. et al. J. Med. Chem. 49, pp. 1624-1634 (2006); Hoshika, S. et al. ChemBioChem. 8, pp. 2133-2138, (2007)).

[0029] Examples of 4'-thio-2'-modified nucleosides include 4'-thio-2'-modified nucleosides bearing 2'-H or 2'-O-methyl (Matsugami, et al. Nucleic Acids Res. 36, 1805 (2008)).

[0030] Examples of bicyclic sugar-modified nucleosides include nucleosides that have a second ring formed by bridging two atoms of the ribose ring. Examples of such nucleosides include 2',4'-BNA / LNA (bridged nucleic acids / locked nucleic acids), in which the 2'-oxygen atom and the 4'-carbon atom are bridged with a methylene chain (Obika, S. et al. Tetrahedron Lett., 38, pp. 8735-(1997); Obika, S. et al., Tetrahedron Lett., 39, pp. 5401-(1998); AA Koshkin, AA et al., Tetrahedron, 54, pp. 3607-(1998); Obika, S. Bioorg. Med. Chem., 9, p. 1001 (2001)), and 2'-O,4'-C-ethylene-bridged nucleic acids (ENA), in which the methylene chain of 2',4'-BNA / LNA is bridged with an ethylene chain extending by one carbon (Morita, K. et al. Bioorg. Med. Chem. Lett., 12, p. 73 (2002); Morita, K. et al. Bioorg. Med. Chem., 11, p. 2211 (2003)). Other examples include AmNA, described in WO2014 / 109384, and S-cEt (2',4'-constrained ethyl), described in the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581).

[0031] As used herein, examples of sugar-modified nucleosides containing 2'-O-methylation include, for example, A t A corresponds to m1t , G t The corresponding one is G m1t , C t The corresponding one is C m1t , 5meC t The corresponding one is 5meC m1t , U t The corresponding one is U m1tThe sugar-modified nucleotides containing the corresponding 2'-O-methyl modifications are sometimes expressed as A p A corresponds to m1p , G p The corresponding one is G m1p , C p The corresponding one is C m1p , 5meC p The corresponding one is 5meC m1p , U p The corresponding one is U m1p It can also be expressed as:

[0032] As used herein, examples of sugar-modified nucleosides containing 2'-O-methoxyethylation include, for example, A t A corresponds to m2t , G t The corresponding one is G m2t , 5meC t The corresponding one is 5meC m2t , T t The corresponding one is T m2t The sugar-modified nucleotides containing the corresponding 2'-O-methoxyethyl modifications are sometimes expressed as A p A corresponds to m2p , G p The corresponding one is G m2p , 5meC p The corresponding one is 5meC m2p , T p The corresponding one is T m2p It can also be expressed as:

[0033] As used herein, examples of sugar-modified nucleosides containing a 4'-CH2-0-2' bridge include, for example, A t A corresponds to 1t , G t The corresponding one is G 1t , 5meC t The corresponding one is C 1t , T t The corresponding one is T 1t The corresponding sugar-modified nucleotides containing a 4'-CH2-0-2' bridge are also represented as A p A corresponds toe1p , G p The corresponding one is G e1p , 5meC p The corresponding one is C e1p , T p The corresponding one is T e1p It can also be expressed as:

[0034] As used herein, examples of sugar-modified nucleosides containing a 4'-(CH2)2-0-2' bridge include, for example, A t A corresponds to 2t , G t The corresponding one is G 2t , 5meC t The corresponding one is C 2t , T t The corresponding one is T 2t The corresponding sugar-modified nucleotides containing a 4'-(CH2)2-0-2' bridge are also represented as A p A corresponds to e2p , G p The corresponding one is G e2p , 5meC p The corresponding one is C e2p , T p The corresponding one is T e2p It can also be expressed as:

[0035] As used herein, the term "modified internucleoside linkage" refers to a linkage that is substituted or altered from a naturally occurring internucleoside linkage (i.e., a phosphodiester internucleoside linkage). That is, an antisense oligonucleotide containing a modified internucleoside linkage contains a modification of the phosphate group of at least one nucleotide. Modified internucleoside linkages include, for example, phosphorothioate linkages, phosphorodithioate linkages, alkylphosphonate linkages, boranophosphate linkages, phosphoramidate linkages, etc.

[0036] In the present specification, the term "phosphorothioate ester" refers to a phosphorothioate ester in place of a phosphate ester of a nucleotide. p A corresponds to s , G pThe corresponding one is G s , C p The corresponding one is C s , 5meC p The corresponding one is 5meC s , T p The corresponding one is T s , U p The corresponding one is U s It can also be expressed as: For sugar-modified nucleotides including 2'-O-methylated and phosphorothioate, s A corresponds to m1s , G s The corresponding one is G m1s , C s The corresponding one is C m1s , 5meC s The corresponding one is 5meC m1s , U s The corresponding one is U m1s It can also be expressed as: For sugar-modified nucleotides including 2'-O-methoxyethylated modifications and phosphorothioates, see A s A corresponds to m2s , G s The corresponding one is G m2s , 5meC s The corresponding one is 5meC m2s , T s The corresponding one is T m2s It can also be expressed as: Sugar-modified nucleotides containing a 4'-CH2-0-2' bridge and phosphorothioate were s A e1s , G s For G e1s , 5meC s C e1s , T s For T e1s It can also be expressed as: Sugar-modified nucleotides containing 4'-(CH2)2-0-2' bridges and phosphorothioates were s A e2s , G s For G e2s , 5meC s Ce2s , T s For T e2s It can also be expressed as:

[0037] A t , G t , 5meC t , C t , T t , U t , A p , G p , 5meC p , C p , T p , U p , A s , G s , 5meC s , C s , T s , U s , A m1t , G m1t , C m1t , 5meC m1t , U m1t , A m1p , G m1p , C m1p , 5meC m1p , U m1p , A m1s , G m1s , C m1s , 5meC m1s , U m1s , A 2t , G 2t , C 2t , T 2t , A e2p , G e2p , C e2p , T e2p , A e2s , G e2s , C e2s , T e2s , A 1t , G 1t , C 1t , T 1t , A e1p , G e1p , C e1p , T e1p , A e1s , G e1s , C e1s , T e1s , A m2t , G m2t , 5meCm2t , T m2t , A m2p , G m2p , 5meC m2p , T m2p , A m2s , G m2s , 5meC m2s , and T m2s The structure of is shown below.

[0038] [ka]

[0039] [ka]

[0040] [ka]

[0041] [ka]

[0042] [ka]

[0043] As used herein, treatment of a disease or condition includes preventing the onset of the disease, suppressing or inhibiting the aggravation or progression of the disease, alleviating one or more symptoms exhibited by an individual suffering from the disease or suppressing the aggravation or progression of the disease, treating secondary diseases, etc.

[0044] 2. Pharmaceutical composition and antisense oligonucleotide of the present invention The present invention provides a pharmaceutical composition for treating xeroderma pigmentosum group F (XP-F group) (hereinafter referred to as the pharmaceutical composition of the present invention), and its active ingredient, an antisense oligonucleotide or a pharmaceutically acceptable salt thereof.

[0045] The pharmaceutical composition of the present invention contains as an active ingredient an antisense oligonucleotide that suppresses abnormal post-transcriptional modifications caused by disease-causing mutations in XP-F group, particularly abnormal post-transcriptional modifications caused by two types of novel intronic mutations, thereby suppressing the decrease in the expression level of the XPF gene in XP-F group carrying these mutations and enabling the treatment of XP-F group carrying these mutations.

[0046] The two mutations newly identified by the present inventors as the cause of XP-F group disease are a T to A mutation at position 196 from the 5' end of intron 1 (position 5412 in NG_011442.1) in the nucleotide sequence of intron 1 of the wild-type XPF gene (positions 5217 to 6874 in the aforementioned NG_011442.1) (corresponding to position 196 in the nucleotide sequence represented by SEQ ID NO: 63), and a C to T mutation at position 326 from the 5' end of intron 8 (position 20913 in NG_011442.1) in the nucleotide sequence of intron 8 of the wild-type XPF gene (positions 20588 to 22609 in the aforementioned NG_011442.1) (corresponding to position 326 in the nucleotide sequence represented by SEQ ID NO: 64). The former mutation causes aberrant splicing of the XPF gene, and the latter mutation causes aberrant polyadenylation of the XPF gene. As a result, it is thought that the expression level of the XPF gene decreases, causing the symptoms of the XP-F group. Therefore, in one embodiment, the pharmaceutical composition of the present invention is applied to the XPF group, whose disease is caused by at least one of these two types of mutations. In the present invention, as long as the XPF gene contains the above-mentioned mutation and the above-mentioned abnormal splicing or abnormal polyadenylation can occur, the XPF gene may also contain a mutation (e.g., deletion, substitution, addition, or insertion of one or several nucleotides).

[0047] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of the present invention has a base sequence capable of hybridizing with a portion of the intron region of the XPF gene. The portion of the intron region, i.e., the scope of the target sequence, is not particularly limited, as long as it can suppress abnormal post-transcriptional modification of the XPF gene. Whether the antisense oligonucleotide or a pharmaceutically acceptable salt thereof of the present invention can suppress abnormal post-transcriptional modification of the XPF gene can be determined by an evaluation method similar to that described in the Examples below, which involves evaluating the amount of XPF mRNA splicing product, protein expression level, or repair activity. If an increase in expression of a correctly post-transcriptionally modified XPF gene or recovery of DNA repair activity is confirmed by any of these evaluation methods, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof is determined to suppress abnormal post-transcriptional modification of the XPF gene.

[0048] In one embodiment, the pharmaceutical composition of the present invention is applicable to XP-F group, a disease caused by the above-mentioned mutation in intron 1. The pharmaceutical composition of the present invention in this embodiment is referred to as Pharmaceutical Composition I of the present invention. Pharmaceutical Composition I of the present invention comprises one or more antisense oligonucleotides that suppress aberrant splicing using the 5' splice site 5' of guanine 193 and the 3' splice site 3' of guanine 1658 in the nucleotide sequence represented by SEQ ID NO: 63 during post-transcriptional modification of the XPF gene having the mutant intron 1 sequence represented by SEQ ID NO: 63. Note that "aberrant splicing using the 5' splice site 5' of guanine 193 and the 3' splice site 3' of guanine 1658 in the nucleotide sequence represented by SEQ ID NO: 63" can also be rephrased as "aberrant splicing that recognizes the region from guanine 1 to adenine 192 as an exon and the region from guanine 193 to guanine 1658 as an intron in the nucleotide sequence represented by SEQ ID NO: 63."

[0049] Whether or not the antisense oligonucleotide in Pharmaceutical Composition I of the present invention suppresses aberrant splicing in intron 1 is determined by the same evaluation methods as those for XPF mRNA splicing product quantity, protein expression level, or repair activity evaluation described in the Examples below. If an increase in expression of correctly spliced XPF gene or restoration of DNA repair activity is confirmed by any of these evaluation methods, the antisense oligonucleotide is deemed to suppress aberrant splicing in intron 1. To evaluate the quantity of XPF mRNA splicing product, droplet digital PCR (ddPCR) is used to confirm whether or not correctly spliced XPF mRNA increases upon transfection of antisense oligonucleotides in cells derived from patients in the XP-F group, whose disease is caused by the above-mentioned mutation in intron 1. ddPCR is a method for absolutely measuring the concentration of a target in a sample by dispersing limiting diluted cDNA into microcompartments, performing PCR amplification, and directly counting the number of microcompartments with positive amplification signals. ddPCR can be performed using commercially available reagents and equipment (e.g., QX100 TM Droplet Digital TM This can be done using a PCR system (Bio-Rad Laboratories, Inc., etc.). Protein expression assessment involves using Western blotting to confirm whether transfection of antisense oligonucleotides into cells derived from XP-F patients, whose disease is caused by the above mutations in intron 1, increases the level of correctly spliced XPF gene products. Repair activity assessment involves using cells derived from XP-F patients, whose disease is caused by the above mutations in intron 1, to confirm whether transfection of antisense oligonucleotides restores the ability to repair DNA damage caused by UV irradiation.

[0050] The antisense oligonucleotide in pharmaceutical composition I of the present invention is not limited to a specific one as long as it suppresses aberrant splicing in intron 1. For example, the antisense oligonucleotide may target a sequence consisting of 15 to 30 consecutive nucleotides (preferably 15 to 23 nucleotides, more preferably 15 to 18 nucleotides) within the base sequence from positions 155 to 217 (preferably positions 175 to 217, more preferably positions 179 to 213, and even more preferably positions 183 to 203) in the base sequence represented by SEQ ID NO: 63. For example, the base sequence targeted by the antisense oligonucleotide in pharmaceutical composition I of the present invention may be a sequence complementary to any one of the base sequences selected from SEQ ID NOS: 65 to 82, 101 to 108, and 124 to 135, preferably a sequence complementary to any one of the base sequences selected from SEQ ID NOS: 65 to 82, 101, and 105 to 108, more preferably a sequence complementary to any one of the base sequences selected from SEQ ID NOS: 65 to 82, and even more preferably a sequence complementary to any one of the base sequences selected from SEQ ID NOS: 75 to 78. In other words, the antisense oligonucleotide in pharmaceutical composition I of the present invention can hybridize with the above sequence (i.e., the target sequence). In a preferred embodiment, the antisense oligonucleotide in pharmaceutical composition I of the present invention can hybridize with the target sequence under highly stringent conditions.

[0051] The antisense oligonucleotide in Pharmaceutical Composition I of the present invention may have a base sequence (tail sequence) at its 5'-end and / or 3'-end that does not contribute to hybridization with the target sequence. Each tail sequence contains 5 or fewer bases (preferably 4, 3, 2, or 1), and it is optimal for the oligonucleotide to have no tail sequence.

[0052] The base sequence of the portion of the antisense oligonucleotide in pharmaceutical composition I of the present invention excluding the tail sequence may have a sequence complementarity with the target sequence of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and optimally be completely complementary, as long as it retains hybridization activity with the target sequence.

[0053] In a preferred embodiment, the antisense oligonucleotide in pharmaceutical composition I of the present invention comprises any one of the nucleotide sequences represented by SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135 (preferably SEQ ID NOs: 65 to 82, 101, and 105 to 108, more preferably SEQ ID NOs: 65 to 82, and even more preferably SEQ ID NOs: 75 to 78). The nucleotide sequences represented by SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135 are described as RNA sequences corresponding to SEQ ID NOs: 1 to 18, 53 to 60, and 112 to 123, respectively, described in the Examples below. Note that uridine residues in the antisense oligonucleotide may be substituted with modified nucleotides based on the corresponding thymidine residues. Therefore, nucleotide sequences in which "u" in the nucleotide sequences represented by SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135 is substituted with "t" are also included in this embodiment.

[0054] Furthermore, the base sequence of the portion of the antisense oligonucleotide in pharmaceutical composition I of the present invention excluding the tail sequence may have a mismatch of 5 bases or less (preferably 4 bases or less, more preferably 3 bases, 2 bases, or 1 base) with the target sequence, as long as it has hybridization activity with the target sequence, but optimally has no mismatch.

[0055] Here, the antisense oligonucleotide in pharmaceutical composition I of the present invention is thought to suppress abnormal splicing by inhibiting the binding of U1 snRNP to the recognition sequence GTAAGTAA (the sequence from bases 193 to 200 in the nucleotide sequence represented by SEQ ID NO: 63) of the newly generated 5' splice site in the mutant intron 1 sequence. U1 snRNP has a sequence nearly complementary to the recognition sequence of the 5' splice site, and base pairs are formed during the splicing process. Therefore, in a preferred embodiment, the antisense oligonucleotide in pharmaceutical composition I of the present invention can target a sequence containing at least one base, e.g., two, three, four, five, six, seven, or eight bases, of the sequence from bases 193 to 200 in the nucleotide sequence represented by SEQ ID NO: 63.

[0056] The length of the antisense oligonucleotide in Pharmaceutical Composition I of the present invention is not particularly limited as long as it suppresses aberrant splicing in intron 1, and can be, for example, 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides.

[0057] In another embodiment, the pharmaceutical composition of the present invention is applicable to the XPF group, whose disease is caused by the above-mentioned mutation in intron 8. The pharmaceutical composition of the present invention in this embodiment is referred to as Pharmaceutical Composition II of the present invention. Pharmaceutical Composition II of the present invention comprises one or more antisense oligonucleotides that suppress aberrant polyadenylation using the poly(A) addition sequence at positions 324 to 329 in the base sequence of SEQ ID NO:64 in the post-transcriptional modification of the XPF gene having the mutant intron 8 sequence represented by SEQ ID NO:64.

[0058] Whether the antisense oligonucleotide in Pharmaceutical Composition II of the present invention suppresses aberrant polyadenylation in intron 8 is determined by the same evaluation methods as those for protein expression level assessment or repair activity assessment described in the Examples below. If either evaluation method confirms an increase in expression of a correctly spliced XPF gene or restoration of DNA repair activity, the antisense oligonucleotide is deemed to suppress aberrant polyadenylation in intron 8. In the protein expression level assessment, transfection of the antisense oligonucleotide in cells derived from patients in the XP-F group, whose disease is caused by the above-mentioned mutation in intron 8, is used to determine whether the XPF gene product polyadenylated at the correct position increases. Furthermore, in the repair activity assessment, transfection of the antisense oligonucleotide in cells derived from patients in the XP-F group, whose disease is caused by the above-mentioned mutation in intron 8, is used to determine whether the activity to repair DNA damage caused by UV irradiation is restored.

[0059] The antisense oligonucleotide in pharmaceutical composition II of the present invention is not limited to a specific one as long as it suppresses aberrant polyadenylation in intron 8. For example, it may target a sequence consisting of 15 to 30, 15 to 23, or 15 to 18 consecutive nucleotides within the base sequence from bases 309 to 343 in the base sequence represented by SEQ ID NO: 64. In other words, the antisense oligonucleotide in pharmaceutical composition II of the present invention can hybridize with the sequence (i.e., the target sequence). In a preferred embodiment, the antisense oligonucleotide in pharmaceutical composition II of the present invention can hybridize with the target sequence under highly stringent conditions.

[0060] The antisense oligonucleotide in pharmaceutical composition II of the present invention may have a base sequence (tail sequence) at its 5'-end and / or 3'-end that does not contribute to hybridization with the target sequence. Each tail sequence contains 5 or fewer bases (preferably 4, 3, 2, or 1), and optimally, the antisense oligonucleotide has no tail sequence.

[0061] The base sequence of the portion of the antisense oligonucleotide in pharmaceutical composition II of the present invention excluding the tail sequence may have a sequence complementarity with the target sequence of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and optimally be completely complementary, as long as it retains hybridization activity with the target sequence.

[0062] In a preferred embodiment, the antisense oligonucleotide in Pharmaceutical Composition II of the present invention comprises the nucleotide sequences represented by SEQ ID NOs: 83 to 100. The nucleotide sequences represented by SEQ ID NOs: 83 to 100 are described as RNA sequences corresponding to SEQ ID NOs: 19 to 36, respectively, described in the Examples below. Note that uridine residues in the antisense oligonucleotides may be substituted with modified nucleotides based on the corresponding thymidine residues. Therefore, nucleotide sequences in which "u" in the nucleotide sequences represented by SEQ ID NOs: 83 to 100 is substituted with "t" are also included in this embodiment.

[0063] Furthermore, the base sequence of the portion of the antisense oligonucleotide in pharmaceutical composition II of the present invention excluding the tail sequence may have a mismatch of 5 bases or less (preferably 4 bases or less, more preferably 3 bases, 2 bases, or 1 base) with the target sequence, as long as it has hybridization activity with the target sequence, but optimally has no mismatch.

[0064] Here, the antisense oligonucleotide in pharmaceutical composition II of the present invention is thought to suppress aberrant polyadenylation by inhibiting the binding of cleavage and polyadenylation factor (CPSF) to the newly generated poly(A) addition sequence AATAAA (the sequence from positions 324 to 329 in the base sequence represented by SEQ ID NO: 64) in the mutant intron 8 sequence. Therefore, in a preferred embodiment, the antisense oligonucleotide in pharmaceutical composition II of the present invention can target a sequence containing at least one base, for example, two, three, four, five, or six bases, of the sequence from positions 324 to 329 in the base sequence represented by SEQ ID NO: 64.

[0065] The length of the antisense oligonucleotide in pharmaceutical composition II of the present invention is not particularly limited as long as it suppresses aberrant polyadenylation in intron 1, and may be, for example, 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides.

[0066] The antisense oligonucleotide in the pharmaceutical composition of the present invention may be DNA, RNA, or a DNA / RNA chimera, which can increase the nuclease resistance of the antisense oligonucleotide and enhance its stability in vivo.

[0067] The antisense oligonucleotide in the pharmaceutical composition of the present invention can contain one or more modifications to increase nuclease resistance and enhance stability in vivo.Modifications include, for example, modification of the sugar moiety of nucleoside and modification of the phosphate group of nucleotide.Examples of sugar-modified nucleoside and modified internucleoside linkage include those described in above 1.

[0068] In a preferred embodiment, the antisense oligonucleotide contains one or more sugar-modified nucleosides. All of the nucleosides contained in the antisense oligonucleotide may be sugar-modified nucleosides. The antisense oligonucleotide may contain two or more types of sugar-modified nucleosides containing different sugar moiety modifications. The sugar-modified nucleoside is 4'-(CH2) n Nucleosides containing an -O-2' bridge (where n is 1 or 2) or 2'-O-methylation are preferred, and nucleosides containing a 4'-(CH2)2-O-2' bridge or 2'-O-methylation are more preferred. When sugar-modified nucleosides containing a 4'-(CH2)2-O-2' bridge are contained in an antisense oligonucleotide, the number thereof is not particularly limited, and may be 1 or more, for example, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more, or 12 or less, for example, 11 or less, 10 or less, or 9 or less.

[0069] In a more preferred embodiment, all of the nucleosides contained in the antisense oligonucleotide are sugar-modified nucleosides, which are sugar-modified nucleosides containing a 4'-(CH2)2-O-2' bridge, sugar-modified nucleosides containing a 2'-O-methylation, or a combination thereof.

[0070] In a preferred embodiment, the antisense oligonucleotide comprises one or more modified internucleoside linkages. Different modified internucleoside linkages may be contained in the antisense oligonucleotide. All the internucleoside linkages in the antisense oligonucleotide may also be modified internucleoside linkages. In a more preferred embodiment, the modified internucleoside linkages are phosphorothioate linkages. In an even more preferred embodiment, all the internucleoside linkages in the antisense oligonucleotide are modified internucleoside linkages, and the modified internucleoside linkages are phosphorothioate linkages.

[0071] In a preferred embodiment, the antisense oligonucleotide in pharmaceutical composition I of the present invention is selected from the group consisting of oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 1 to 18, 37 to 60, and 112 to 123. The nucleosides contained in these oligonucleotides are either sugar-modified nucleosides containing a 4'-(CH2)2-O-2' bridge or sugar-modified nucleosides containing 2'-O-methylation. Furthermore, all internucleoside linkages in these oligonucleotides are phosphorothioate linkages. The oligonucleotides shown below are more suitable as antisense oligonucleotides in pharmaceutical composition I of the present invention.

[0072] Example 11 (XPF-int1-011): HO-C m1s -C e2s -C m1s -U m1s -T e2s -A m1s -C m1s -T e2s -U m1s -A m1s -C e2s -G m1s -U m1s -C e2s -U m1s -G m1s -T e2s -G m1t -H (SEQ ID NO: 11) Example 12 (XPF-int1-012): HO-C m1s -C e2s -U m1s -U m1s -A e2s -C m1s -U m1s -T e2s -A m1s -C m1s -G e2s -U m1s -C m1s -T e2s -G m1s -U m1s -G e2s -U m1t -H (SEQ ID NO: 12) Example 13 (XPF-int1-013): HO-C m1s -Te2s -U m1s -A m1s -C e2s -U m1s -U m1s -A e2s -C m1s -G m1s -T e2s -C m1s -U m1s -G e2s -U m1s -G m1s -T e2s -U m1t -H (array number 13) Example 14 (XPF-int1-014): HO-U m1s -T e2s -A m1s -C m1s -T e2s -U m1s -A m1s -C e2s -G m1s -U m1s -C e2s -U m1s -G m1s -T e2s -G m1s -U m1s -T e2s -C m1t -H (array number 14) Example 45 (XPF-int1-013_1): HO-C m1s -T e2s -U m1s -A m1s -C e2s -U m1s -U m1s -A m1s -C m1s -G m1s -T e2s -C m1s -U m1s -G m1s -U m1s -G m1s -T e2s -U m1t -H (array number 45) Example 46 (XPF-int1-013_2): HO-C m1s -T e2s -U m1s -A m1s -Ce2s -U m1s -U m1s -A e2s -C m1s -G m1s -T e2s -C m1s -U m1s -G m1s -U m1s -G m1s -T e2s -U m1t -H (array number 46) Example 62 (XPF-int1-013_5): HO-C m1s -T e2s -U m1s -A m1s -C e2s -U m1s -U m1s -A e2s -C m1s -G m1s -T e2s -C m1s -T e2s -G m1s -U m1s -G m1s -T e2s -U m1t -H (array number 112) Example 63 (XPF-int1-013_6): HO-C m1s -T e2s -U m1s -A m1s -C e2s -U m1s -U m1s -A e2s -C m1s -G m1s -T e2s -C m1s -U m1s -G m1s -T e2s -G m1s -T e2s -U m1t -H (array number 113)

[0073] In a preferred embodiment, the antisense oligonucleotide in Pharmaceutical Composition II of the present invention is selected from the group consisting of oligonucleotides consisting of the base sequences represented by SEQ ID NOS: 19 to 36. The nucleosides contained in these oligonucleotides are either sugar-modified nucleosides containing a 4'-(CH2)2-O-2' bridge or sugar-modified nucleosides containing 2'-O-methylation. Furthermore, all internucleoside linkages in these oligonucleotides are phosphorothioate linkages.

[0074] The method for preparing antisense oligonucleotides is not particularly limited, and known chemical synthesis methods (such as the phosphate triester method, phosphoramidite method, and H-phosphonate method) can be used. Synthesis can also be performed using commercially available nucleic acid synthesizers and reagents used in commercially available DNA / RNA synthesis. For example, antisense oligonucleotides having phosphorothioate bonds can be synthesized by coupling a phosphoramidite reagent followed by reaction with sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0075] The oligonucleotides (antisense oligonucleotides) of the present invention may have a group at the 5'-end and / or 3'-end having a desired chemical structure for controlling the physical properties and pharmacokinetics of the oligonucleotides. For example, an aminoalkyl group can be introduced at the 5'-end and / or 3'-end of the oligonucleotides (antisense oligonucleotides) of the present invention, and the desired chemical structure can be added thereto.

[0076] Introduction of an aminoalkyl group into an oligonucleotide can be performed by methods known in the art, or using commercially available reagents. For example, after chain elongation of an oligonucleotide having a target sequence is completed, the oligonucleotide can be reacted with an amino-modifying reagent such as 5'-Amino-Modifier C6 (Glen Research), 5'-TFA-Amino-Modifier C6-CE Phosphoramidite, or 5'-TFA-Amino-Modifier-C5-CE Phosphoramidite (Link Technologies) to synthesize an oligonucleotide having an aminoalkyl phosphate group attached to the 5' end. Alternatively, the oligonucleotide can be reacted with an amino-modifying reagent such as 3'-amino-Modifier C3 CPG or 3'-amino-Modifier C7 CPG (Glen Research) to synthesize an oligonucleotide having an aminoalkyl group attached to the 3' end.

[0077] Chemical structures that can be introduced into the 5' and / or 3' ends of the oligonucleotides (antisense oligonucleotides) of the present invention can be any chemical structure known in the art to be capable of controlling the physical properties and pharmacokinetics of oligonucleotides, such as fatty acids, cholesterol, and GalNAc structures. For example, it is known that antisense oligonucleotides can be synthesized using amidite compounds containing fatty acids that are useful for the transfer of nucleic acids to muscle tissue, and that this can promote the transfer of antisense oligonucleotides to muscle tissue (e.g., Nucleic Acids Res. (2020) 47, 6029-6044, Link Technologies products such as 5'-Palmitate-C6-CE Phosphoramidite, etc.). Furthermore, the function of cholesterol-conjugated siRNA in the brain has been described in publicly known literature (e.g., Mol. Cancer Ther. (2018) 17, 1251-1258). It is also known that antisense oligonucleotides can be synthesized using amidite compounds containing a GalNAc structure, which is useful for the transfer of nucleic acids into liver cells, and that antisense oligonucleotides can be delivered specifically to liver cells (e.g., Methods in Enzymology, 1999, Vol. 313, pp. 297-321; WO2009 / 073809; WO2014 / 076196; WO2014 / 179620; WO2015 / 006740; WO2015 / 105083; WO2016 / 055601; WO2017 / 023817; WO2017 / 084987; WO2017 / 131236; WO2019 / 172286).

[0078] The antisense oligonucleotide in the pharmaceutical composition of the present invention may be in the form of its pharmaceutically acceptable salt. "Its pharmaceutically acceptable salt" refers to a salt of oligonucleotide (antisense oligonucleotide), and examples of such salts include metal salts such as alkali metal salts (sodium salt, potassium salt, lithium salt), alkaline earth metal salts (calcium salt, magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc.; inorganic salts (ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzoylethylenediamine salt, ... Examples of suitable salts include amine salts, such as organic salts like diphenylphenethylamine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; hydrohalides like hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts like nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates like methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates like benzenesulfonate and p-toluenesulfonate; organic acid salts like acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts like glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. These salts can be prepared by known methods.

[0079] Furthermore, the antisense oligonucleotides and pharmaceutically acceptable salts thereof in the pharmaceutical compositions of the present invention may exist as solvates (for example, hydrates), and may be in the form of such solvates.

[0080] The pharmaceutical composition of the present invention can be formulated by mixing the antisense oligonucleotide with appropriate pharmaceutically acceptable additives. For example, the pharmaceutical composition of the present invention can be administered orally as a tablet, capsule, granule, or parenterally as an injection, transdermal agent, or the like.

[0081] These formulations can be manufactured by well-known methods using additives such as excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, diluents, solvents for injections, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, preservatives, and antioxidants.

[0082] Examples of excipients include organic excipients and inorganic excipients. Examples of organic excipients include sugar derivatives such as lactose and sucrose, starch derivatives such as corn starch and potato starch, cellulose derivatives such as crystalline cellulose, and gum arabic. Examples of inorganic excipients include sulfates such as calcium sulfate.

[0083] Examples of binders include the above-mentioned excipients, gelatin, polyvinylpyrrolidone, polyethylene glycol, and the like.

[0084] Disintegrants include, for example, the above-mentioned excipients; chemically modified starch or cellulose derivatives such as croscarmellose sodium and carboxymethyl starch sodium; cross-linked polyvinylpyrrolidone; and the like.

[0085] Examples of lubricants include talc; stearic acid; colloidal silica; waxes such as beeswax and lime; sulfates such as sodium sulfate; lauryl sulfates such as sodium lauryl sulfate; and starch derivatives of the above excipients.

[0086] Examples of emulsifiers include colloidal clays such as bentonite and veegum; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers.

[0087] Examples of the stabilizer include parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol; and phenols such as phenol and cresol.

[0088] Examples of diluents include water, ethanol, and propylene glycol.

[0089] Solvents for injections include, for example, water, ethanol, glycerin, etc.

[0090] Examples of solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate.

[0091] Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

[0092] Examples of isotonic agents include sodium chloride, glycerin, and D-mannitol.

[0093] Examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0094] An example of a soothing agent is benzyl alcohol.

[0095] Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0096] Antioxidants include, for example, sulfites and ascorbic acid.

[0097] The subject to which the pharmaceutical composition of the present invention is administered is a human.

[0098] The pharmaceutical composition of the present invention may be administered orally or parenterally, and a suitable route may be selected depending on the target symptoms. The route of administration may be systemic or local. Examples of parenteral administration include intravenous, intraarterial, intrathecal, intramuscular, intradermal, subcutaneous, intraperitoneal, transdermal, intraosseous, and intraarticular administration. For example, transdermal and subcutaneous administration may be selected for cutaneous symptoms, and intrathecal administration may be selected for neurological symptoms.

[0099] The pharmaceutical composition of the present invention is administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount that produces a therapeutic effect for a specific disease, administration form, and administration route, and is determined appropriately depending on the subject's species, type of disease, symptoms, sex, age, chronic illnesses, and other factors.

[0100] The dosage of the pharmaceutical composition of the present invention can be determined appropriately depending on the species of the subject, the type of disease, symptoms, sex, age, chronic illnesses, and other factors.

[0101] The pharmaceutical compositions of the present invention may be used in combination with at least one known therapeutic agent or therapy.

[0102] The present invention also provides a method for treating xeroderma pigmentosum Group F, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.

[0103] The term "therapeutically effective amount" as used herein means an amount that produces a therapeutic effect for a specific disease or symptom, dosage form, and administration route, and is determined appropriately depending on the subject's species, type of disease or symptom, symptoms, sex, age, chronic illnesses, and other factors. [Example]

[0104] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0105] Example 1 HO-U m1s -C e2s -C m1s -U m1s -A e2s -G m1s -C m1s -G e2s -A m1s -C m1s -C e2s -C m1s -C m1s -T e2s -U m1s -A m1s -C e2s -U m1t -H(XPF-int1-001) (SEQ ID NO: 1) Synthesis was carried out using an automated nucleic acid synthesizer (BioAutomation MerMade 192X) using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)). The reagents used were activator solution-3 (0.25 mol / L 5-benzylthio-1H-tetrazole in acetonitrile, Wako Pure Chemical Industries, Ltd., product No. 013-20011), CAP A for AKTA (1-methylimidazole in acetonitrile, Sigma-Aldrich, product No. L040050), Cap B1 for AKTA (acetic anhydride in acetonitrile, Sigma-Aldrich, product No. L050050), Cap B2 for AKTA (pyridine in acetonitrile, Sigma-Aldrich, product No. L050150), DCA Deblock (dichloroacetic acid in toluene, Sigma-Aldrich, product No. The thiolation reagent used to form phosphorothioate bonds was phenylacetyl disulfide (Carbosynth, product no. FP07495) dissolved in a 1:1 (v / v) solution of acetonitrile (dehydrated, Kanto Chemical, product no. 01837-05) and pyridine (dehydrated, Kanto Chemical, product no. 11339-05). The amidite reagents used were 2'-O-Me nucleoside phosphoramidites (adenosine product no. ANP-5751, cytidine product no. ANP-5752, guanosine product no. ANP-5753, and uridine product no. ANP-5754) from ChemGenes.The non-natural phosphoramidites are those described in JP-A 2000-297097, including Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-6-N-benzoyladenosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-2-N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), and Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite). The compounds used were Example 1 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) and Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite). The compound of Example 1 was synthesized using Glen Unysupport FC 96-well format 0.2 μmol (manufactured by GlenResearch) as the solid phase support. However, the time required for condensation of the amidite was approximately 9 minutes.

[0106] The protected oligonucleotide analogues containing the target sequence were treated with 600 μL of concentrated aqueous ammonia to cleave the oligomer from the support and remove the cyanoethyl protecting group on the phosphorus atom and the protecting groups on the nucleobases. The oligomer mixture was mixed with 300 μL of Clarity QSP DNA Loading Buffer (Phenomenex) and loaded onto a Clarity SPE 96-well plate (Phenomenex). 1 mL of a 1:1 Clarity QSP DNA Loading Buffer:water solution, 3 mL of water, 3 mL of 3% dichloroacetic acid (DCA) solution, and 6 mL of water were added in that order. The components extracted with a 9:1 solution of 20 mM Tris aqueous solution:acetonitrile were then collected. The target compound was obtained after evaporation. This compound was analyzed by reverse-phase HPLC (Phenomenex Clarity 2.6 μm Oligo-MS 100A (2.1 × 50 mm) column, Solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM aqueous triethylamine solution, Solution B: methanol, B%: 10% → 25% (4 min, linear gradient); 60 °C; 0.5 mL / min; 260 nm) and eluted at 2.78 min. The compound was identified by negative ion electrospray ionization mass spectrometry.

[0107] The base sequence of this compound is complementary to the sequence from bases 412 to 429 of the mRNA (corresponding to bases 5001 to 37192 of NG_011442.1) encoded by Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4) (NCBI-GenBank accession no. NG_011442.1) in which T at base 412 has been mutated to A.

[0108] (Examples 2 to 36) The compounds of Examples 2 to 36 were synthesized in the same manner as the compound of Example 1. Information on the compounds of Examples 1 to 36 is shown in Table 1.

[0109] [Table 1]

[0110] In the "Sequence" in the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMe RNA. All internucleoside bonds are phosphorothioate bonds. "Start" and "End" in the table indicate nucleotide numbers in the base sequence represented by SEQ ID NO: 63 for Examples 1 to 18 and SEQ ID NO: 64 for Examples 19 to 36. "Sequence" in the table indicates a sequence complementary to the base sequence from "Start" to "End" except for Example 17. The "Sequence" in Example 17 indicates a sequence in which "c" has been added to the 3' end of the sequence complementary to the base sequence from "Start" to "End". The "Molecular weight" in the table indicates the actual value measured by negative ion ESI mass spectrometry.

[0111] (Examples 37 to 60) The compounds of Examples 37 to 60 were synthesized in the same manner as in Example 1. Information on the compounds of Examples 37 to 60 is shown in Table 2.

[0112] [Table 2]

[0113] In the "Sequence" section of the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMe RNA. All internucleoside bonds are phosphorothioate bonds. "Start" and "End" in the table indicate the nucleotide numbers of the base sequence represented by SEQ ID NO: 63, and "Sequence" in the table indicates the sequence complementary to the base sequence from "Start" to "End." The "Molecular weight" in the table indicates the actual value measured by negative ion ESI mass spectrometry.

[0114] Example 61 HO-C e1s -C s -T e1s -T s -A e1s -C s -T e1s -T s -A e1s -Cs -G e1s -T s -C e1s -T s -G 1t -H(LNA-int1-001) (SEQ ID NO: 61) The compound of Example 61 was synthesized using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)). The LNA moiety was synthesized using the phosphoramidite described in WO99 / 14226. Hereinafter or in the figures, it may be referred to as "LNA."

[0115] This compound was analyzed by reverse-phase HPLC (column (X-Bridge C18 2.5 μm (4.6 × 75 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, solution B: methanol, B%: 5% → 30% (20 min, linear gradient); 60 °C; 1 mL / min; 260 nm), and eluted at 10.23 min. The compound was identified by negative ion electrospray ionization mass spectrometry (observed value: 4972.26).

[0116] (Reference example 1) HO-G e1s -T s -T e1s -C s -A e1s -T s -C e1s -C s -G e1s -T s -A e1s -C s -T e1s -T s -C 1t -H(LNA-int1-001S) (SEQ ID NO: 62) The compound of Reference Example 1 was synthesized in the same manner as the compound of Example 61. Hereinafter or in the figures, it may be referred to as "control DNA."

[0117] This compound was analyzed by reverse-phase HPLC (column (X-Bridge C18 2.5 μm (4.6 × 75 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, solution B: methanol, B%: 5% → 30% (20 min, linear gradient); 60 °C; 1 mL / min; 260 nm), and eluted at 10.46 min. The compound was identified by negative ion electrospray ionization mass spectrometry (observed value: 4971.16).

[0118] (Test Example 1) Analysis of XPF mRNA splicing product amount, XPF protein expression amount, and DNA damage repair activity by Example compounds (1) 1-1. Evaluation of XPF mRNA splicing product quantity Patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7(6), 969-974) were cultured in a 6-well plate at 5 x 10 cells per well. 4 After 24 hours, LNA was transfected at a final concentration of 40 nM using Lipofectamine 2000 (Thermo Fisher Scientific). After 4 hours, the medium was replaced with fresh medium (DMEM 10% FBS). 24 hours after transfection, the cells were harvested. TM Total RNA was extracted using RNA MiniPrep (ZYMO RESEARCH). cDNA was obtained from 100 ng of total RNA using SuperScript® IV (Thermo Fisher Scientific). TM Droplet Digital TM Splicing products were quantified using droplet digital PCR (ddPCR) using a PCR system (Bio-Rad Laboratories, Inc.). Splicing products were also quantified in healthy human fibroblasts (48BR) in the same manner.

[0119] ddPCR was performed according to the following procedure. 10 μl of QX200 Eva Green ddPCR Supermix (Bio-Rad Laboratories, Inc.) was added, along with forward and reverse primers each at a final concentration of 100 nM. 5 μl of sample cDNA (total RNA adjusted to 100 ng) and an appropriate amount of water were added to bring the total volume to 20 μl. QX200 TM / QX100 TM Place the sample and 70 μl of Droplet Generation oil for EvaGreen into a DG8 cartridge for droplet generator and then use the QX100 TM The suspension was placed in a droplet generator and placed in a 96-well plate (Bio-Rad Laboratories, Inc.) and sealed using a PX1 PCR plate sealer. TM Analysis was performed using a Droplet Reader. Correct splicing products were quantified using ddPCR with the primer combination XPF ex1-F and XPF ex2-R, while aberrant splicing products were quantified using ddPCR with the primer combination XPF int1-F and XPF ex2-R. Forward primer XPF ex1-F: 5'-CTCCTCTACCACTTTCTCCAGCTG-3' (SEQ ID NO: 109) XPF int1-F: 5'-CGCGATGACACAGAGAAGGATG-3' (SEQ ID NO: 110) Reverse primer XPF ex2-R: 5'-GAGGGAGGTGTTCAACTCCTTC-3' (SEQ ID NO: 111)

[0120] The results are shown in Figure 1A. Analysis of mRNA from patient-derived cells revealed that the amount of correctly spliced product was low, whereas an increase in the amount of correctly spliced product was observed in cells transfected with the compound of Example 61 (LNA-int1-001).

[0121] 1-2. Protein expression level evaluation Patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7(6), 969-974) were cultured in a 6-well plate at a density of 16 x 10 cells per well. 4 Cells were seeded at 1000 x 1000 cells / well. 24 hours later, the Example Compound or Reference Example Compound was transfected at a final concentration of 40 nM using Lipofectamine 2000 (Thermo Fisher Scientific) (transfection was performed according to the manufacturer's instructions). 4 hours later, the medium was replaced with fresh medium. 24 hours after transfection, proteins were collected, and protein expression levels were examined by Western blotting (using XPF Ab-1 (Cat. #MS-1381, Thermo Fisher Scientific) and β-Actin antibody (sc-47778, Santa Cruz)).

[0122] The results are shown in Figure 1B. Analysis of the XPF protein levels in patient-derived fibroblasts revealed lower levels compared to healthy donor-derived fibroblasts (48BR). An increase in XPF protein levels was observed in cells transfected with the compound of Example 61 (LNA-int1-001). On the other hand, no increase in XPF protein levels was observed in cells transfected with the compound of Reference Example 1 (LNA-int1-001S, control DNA). Furthermore, as shown in Figure 2B, patient-derived cells were similarly transfected with the compounds of Examples 1 to 18 (XPF-int1-001 to XPF-int1-018), and the XPF protein levels were analyzed. An increase in XPF protein levels was observed for all compounds of Examples 1 to 18. The increase was particularly large when the compounds of Examples 11 to 14 were used.

[0123] 1-3. Repair activity evaluation Patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7(6), 969-974) were cultured in a 96-well plate at 1x10 cells per well. 4 Cells derived from the same patient were seeded in a total of 10 wells (2 sets of 5 wells). After 24 hours, the Example Compound or Reference Example Compound was transfected at a final concentration of 40 nM using Lipofectamine 2000 (Thermo Fisher Scientific) (the transfection method was performed according to the manufacturer's instructions). After 4 hours, the medium was replaced with fresh DMEM 10% FBS. After 24 hours from transfection, 1 set of 5 wells was irradiated with UV-C at 20 J / m 2The cells were irradiated (the remaining 5-well set was not irradiated). The medium was discarded, washed once with PBS, and then the PBS was discarded. The cells were then irradiated with UV light while the liquid was still in the empty wells. After UV irradiation, serum-free medium containing 5-ethynyl-2'-deoxyuridine (EdU) at a final concentration of 5 μM was added to the cells. After 4 hours of incubation in the EdU-containing medium, the cells were washed once with PBS, and a fixative solution (300 mM sucrose, 2% formalin, 0.5% Triton X-100 in PBS) was added and the cells were fixed at 4°C for 20 minutes. The cells were then washed three times with PBS. A staining solution (50 mM Tris pH 7.3, 4 mM CuSO4, 10 mM sodium L-ascorbate, 10 μM fluorescent dye 488 azide, 0.03 mg / ml DAPI solution) was added, and the cells were left at room temperature for 1 hour, protected from light. The cells were washed three times with PBS-T (0.05% Tween 20 in PBS). Fixative (3.7% formalin in PBS) was added and the cells were left to stand for 20 minutes. The fixative was discarded and PBS was added. The mean fluorescence intensity of 488 azide in the nucleus was calculated. The fluorescence value indicates repair activity. Repair activity was also evaluated in healthy human fibroblasts (48BR) in the same manner.

[0124] The results are shown in Figure 1C. Analysis of the repair activity of patient-derived cells revealed that it was lower than that of cells derived from healthy individuals. Patient-derived cells transfected with the compound of Example 61 (LNA-int1-001) showed increased repair activity compared to patient-derived cells transfected with the compound of Reference Example 1 (LNA-int1-001S, control DNA). Furthermore, as shown in Figure 2A, patient-derived cells were similarly transfected with the compounds of Examples 1 to 18 (XPF-int1-001 to XPF-int1-018) and the repair activity was analyzed. Increased repair activity was observed for all compounds of Examples 1 to 18.

[0125] (Examples 62 to 73) The compounds of Examples 62 to 73 were synthesized in the same manner as in Example 1. Examples 62 to 73 are listed in Table 3.

[0126] [Table 3]

[0127] In the "Sequence" section of the table, uppercase letters indicate ENA, and lowercase letters indicate 2'-OMe RNA. All internucleoside bonds are phosphorothioate. "Start" and "End" in the table indicate the nucleotide number of the sequence in which T at position 412 of Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4), RefSeqGene (LRG_463) on chromosome 16 (NCBI-GenBank accession no. NG_011442.1) has been mutated to A. "Sequence" in the table indicates the sequence complementary to the nucleotide sequence from "Start" to "End." "Molecular weight" in the table indicates the actual value measured by negative ion electrospray ionization mass spectrometry.

[0128] (Reference example 2) The compound of Reference Example 2 was also synthesized in the same manner as in Example 1. The compound of Reference Example 2 is shown in Table 4.

[0129] [Table 4]

[0130] In the "sequence" in the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMe RNA. All internucleoside bonds are phosphorothioate bonds. The sequence of the compound of Reference Example 2 is complementary to the nucleotide sequence from nucleotides 223 to 240 of Mus musculus strain mdx dystrophin genes, partial cds (NCBI-GenBank accession no. AH007099.2).

[0131] (Test Example 2) Analysis of XPF mRNA splicing product amount, XPF protein expression amount, and DNA damage repair activity by Example compounds (2) 2-1. Protein expression level evaluation Protein expression levels of the example compounds were evaluated in the same manner as in Tests 1-2. Patient-derived cells were transfected with the compounds of Examples 37, 38, 44 to 46, and 53 to 60 (XPF-int1-011_1, XPF-int1-011_2, XPF-int1-012_4, XPF-int1-013_1, XPF-int1-013_2, XPF-int1-0-1, XPF-int1-0-2, XPF-int1-0-3, XPF-int1-0-4, and XPF-int1-19 to 22) and the compounds of Examples 11 and 14 (XPF-int1-11 and XPF-int1-14), and the amount of XPF protein was analyzed. The compound of Reference Example 2 was used as a negative control. The results are shown in Figure 3B. The compounds of Examples 37, 38, 44 to 46, 53, 57 to 60, 11, and 14 were found to increase the amount of XPF protein compared to the negative control (negative con. in the figure) and the control to which no example compound was added (w / o in the figure). 2-2. Repair activity evaluation The repair activity of the example compounds was evaluated in the same manner as in Tests 1-3. The compounds of Examples 37 to 60 (XPF-int1-011_1 to 4, XPF-int1-012_1 to 4, XPF-int1-013_1 to 4, XPF-int1-014_1 to 4, XPF-int1-0-1 to 4, XPF-int1-019 to 022) and the compounds of Examples 11 to 14 (XPF-int1-11 to 14) were transfected into patient-derived cells, and the repair activity was analyzed. The compound of Reference Example 2 was used as a negative control. The results are shown in Figure 3A. The compounds of Examples 37 to 53, 57 to 60, and 11 to 14 exhibited increased repair activity compared to the negative control (negative control in the figure) and the control without the example compound (w / o in the figure). [Industrial Applicability]

[0132] The pharmaceutical composition of the present invention can treat a specific XP-F group for which no therapeutic means existed previously. [Sequence List Free Text]

[0133] SEQ ID NOs: 1 to 61: Oligonucleotide sequences of Example Compounds 1 to 61 SEQ ID NO: 62: Oligonucleotide sequence of Reference Example Compound 1 SEQ ID NO: 63: DNA sequence of mutant intron 1 of the XPF gene SEQ ID NO: 64: DNA sequence of mutant intron 8 of the XPF gene SEQ ID NOs: 65 to 100: RNA sequences corresponding to SEQ ID NOs: 1 to 36 SEQ ID NOs: 101 to 108: RNA sequences corresponding to SEQ ID NOs: 53 to 60 SEQ ID NOs: 109 to 111: DNA sequences of primers used in ddPCR SEQ ID NOs: 112 to 123: Oligonucleotide sequences of Example Compounds 62 to 73 SEQ ID NOs: 124 to 135: RNA sequences corresponding to SEQ ID NOs: 112 to 123 SEQ ID NO: 136: Oligonucleotide sequence of Reference Example Compound 2 SEQ ID NO: 137: RNA sequence corresponding to SEQ ID NO: 136 In the sequence listing, Am1s, Gm1s, Cm1s, Um1s, Ae2s, Ge2s, Ce2s, Te2s, Ae1s, Ge1s, Ce1s, Te1s, Cs, Ts, Am1t, Gm1t, Cm1t, Um1t, T2t, G1t, and C1t represent A, G, and C, respectively. m1s , G m1s , C m1s , U m1s , A e2s , G e2s , C e2s , T e2s , A e1s , G e1s , C e1s , T e1s , C s , T s , A m1t , G m1t , C m1t , U m1t , T 2t , G 1t , and C 1t Represents.

Claims

1. An antisense oligonucleotide having a base sequence capable of hybridizing with a portion of the intron region of the XPF gene and having activity of suppressing abnormal post-transcriptional modification of the XPF gene, wherein the 5'-end and / or 3'-end may be chemically modified, or a pharmaceutically acceptable salt thereof.

2. 2. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, which suppresses abnormal splicing using the 5' splice site 5' of the 193rd guanine and the 3' splice site 3' of the 1658th guanine in the base sequence represented by SEQ ID NO: 63 in the post-transcriptional modification of the XPF gene having a mutant intron 1 sequence represented by SEQ ID NO:

63.

3. 3. The antisense oligonucleotide according to claim 1 or 2, which is capable of hybridizing to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from the 155th to the 217th bases in the base sequence represented by SEQ ID NO: 63, or a pharmaceutically acceptable salt thereof.

4. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, comprising a sequence that is 90% or more complementary to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence of 155 to 217 in the base sequence represented by SEQ ID NO:

63.

5. The antisense oligonucleotide according to any one of claims 1 to 4, comprising a base sequence represented by any one of SEQ ID NOs: 65 to 82 and 101 to 108 (in the sequences, u may be replaced with t), or a pharmaceutically acceptable salt thereof.

6. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, comprising one or more sugar-modified nucleosides.

7. The sugar-modified nucleoside is 4'-(CH ２ ) ｎ 7. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 6, which is a nucleoside containing an -O-2' bridge (wherein n is 1 or 2) or a 2'-O-methylation.

8. 8. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, comprising one or more modified internucleoside linkages.

9. 9. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, wherein the modified internucleoside bond is a phosphorothioate bond.

10. The antisense oligonucleotide according to any one of claims 1 to 9, which consists of a base sequence represented by any one of SEQ ID NOs: 1 to 18, 37 to 60, and 112 to 123, or a pharmaceutically acceptable salt thereof.

11. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, which suppresses abnormal polyadenylation using the poly A addition sequence at positions 324 to 329 in the base sequence represented by SEQ ID NO: 64 in the post-transcriptional modification of the XPF gene having a mutant intron 8 sequence represented by SEQ ID NO:

64.

12. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 11, which is capable of hybridizing to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from the 309th to the 343rd base in the base sequence represented by SEQ ID NO:

64.

13. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1, 11 and 12, comprising a sequence that is 90% or more complementary to a sequence consisting of 15 to 30 consecutive nucleotides within the base sequence from positions 309 to 343 in the base sequence represented by SEQ ID NO:

64.

14. The antisense oligonucleotide according to any one of claims 1 and 11 to 13, or a pharmaceutically acceptable salt thereof, comprising a base sequence represented by any one of SEQ ID NOs: 83 to 100 (in the sequences, u may be replaced with t).

15. 15. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 and 11 to 14, comprising one or more sugar-modified nucleosides.

16. The sugar-modified nucleoside is 4'-(CH ２ ) ｎ 16. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 15, which is a nucleoside comprising an -O-2' bridge (wherein n is 1 or 2) and 2'-O-methylation.

17. 17. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 and 11 to 16, comprising one or more modified internucleoside linkages.

18. 18. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 17, wherein the modified internucleoside linkage is a phosphorothioate linkage.

19. The antisense oligonucleotide according to any one of claims 1 and 11 to 18, which consists of a base sequence represented by any one of SEQ ID NOs: 19 to 36, or a pharmaceutically acceptable salt thereof.

20. 20. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 19, wherein the chemical modification is the addition of a molecular structure suitable for transport of the oligonucleotide.

21. A pharmaceutical composition for treating xeroderma pigmentosum group F, comprising the antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof.

22. A method for treating xeroderma pigmentosum group F, comprising administering to a patient an effective amount of the antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20.

23. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, for use in the treatment of xeroderma pigmentosum group F.

24. Use of the antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20 for the manufacture of a pharmaceutical composition for treating xeroderma pigmentosum group F.

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