WO2021112106A1

WO2021112106A1 - Xeroderma pigmentosum group f therapeutic agent

Info

Publication number: WO2021112106A1
Application number: PCT/JP2020/044768
Authority: WO
Inventors: 朋男荻; 由華中沢; 小泉　誠; 貴生小路
Original assignee: 第一三共株式会社; 国立大学法人東海国立大学機構
Priority date: 2019-12-02
Filing date: 2020-12-02
Publication date: 2021-06-10
Also published as: TW202134438A; JPWO2021112106A1

Abstract

To provide a means for treating xeroderma pigmentosum group F, for which no disease-causing mutation has been identified to date.　The present invention provides: an antisense oligonucleotide having a base sequence capable of hybridizing with a portion of the intron region of an XPF gene and having activity to inhibit abnormal posttranscriptional modification of the XPF gene, or a pharmaceutically acceptable salt of said antisense oligonucleotide; and a therapeutic agent or a therapeutic composition for xeroderma pigmentosum group F containing said antisense oligonucleotide or pharmaceutically acceptable salt thereof as an active ingredient.

Description

Xeroderma pigmentosum group F therapeutic agent

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

Xeroderma pigmentosum (XP) is a congenital gene involved in nucleotide excision repair mechanism (NER) that removes photoDNA damage caused by ultraviolet rays in sunlight from the genome and damage bypass synthesis (TLS). It develops due to an abnormality. XP patients have photosensitivity, xeroderma pigmentosum in the sun-exposed area, high cancer incidence, and neurological symptoms, and the prevalence of Japanese is estimated to be about 1 in 25,000 births. .. As genes for which disease-causing mutations have been reported so far, in addition to the XPA to XPG genes, the POHL gene encoding TLS polymerase is known. XP cases with each mutation are classified into the complementarity group of XP-A to XP-G group and XP-V (variant) group. The proportion of each complementary group in XP cases varies depending on the race, but in the Japanese population, there are many abnormalities in the XP-A group and then in the XP-V group.

Of the XP complementarity group, except for the variant group that is TLS deficient, NER deficiency is shown. NER is divided into whole genome repair (GG-NER) and transcriptional conjugated repair (TC-NER) according to the recognition form of DNA damage. The XP-C / E group shows a deficiency of only GG-NER, and the XP-A / B / D / F / G group shows a deficiency of both GG-NER and TC-NER. In XP cases, DNA damage accumulates in the genome due to these NER deficiencies, leading to genome instability, and skin cancer occurs frequently at a young age. Neurological symptoms differ in onset time depending on the complementary group and disease-causing mutation, but especially in cases with group A Japanese founder mutation (IVS3-1G> C), loss of XPA protein expression results in the loss of XPA protein expression and the full function of NER. And show severe neurological symptoms.

XP is a systemic hereditary disease, and no effective treatment has been developed at present. XP is often diagnosed by visiting a dermatologist after a severe sunburn in infancy. In addition, a rough prognosis can be determined by genetic testing. Although it is possible to avoid exposure to sunlight and try to prevent skin cancer, there is no effective way to alleviate the progression of neurological symptoms that have a significant effect on prognosis and quality of life.

For the definitive diagnosis of XP, a method for measuring DNA repair activity using fibroblasts established from a patient's skin patch is known. These include the Irregular DNA Synthesis (UDS) test, which measures GG-NER activity by detecting repair DNA synthesis after removal of photoDNA damage, and RNA synthesis in regions of high transcriptional activity after DNA damage. There is a method for evaluating TC-NER activity by examining resilience (RRS) (Non-Patent Documents 1 and 2). Complementarity groups can be identified by evaluating UDS / RRS in patient-derived cells and investigating which known XP genes restore these activities (Non-Patent Documents 3 and 4).

Regarding gene mutations carried by XP-F group patients, mutations in the exon region of the XPF gene (ERCC4) are reported as individual patient mutations in Non-Patent Document 5. However, mutations in the intron region of the XPF gene are not disclosed.

For the XP-F group, there were cases in which the disease-causing mutation was not identified, and it was not possible to treat it so far. An object of the present invention is to provide a therapeutic means for such XP-F group.

The present inventors have identified two novel internal mutations in introns that are the cause of the disease in patients in the XP-F group. The first is a novel Japanese founder mutation that exists deep in the intron (about 100 bp downstream from the exon 1-intron 1 boundary) and gives rise to a new U1 nuclear small ribonuclear protein (U1 snRNP) binding sequence. It was shown that this caused a decrease in gene expression due to abnormal alternative splicing. The second is a single base substitution mutation in the deep intron (several hundred bp downstream from the exon 8-intron 8 boundary site), which forms a pathological polyA addition sequence (cleaved / polyadenylation factor binding sequence). It has been shown that incomplete transcription termination and mRNA destabilization lead to disease onset. The present inventors have further studied and completed the present invention.

That is, the present invention includes the following inventions.
[1] An antisense oligonucleotide having a base sequence capable of hybridizing with a part of the intron region of the XPF gene and having an activity of suppressing abnormal post-transcriptional modification of the XPF gene, the 5'end thereof. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which may be chemically modified at the 3'end.
[2] In post-transcriptional modification of the XPF gene having the mutant intron 1 sequence represented by SEQ ID NO: 63, the 5'splice site on the 5'side of the 193rd guanine and 1658 in the base sequence represented by SEQ ID NO: 63. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to [1], which suppresses abnormal splicing using the 3'splice site on the 3'side of the second guanine.
[3] Described in [1] or [2], which can hybridize with a sequence consisting of consecutive 15 to 30 nucleotides in the 155th to 217th base sequences in the base sequence represented by SEQ ID NO: 63. Antisense oligonucleotides or pharmaceutically acceptable salts thereof.
[4] 90% or more, preferably 95% or more, optimally completely complementary to the sequence consisting of 15 to 30 nucleotides in the 155th to 217th base sequences in the base sequence represented by SEQ ID NO: 63. The antisense oligonucleotide according to any one of [1] to [3] or a pharmaceutically acceptable salt thereof, which comprises a specific sequence.
[5] Described in any of [1] to [4], which comprises a base sequence represented by any of SEQ ID NOs: 65 to 82 and 101 to 108 (u may be replaced with t in the sequence). Antisense oligonucleotide or pharmaceutically acceptable salt thereof.
[6] The antisense oligonucleotide according to any one of [1] to [5] or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides.
[7] The sugar-modified nucleoside is a nucleoside _{containing 4'-(CH 2} ) _n- O-2'cross-linking (where n is 1 or 2 in the formula) or 2'-O-methylation, [6] ] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof.
[8] The antisense oligonucleotide according to any one of [1] to [7] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
[9] The antisense oligonucleotide according to [8] or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
[10] The antisense oligonucleotide according to any one of [1] to [9] or pharmaceutically acceptable thereof, which comprises the base sequence represented by any of SEQ ID NOs: 1 to 18, 37 to 60 and 112 to 123. Salt to be done.
[11] In post-transcriptional modification of the XPF gene having the mutant intron 8 sequence represented by SEQ ID NO: 64, abnormal polyadenylation using the 324th to 329th polyA addition sequence in the nucleotide sequence represented by SEQ ID NO: 64. The antisense oligonucleotide according to [1] or a pharmaceutically acceptable salt thereof.
[12] The description in [1] or [11], which can hybridize with a sequence consisting of 15 to 30 nucleotides in a row in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64. Antisense oligonucleotides or pharmaceutically acceptable salts thereof.
[13] 90% or more, preferably 95% or more, optimally completely complementary to the sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64. The antisense oligonucleotide according to any one of [1], [11] and [12] or a pharmaceutically acceptable salt thereof, which comprises a specific sequence.
[14] Described in any of [1] and [11] to [13], which comprises a base sequence represented by any of SEQ ID NOs: 83 to 100 (u may be replaced with t in the sequence). Antisense oligonucleotide or pharmaceutically acceptable salt thereof.
[15] The antisense oligonucleotide according to any one of [1] and [11] to [14] or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides.
[16] The sugar-modified nucleoside is a nucleoside _{containing 4'-(CH 2} ) _n- O-2'cross-linking (where n is 1 or 2 in the formula) and 2'-O-methylation, [15] ] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof.
[17] The antisense oligonucleotide according to any one of [1] and [11] to [16] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
[18] The antisense oligonucleotide of [17] or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
[19] The antisense oligonucleotide according to any one of [1] and [11] to [18] or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by any of SEQ ID NOs: 19 to 36.
[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 transporting the oligonucleotide.
[21] A pharmaceutical composition for treating xeroderma pigmentosum F group, which comprises the antisense oligonucleotide according to any one of [1] to [20] or a pharmaceutically acceptable salt thereof.
[22] Xeroderma pigmentosum F, which comprises administering to a patient an effective amount of the antisense oligonucleotide according to any one of [1] to [20] or a pharmaceutically acceptable salt thereof. How to treat the group.
[23] The antisense oligonucleotide according to any one of [1] to [20] or a pharmaceutically acceptable salt thereof for use in the treatment of xeroderma pigmentosum group F.
[24] The antisense oligonucleotide according to any one of [1] to [20] or a pharmaceutically acceptable salt thereof for producing a therapeutic pharmaceutical composition for group F xeroderma pigmentosum. use.

The present invention also provides the following [A1] to [A20].
[A1] An antisense oligonucleotide or a pharmaceutically acceptable antisense oligonucleotide having a base sequence capable of hybridizing with a part of the intron region of the XPF gene and having an activity of suppressing abnormal post-transcriptional modification of the XPF gene. salt.
[A2] In the post-transcriptional modification of the XPF gene having the mutant intron 1 sequence represented by SEQ ID NO: 63, the 5'splice site on the 5'side of the 193rd guanine and 1658 in the base sequence represented by SEQ ID NO: 63. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to [A1], which suppresses abnormal splicing using the 3'splice site on the 3'side of the second guanine.
[A3] Described in [A1] or [A2], which can hybridize with a sequence consisting of 15 to 30 consecutive nucleotides in the 175th to 217th base sequences in the base sequence represented by SEQ ID NO: 63. Antisense oligonucleotides or pharmaceutically acceptable salts thereof.
[A4] 90% or more, preferably 95% or more, optimally completely complementary to the sequence consisting of 15 to 30 nucleotides in the 175th to 217th base sequences in the base sequence represented by SEQ ID NO: 63. The antisense oligonucleotide according to any one of [A1] to [A3] or a pharmaceutically acceptable salt thereof, which comprises a specific sequence.
[A5] Described in any of [A1] to [A4], which comprises a base sequence represented by any of SEQ ID NOs: 65 to 82 and 101 to 108 (u may be replaced with t in the sequence). Antisense oligonucleotide or pharmaceutically acceptable salt thereof.
[A6] The antisense oligonucleotide according to any one of [A1] to [A5] or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides.
[A7] The sugar-modified nucleoside is a nucleoside _{containing 4'-(CH 2} ) _n- O-2'cross-linking (where n is 1 or 2 in the formula) or 2'-O-methylation, [A6]. ] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof.
[A8] The antisense oligonucleotide according to any one of [A1] to [A7] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
[A9] The antisense oligonucleotide according to [A8] or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
[A10] The antisense oligonucleotide according to any one of [A1] to [A9] or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by any of SEQ ID NOs: 1 to 18 and 37 to 60.
[A11] In post-transcriptional modification of the XPF gene having the mutant intron 8 sequence represented by SEQ ID NO: 64, abnormal polyadenylation using the 324th to 329th polyA addition sequence in the nucleotide sequence represented by SEQ ID NO: 64. The antisense oligonucleotide according to [A1] or a pharmaceutically acceptable salt thereof.
[A12] Described in [A1] or [A11], which can hybridize with a sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64. Antisense oligonucleotides or pharmaceutically acceptable salts thereof.
[A13] 90% or more, preferably 95% or more, optimally completely complementary to the sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64. The antisense oligonucleotide according to any one of [A1], [A11] and [A12] or a pharmaceutically acceptable salt thereof, which comprises a specific sequence.
[A14] Described in any of [A1] and [A11] to [A13], which comprises a base sequence represented by any of SEQ ID NOs: 83 to 100 (u may be replaced with t in the sequence). Antisense oligonucleotide or pharmaceutically acceptable salt thereof.
[A15] The antisense oligonucleotide according to any one of [A1] and [A11] to [A14] or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides.
[A16] The sugar-modified nucleoside is a nucleoside _{containing 4'-(CH 2} ) _n- O-2'cross-linking (where n is 1 or 2 in the formula) and 2'-O-methylation, [A15]. ] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof.
[A17] The antisense oligonucleotide according to any one of [A1] and [A11] to [A16] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
[A18] The antisense oligonucleotide according to [A17] or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
[A19] The antisense oligonucleotide according to any one of [A1] and [A11] to [A18] or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by any of SEQ ID NOs: 19 to 36.
[A20] A therapeutic pharmaceutical composition for group F of xeroderma pigmentosum, which comprises the antisense oligonucleotide according to any one of [A1] to [A19] or a pharmaceutically acceptable salt thereof.

According to the present invention, it is possible to treat a specific XP-F group for which a therapeutic means has not existed in the past.

FIG. 1A shows an increase in the amount of XPF mRNA splicing product in cells derived from an XPF patient transfected with the compound of Example 61. Black bars represent the correct amount of splicing products and white bars represent the amount of abnormal splicing products. FIG. 1B shows an increase in XPF protein expression in cells derived from XPF patients transfected with the compound of Example 61. FIG. 1C shows that DNA repair activity after UV irradiation is increased in cells derived from XPF patients transfected with the compound of Example 61. FIG. 2A shows that DNA repair activity after UV irradiation is increased in cells derived from XPF patients transfected with the compounds of Examples 1-18. FIG. 2B shows an increase in XPF protein expression in cells derived from XPF patients transfected with the compounds of Examples 1-18. FIG. 3A shows that DNA repair activity after UV irradiation is increased in cells derived from XPF patients transfected with the compounds of Examples 11-14 and 37-60. Black bars represent the correct amount of splicing products and white bars represent the amount of abnormal splicing products. FIG. 3B shows increased XPF protein expression levels in cells derived from XPF patients transfected with the compounds of Examples 11, 13, 37, 38, 44-46, and 53-60. The upper panel shows the expression level of XPF protein, and the lower panel shows the expression level of β-actin.

1. 1. Definitions In the present specification, "xeroderma pigmentosum F group" (also referred to as XP-F group) is one of the complementary groups of xeroderma pigmentosum, and both GG-NER and TC-NER. Shows a defect. The gene for which the causative mutation has been reported is the XPF gene.

In the present specification, the "XPF gene" is a human gene encoding an endonuclease involved in the nucleotide excision and 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 as, for example, Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4), RefSeqGene (LRG_463) on chromosome 16 (NCBI-GenBank accession No. NG_011442.1).

In the present specification, the "mutant intron 1 sequence" is the base sequence of intron 1 of the wild-type XPF gene (base sequence 5217 to 6874 in the above NG_011442.1), which is 196th from the 5'end of intron 1 (the base sequence of intron 1). In NG_011442.1, T of 5412) is a sequence mutated to A, and is a base sequence represented by SEQ ID NO: 63. The mutation site corresponds to the 196th position in the base sequence represented by SEQ ID NO: 63.

In the present specification, the "mutant intron 8 sequence" refers to the 326th from the 5'end of the intron 8 in the base sequence of the wild-type XPF gene intron 8 (the base sequence of 20588 to 22609 in the above NG_011442.1). In NG_011442.1, C is the sequence mutated to T (20913th), and is the base sequence represented by SEQ ID NO: 64. The mutation site corresponds to the 326th position in the base sequence represented by SEQ ID NO: 64.

As used herein, "post-transcriptional modification" refers to the modification that pre-mRNA undergoes in the process of becoming a mature mRNA, which includes cap addition to the 5'end, polyadenylation at the 3'end, and splicing. included.

In the present specification, "abnormal post-transcriptional modification" refers to post-transcriptional modification that is not normally observed in post-transcriptional modification of wild-type genes, and includes, for example, abnormal splicing and abnormal polyadenylation.

In the present specification, "abnormal splicing" refers to splicing that is not normally found in post-transcriptional modification of wild-type genes. mRNA splicing proceeds by recognizing the 5'splice site, branch site, and 3'splice site in the intron by the splicing factor, the small nuclear ribonuclear protein (snRNP). In the post-transcriptional modification of the XPF gene having the mutant intron 1 sequence, the mutation at position 196 in the nucleotide sequence represented by SEQ ID NO: 63 gives rise to a new recognition sequence for the splicing factor U1 snRNP (GTATGTAA), which is number 193. The 5'side of guanine is the 5'splice site. As a result, abnormal splicing occurs 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 nucleotide sequence represented by SEQ ID NO: 63.

In the present specification, "abnormal polyadenylation" refers to polyadenylation that is not normally observed in post-transcriptional modification of wild-type genes. Polyadenylation of mRNA occurs depending on the polyA addition sequence (also referred to as cleavage / polyadenylation factor binding sequence or polyadenylation signal). Poly-A addition sequences in humans usually include AATAAA. In post-transcriptional modification of the XPF gene having a mutant intron 8 sequence, a new poly A addition sequence (AATAAA) is generated by the 326th mutation in the nucleotide sequence represented by SEQ ID NO: 64. As a result, abnormal polyadenylation using the 324th to 329th polyA addition sequences in the nucleotide sequence represented by SEQ ID NO: 64 occurs.

In the present specification, the "antisense oligonucleotide" is a single-stranded oligonucleotide having an ability to hybridize to a nucleotide containing a target base sequence. “Able to hybridize” means double-stranded with a target nucleotide by interaction between bases (AG (adenine-guanine) and CT / U (cytosine-thymine / uracil)). Means that can be formed. The antisense oligonucleotide need only have sequence complementarity with the target sequence to the extent that it can hybridize, and does not have to be a completely complementary sequence. Also, the antisense oligonucleotide can be a DNA, RNA, and DNA / RNA chimera. The antisense oligonucleotide may also contain modifications such as modified nucleosides and bonds between modified nucleosides.

"Hybridizing" includes hybridizing under low stringent conditions, hybridizing under medium stringent conditions, and hybridizing under high stringent conditions. "Low stringent conditions" can be, for example, 5 x SSC, 5 x Denhardt solution, 0.5% SDS, 50% formamide, 32 ° C., or equivalent. "Medium stringent conditions" are, for example, 5 x SSC, 5 x Denhardt solution, 0.5% SDS, 50% formamide, 42 ° C, or 5 x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5). , 50% formamide, 42 ° C., or equivalent. “High stringent conditions” are, for example, 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 50 ° C, or 0.2 × SSC, 0.1% SDS, 65 ° C, or equivalent conditions. Can be. Multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art will appropriately select these factors. By doing so, it is possible to achieve similar stringency. The above conditions can also be realized by using a commercially available hybridization reagent. For example, "high stringent conditions" can be defined ^{as 0.7 in a commercially available hybridization solution ExpressHyb TM} hybridization solution (manufactured by Clontech) by hybridizing at 68 ° C. or by using a filter on which DNA is fixed. After hybridization at 68 ° C. in the presence of −1.0 M NaCl, a 0.1-2 times concentration SSC solution (1 time concentration SSC consists of 150 mM NaCl and 15 mM sodium citrate) was used at 68 ° C. It can be realized by cleaning with.

In the present specification, nucleosides include natural nucleosides and modified nucleosides. "Natural nucleosides" are 2'-deoxy such as 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, thymidine, 2'-deoxyuridine, etc. Ribonucleosides such as nucleosides, adenosine, guanosine, thymidine, 5-methylcytidine, and uridine. Of the nucleobases, uracil (U) or (u) and thymine (T) or (t) are compatible, and either uracil (U) or (u) and thymine (T) or (t) It can be used for base pairing with the complementary strand adenine (A) or (a).

Herein, 2'-deoxy adenosine ^A t, 2'-deoxyguanosine and ^G t, 2'-deoxycytidine and ^C t, 2'-deoxy-5-methylcytidine 5meC ^t, thymidine ^T t, 2'-deoxyuridine may represent a ^{U t.} As nucleotides corresponding thereto, in the present specification, 2'-deoxyadenosine nucleotide ^A p, 2'-deoxyguanosine nucleotides ^G p, 2'-deoxycytidine nucleotides ^C p, 2'-deoxy - 5meC 5-methyl cytidine nucleotides ^p, the thymidine nucleotides ^T p, sometimes a 2'-deoxyuridine nucleotide represented as ^{U p.}

In the present specification, the "sugar-modified nucleoside" means a nucleoside in which the sugar portion of the nucleoside is modified. Sugar-modified nucleosides include all forms of sugar modification known in the art to which the present invention belongs. Sugar-modified nucleosides include, for example, 2'-modified nucleosides, 4'-thio-modified nucleosides, 4'-thio-2'-modified nucleosides and bicyclic sugar-modified nucleosides.

Examples of 2'-modified nucleotides are halo, allyl, amino, azide, O-allyl _{, OC 1-} C ₁₀ alkyl, OCF ₃ , O- (CH ₂ ) _2- O-CH ₃ , 2'-. O (CH ₂ ) ₂ SCH ₃ , O- (CH ₂ ) ₂ -ON (R _m ) (R _n ), or O-CH _2- C (= O) -N (R _m ) (R _n ) Each R _m and R _n is H, an amino protecting group, or a substituted or unsubstituted C _1- C ₁₀ alkyl individually. 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 are described in the literature (Blommers et al. Biochemistry (1998), 37). , 17714-17725.). 2'-O-propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine, and 2'-O-propyluridine are described in the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832). -7838.) Can be synthesized according to. Commercially available reagents can be used for 2'-O-allyl guanosine, 2'-O-allyl adenosine, 2'-O-allyl cytidine, and 2'-O-allyl uridine. 2'-O-Methoxyethyl guanosine, 2'-O-Methoxyethyl adenosine, 2'-O-Methoxyethyl cytidine, and 2'-O-Methoxyethyl uridine are available in the patent (US6261840) or in the literature (Martin, P. et al. Synthesized according to Helv. Chim. Acta. (1995) 78, 486-504. 2'-O-butylguanosine, 2'-O-butyladenosine, 2'-O-butylcytidine, and 2'-O-butyl Uridine 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.). 2'-O-propargylguanosine, 2'-O-propargyl adenosine. , 2'-O-propargylcytidine, and 2'-O-propargyluridine, commercially available reagents can be used.

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

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

Examples of bicyclic sugar-modified nucleosides include nucleosides that retain a second ring formed by cross-linking two atoms of the ribose ring, and examples of such nucleosides are 2'-. 2', 4'-BNA / LNA (bridged nucleoside acids / locked nucleoside acids) (Obika, S. et al. Tetrahedron Lett., 38, p. 8735-) in which an oxygen atom and a 4'-carbon atom are crosslinked with a methylene chain. (1997) .; Obika, S. et al., Tetrahedron Lett., 39, p.5401- (1998) .; A.A. Koshkin, A.A. et al. Tetrahedron, 54, p.3607 (1998). ) .; Obika, S. Bioorg. Med. Chem., 9, p.1001 (2001).), ENA (2) in which the methylene chain of 2', 4'-BNA / LNA is cross-linked with an ethylene chain extended by one carbon. '-O, 4'-C-ethylene-bridged nucleic acids) can be mentioned (Morita, K. et al. Bioorg. Med. Chem. Lett., 12, p.73 (2002) .; Morita, K. . Et al. Bioorg. Med. Chem., 11, p. 2211 (2003).). In addition, AmNA described in WO2014 / 109384 or S-cEt (2', 4'-constrained ethyl) described in the literature (Seth, PP et al. J.Org.Chem (2010), 75, 1569-1581.) Can also be given as an example.

In the specification, a sugar-modified nucleosides that include a 2'-O- methylation modification, for example, those corresponding to ^{^A t A m1t,} ^G ^m1t those corresponding to ^{G t,} those corresponding to the ^{C t} C ^M1T, ^{may 5meC} those corresponding to 5meC ^t M1T, those corresponding to ^{U t} is expressed as like ^{U M1T.} As for the sugar modified nucleotides containing 2'-O- methylation modification corresponding to them, those corresponding to ^{^A p A} m1p, ^G m1p those corresponding to ^{G p,} those corresponding to the ^{C p} ^{C m1p} , may represent ^5meC m1p those corresponding to 5meC ^p, those corresponding to ^{U p} and ^{U m1p.}

In the specification, a sugar-modified nucleosides that include a 2'-O- methoxyethyl of modifications, for example, those corresponding to ^{^A t A} m2t, ^G m2t those corresponding to ^{G t,} those corresponding to 5meC ^t 5meC ^m2t, those corresponding to ^{T t} may be represented by such ^{T m2t.} As for the sugar modified nucleotides containing 2'-O- methoxyethyl of modifying their corresponding, 5meC those corresponding ones corresponding to ^{^A p A} m2p, those corresponding to the ^{G p} ^G m2p, the 5meC ^p ^m2p, sometimes those corresponding to ^{T p} represents a ^{T m2p.}

In the present specification, the sugar modified nucleosides containing _4'-CH 2 -0-2 'bridge, for example, correspond to one corresponding to ^{A t} ^A 1t, those corresponding to the ^{G t} in ^G 1t, 5meC ^t sometimes represent things ^C 1t, those corresponding to ^{T t} and ^{T 1t.} As for the sugar-modified nucleotides comprising a _4'-CH 2 -0-2 'bridge their corresponding, which corresponds to that corresponding to ^{A p} ^A E1p, those corresponding to the ^{G p} ^G E1p, the 5meC ^p certain ^C E1p, also those corresponding to ^{T p} represents a ^{T E1p.}

Herein, 4 _- as a sugar-modified nucleosides that include a _'(CH 2) 2 -0-2' bridge, for example, those corresponding to ^{A t} ^A 2t, those corresponding to the ^{^G ^t G} 2t, 5meC ^t The one corresponding to T ^t may be expressed ^{as C 2t} , and the one corresponding to T ^{t may be expressed as T 2t.} The 4 corresponding to them _- for _'(CH 2) 2 -0-2' sugar modified nucleotides comprising a bridge, the one corresponding to ^{A p} ^A e2p, those corresponding to the ^{G p} ^G e2p, the 5meC ^p It may represent corresponding ones ^C e2p, those corresponding to ^{T p} and ^{T e2p.}

In the present specification, the "modified nucleoside bond" refers to a bond in which a naturally occurring nucleoside bond (that is, a phosphodiester nucleoside bond) is replaced or changed. That is, the antisense oligonucleotide containing the modified nucleoside linkage comprises the modification of the phosphate group of at least one nucleotide. Examples of the modified nucleoside bond include a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a boranophosphate bond, a phosphoramidate bond and the like.

In the present specification, phosphorothioate esters has a phosphorothioate ester in place of phosphoric acid esters of nucleotides, the one corresponding to A ^p A ^s, a G ^s which corresponds to G ^{^p,} those corresponding to the C ^p some C ^s, 5meC those corresponding to 5meC ^p ^s, the ^T s corresponds to ^{T p,} also those corresponding to ^{U p} expressed as ^{U s.}
2'-O- For methylation modifications and sugar modified nucleotides containing phosphorothioate, ^{A s} to the corresponding ones ^A m1s, those corresponding to the ^{G s} ^G m1s, those corresponding to the ^{^C ^s C} m1s, 5meC ^s ^5meC m1s those corresponding to, sometimes those corresponding to ^{U s} expressed as ^{U m1s.}
For 2'-O- methoxyethyl of modified and sugar-modified nucleotides comprising a phosphorothioate, those corresponding to ^{A s} ^A m2s, those corresponding to the ^{G s} ^G m2s, ^5meC those corresponding to 5meC ^s ^m2s, T ^The one corresponding to s may be expressed ^{as T m2s.}
T the _4'-CH 2 -0-2 'bridge and sugar modified nucleotides containing phosphorothioate, ^A E 1 s relative to ^{A s,} ^G ^e1s against ^{G s,} ^C e1s respect 5meC ^s, with respect to ^{T s} It may also be expressed as ^e1s.
4 _- a _'(CH 2) 2 -0-2' bridge and sugar modified nucleotides containing phosphorothioate, ^A E2S against ^{A s,} ^G ^e2s against ^{G s,} ^C e2s respect 5meC ^s, the ^{T s} On the other hand, it may be expressed as ^Te2s.

^{^{^{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, 5meC m2t, T m2t, A m2p, G m2p, 5meC m2p, T m2p, A m2s, G m2s , 5meC ^m2s , and T ^m2s are shown below.

As used herein, the treatment of a disease or symptom includes prevention of the onset of the disease, suppression or inhibition of exacerbation or progression, alleviation or exacerbation or suppression of progression of one or more symptoms exhibited by an individual suffering from the disease. Includes treatment of secondary illnesses.

2. The pharmaceutical composition of the present invention and the antisense oligonucleotide The present invention relates to a pharmaceutical composition for treating xeroderma pigmentosum F group (XP-F group) (hereinafter referred to as the pharmaceutical composition of the present invention), and Provided is an antisense oligonucleotide which is an active ingredient thereof or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the present invention contains an antisense oligonucleotide that suppresses abnormal post-transcriptional modifications caused by disease-causing mutations in the XP-F group, particularly abnormal post-transcriptional modifications caused by two types of novel intron internal mutations. By containing the mutation, it is possible to suppress a decrease in the expression level of the XPF gene in the XP-F group having the mutation and to treat the XP-F group having the mutation.

The two mutations that are the cause of the disease in the XP-F group newly identified by the present inventors are the introns in the base sequence of intron 1 of the wild XPF gene (base sequence 5217 to 6874 in the above NG_011442.1). The mutation from the 5'end of 1 to the 196th position (5412th in NG_011442.1) from T to A (corresponding to the 196th position in the nucleotide sequence represented by SEQ ID NO: 63), and the base of Intron 8 of the wild XPF gene. Mutation from C to T (base sequence represented by SEQ ID NO: 64) at position 326 from the 5'end of Intron 8 (position 20913 in NG_011442.1) in the sequence (base sequence 20588 to 22609 in the above NG_011442.1) It corresponds to the 326th in.). The former causes abnormal splicing of the XPF gene, and the latter causes abnormal polyadenylation of the XPF gene. As a result, the expression level of the XPF gene is reduced, which is considered to cause the symptoms of the XP-F group. Therefore, in one aspect, the application target of the pharmaceutical composition of the present invention is the XP-F group in which at least one of these two types of mutations is the cause of the disease. In the present invention, the XPF gene contains the above-mentioned mutation, and as long as the above-mentioned abnormal splicing or abnormal polyadenylation can occur, the XPF gene is further mutated (for example, deletion or substitution of one or several nucleotides). Addition or insertion) may be included.

The antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof has a base sequence capable of hybridizing with a part of the intron region of the XPF gene. As long as the abnormal post-transcriptional modification of the XPF gene can be suppressed, a part of the intron region, that is, the range of the target sequence is not particularly limited. Whether or not the antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress abnormal post-transcriptional modification of the XPF gene is described in the following Examples, Evaluation of XPF mRNA Splicing Product Amount, Protein. It is determined by the same evaluation method as the expression level evaluation or the repair activity evaluation. If either evaluation method confirms increased expression of the correctly post-transcriptionally modified XPF gene or recovery of DNA repair activity, the antisense oligonucleotide or its pharmaceutically acceptable salt is abnormally transcribed of the XPF gene. It is judged to suppress post-modification.

In one aspect, the pharmaceutical composition of the present invention applies to the XP-F group whose disease cause is the above-mentioned mutation in intron 1. The pharmaceutical composition of the present invention in this embodiment is referred to as the pharmaceutical composition I of the present invention. In the post-transcriptional modification of the XPF gene having the mutant intron 1 sequence represented by SEQ ID NO: 63, the pharmaceutical composition I of the present invention is located on the 5'side of the 193rd guanine in the nucleotide sequence represented by SEQ ID NO: 63. It contains one or more antisense oligonucleotides that suppress abnormal splicing using the 5'splice site and the 3'splice site on the 3'side of position 1658 guanine. In addition, "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 nucleotide sequence represented by SEQ ID NO: 63" is referred to as "SEQ ID NO: It can also be rephrased as "abnormal splicing in which the 1st to 192nd adenine in the base sequence represented by 63 is recognized as an exon and the 1658th guanine from the 193rd guanine is recognized as an intron".

Whether or not the antisense oligonucleotide in the pharmaceutical composition I of the present invention suppresses abnormal splicing in intron 1 is determined by the XPF mRNA splicing product amount evaluation, protein expression level evaluation, or repair activity evaluation described in the following Examples. It is determined by a similar evaluation method. If an increase in the expression of the correctly spliced XPF gene or a recovery in the DNA repair activity is confirmed by any of the evaluation methods, it is judged that the antisense oligonucleotide suppresses the abnormal splicing in the intron 1. In the XPF mRNA splicing product amount evaluation, whether or not correctly spliced XPF mRNA is increased by transfecting antisense oligonucleotide in cells derived from patients in the XP-F group whose disease is caused by the above mutation in intron 1. This is confirmed by Droplet digital PCR (ddPCR). In ddPCR, the concentration of the target in the sample is absolutely measured by dispersing the limitingly diluted cDNA in the micro-compartment, performing PCR amplification, and directly counting the number of micro-compartments in which the amplification signal is positive. The method. ddPCR can be performed using commercially available reagents and equipment (eg, QX100 ^TM Droplet Digital ^TM PCR system (Bio-Rad Laboratories, Inc.), etc.). In protein expression assessment, whether or not transfection of antisense oligonucleotides increases correctly spliced XPF gene products in cells derived from patients in the XP-F group whose disease is caused by the above mutation in intron 1. Is confirmed by Western blotting. In the repair activity evaluation, in cells derived from patients in the XP-F group caused by the above mutation in intron 1, the activity of repairing DNA damage caused by UV irradiation by transfecting an antisense oligonucleotide was found. Check if it recovers.

The antisense oligonucleotide in the pharmaceutical composition I of the present invention is not limited to a specific one as long as it suppresses abnormal splicing in intron 1, but for example, it is located at positions 155 to 217 (preferably) in the base sequence represented by SEQ ID NO: 63. Is a contiguous 15-30 nucleotides (preferably 15-23 nucleotides, more preferably 15) in the 175th-217th, more preferably 179th-213rd, even more preferably 183rd-203rd base sequence. A sequence consisting of (~ 18 nucleotides) can be targeted. For example, the base sequence targeted by the antisense oligonucleotide in the pharmaceutical composition I of the present invention is a base sequence represented by any one selected from SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135. Can be a sequence complementary to, preferably a sequence complementary to the base sequence represented by any one selected from SEQ ID NOs: 65-82, 101, and 105-108, more preferably a sequence. It can be a sequence complementary to the base sequence represented by any one selected from Nos. 65 to 82, and even more preferably the base sequence represented by any one selected from SEQ ID NOs: 75 to 78. Can be a complementary sequence to. In other words, the antisense oligonucleotide in the pharmaceutical composition I of the present invention can hybridize with the sequence (ie, the target sequence). In a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition I of the present invention can hybridize with the target sequence under highly stringent conditions.

The antisense oligonucleotide in the pharmaceutical composition I of the present invention may have a base sequence (tail sequence) that does not contribute to hybridization with the target sequence at its 5'end and / or 3'end. The number of bases contained in each tail sequence is 5 or less (preferably 4, 3, 2 or 1), and those having no tail sequence are optimal.

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

In a preferred embodiment, the antisense oligonucleotides in pharmaceutical composition I of the present invention are SEQ ID NOs: 65-82, 101-108, and 124-135 (preferably SEQ ID NOs: 65-82, 101, and 105-108, more preferably. Includes a base sequence represented by any one selected from SEQ ID NOs: 65-82, and even more preferably SEQ ID NOs: 75-78). The nucleotide sequences represented by SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135 are RNA sequences corresponding to SEQ ID NOs: 1 to 18, 53 to 60, and 112 to 123, respectively, which are described in Examples described later. It is described as. The uridine residue in the antisense oligonucleotide may be replaced with a modified nucleotide based on the corresponding thymidine residue. Therefore, the base sequence in which "u" is replaced with "t" in the base sequences represented by SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135 is also included in this embodiment.

In addition, the base sequence of the portion of the pharmaceutical composition I of the present invention excluding the tail sequence of the antisense oligonucleotide is 5 bases or less (preferably) with the target sequence as long as it has hybrid activity with the target sequence. There may be a mismatch of 4 bases or less, more preferably 3 bases, 2 bases or 1 base), but optimally there is no mismatch.

Here, the antisense oligonucleotide in the pharmaceutical composition I of the present invention is the recognition sequence GTAAGTAA of the 5'splice site newly generated in the mutant intron 1 sequence (No. 193 to 200 in the base sequence represented by SEQ ID NO: 63). It is considered that abnormal splicing is suppressed by inhibiting the binding of U1snRNP to (sequence). U1 snRNP has a sequence that is almost complementary to the recognition sequence at the 5'splice site, and base pairs are formed during the splicing process. Therefore, in a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition I of the present invention is at least one base of the 193 to 200th sequence in the base sequence represented by SEQ ID NO: 63, for example, 2 bases, 3 bases, 4 bases. Sequences containing 5, 6 bases, 7 bases, or 8 bases can be targeted.

The length of the antisense oligonucleotide in the pharmaceutical composition I of the present invention is not particularly limited as long as it suppresses abnormal splicing in intron 1, and is, for example, 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides. obtain.

In yet another aspect, the pharmaceutical composition of the present invention applies to the XP-F group whose disease cause is the above-mentioned mutation in intron 8. The pharmaceutical composition of the present invention in this embodiment is referred to as the pharmaceutical composition II of the present invention. In the post-transcriptional modification of the XPF gene having the mutant intron 8 sequence represented by SEQ ID NO: 64, the pharmaceutical composition II of the present invention adds polyA at positions 324 to 329 in the base sequence represented by SEQ ID NO: 64. Includes one or more antisense oligonucleotides that suppress aberrant polyadenylation using sequences.

Here, whether or not the antisense oligonucleotide in the pharmaceutical composition II of the present invention suppresses abnormal polyadenylation in intron 8 is determined by the same evaluation method as the protein expression level evaluation or repair activity evaluation described in the following Examples. It is determined. If an increase in the expression of the correctly spliced XPF gene or a recovery in the DNA repair activity is confirmed by any of the evaluation methods, it is judged that the antisense oligonucleotide suppresses the abnormal polyadenylation in the intron 8. In protein expression assessment, transfection of antisense oligonucleotides increases the polyadenylated XPF gene product at the correct location in cells from patients in the XP-F group whose disease is caused by the mutation in intron 8. Check if it is. In the evaluation of repair activity, cells derived from patients in the XP-F group caused by the above mutation in intron 8 were found to have the activity of repairing DNA damage caused by UV irradiation by transfecting antisense oligonucleotides. Check if it recovers.

The antisense oligonucleotide in the pharmaceutical composition II of the present invention is not limited to a specific one as long as it suppresses abnormal polyadenylation in intron 8, but for example, the 309th to 343rd nucleotides in the nucleotide sequence represented by SEQ ID NO: 64. A sequence consisting of 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides in a sequence within a base sequence can be targeted. In other words, the antisense oligonucleotide in the pharmaceutical composition II of the present invention can hybridize with the above sequence (ie, the target sequence). In a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition II of the present invention can hybridize with the target sequence under highly stringent conditions.

The antisense oligonucleotide in the pharmaceutical composition II of the present invention may have a base sequence (tail sequence) that does not contribute to hybridization with the target sequence at its 5'end and / or 3'end. The number of bases contained in each tail sequence is 5 or less (preferably 4, 3, 2 or 1), and those having no tail sequence are optimal.

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

In a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition II of the present invention comprises the base sequence represented by SEQ ID NOs: 83-100. The base sequences represented by SEQ ID NOs: 83 to 100 are described as RNA sequences corresponding to SEQ ID NOs: 19 to 36 described in Examples described later. The uridine residue in the antisense oligonucleotide may be replaced with a modified nucleotide based on the corresponding thymidine residue. Therefore, the base sequence in which "u" is replaced with "t" in the base sequence represented by SEQ ID NOs: 83 to 100 is also included in this embodiment.

In addition, the base sequence of the portion of the pharmaceutical composition II of the present invention excluding the tail sequence of the antisense oligonucleotide is 5 bases or less (preferably) with the target sequence as long as it has hybrid activity with the target sequence. There may be a mismatch of 4 bases or less, more preferably 3 bases, 2 bases or 1 base), but optimally there is no mismatch.

Here, the antisense oligonucleotide in the pharmaceutical composition II of the present invention is a polyA addition sequence AATAAA newly generated in the mutant intron 8 sequence (sequences 324 to 329 in the base sequence represented by SEQ ID NO: 64). It is thought that abnormal polyadenylation is suppressed by inhibiting cleavage and binding of polyadenylation factor (CPSF) to. Therefore, in a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition II of the present invention is at least one base of the 324 to 329th sequence in the base sequence represented by SEQ ID NO: 64, for example, 2 bases, 3 bases, 4 bases. Sequences containing 5, or 6 bases can be targeted.

The length of the antisense oligonucleotide in the pharmaceutical composition II of the present invention is not particularly limited as long as it suppresses abnormal polyadenylation in intron 1, but is, for example, 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides. possible.

The antisense oligonucleotide in the pharmaceutical composition of the present invention may be any of DNA, RNA, and DNA / RNA chimera. By making it a DNA / RNA chimera, the nuclease resistance of the antisense oligonucleotide can be increased and the stability in vivo can be enhanced.

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

In a preferred embodiment, the antisense oligonucleotide comprises 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 sugar-modified nucleosides containing modifications of different sugar moieties. The sugar-modified nucleoside is _{preferably a nucleoside containing 4'-(CH 2} ) _n- O-2'cross-linking (where n is 1 or 2 in the formula) or 2'-O-methylation. -(CH ₂ ) ₂ -O-2'crosslinking or 2'-O-methylation-containing nucleosides are more preferred. When the antisense oligonucleotide contains a sugar-modified nucleoside containing a 4'-(CH ₂ ) _2- O-2'crosslink, the number is not particularly limited, but one or more, for example, two or more, or three. It can be 4 or more, 5 or more, or 6 or more, 12 or less, for example, 11 or less, 10 or less, or 9 or less.

In a more preferred embodiment, all of the nucleosides contained in the antisense oligonucleotide are sugar-modified nucleosides, where the sugar-modified nucleoside is a _{sugar-modified nucleoside containing a 4'-(CH 2} ) _2- O-2'bridge, 2'-. A sugar-modified nucleoside containing O-methylation, or a combination thereof.

In a preferred embodiment, the antisense oligonucleotide comprises one or more modified nucleoside linkages. Different modified nucleoside linkages may be included in the antisense oligonucleotide. Further, all the nucleoside linkages in the antisense oligonucleotide may be modified nucleoside linkages. In a more preferred embodiment, the modified nucleoside bond is a phosphorothioate bond. In an even more preferred embodiment, all nucleoside linkages in the antisense oligonucleotide are modified nucleoside linkages and the modified nucleoside linkages are phosphorothioate linkages.

In a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition I of the present invention is selected from the group consisting of oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 1-18, 37-60 and 112-123. The nucleosides contained in these oligonucleotides are _{either sugar-modified nucleosides containing 4'-(CH 2} ) ₂ -O-2'crosslinks or sugar-modified nucleosides containing 2'-O-methylation. Moreover, all the nucleoside-to-nucleoside bonds in these oligonucleotides are phosphorothioate bonds. More suitable antisense oligonucleotides in the pharmaceutical composition I of the present invention are the oligonucleotides shown below.

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 (SEQ ID NO: 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 (SEQ ID NO: 14)
Example 45 (XPF-int1-013_1): HO-C ^m1s - ^Te2s- 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 - ^Te2s- U ^m1t- H (SEQ ID NO: 45)
Example 46 (XPF-int1-013_2): 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- U ^m1s- G ^m1s - ^Te2s- U ^m1t- H (SEQ ID NO: 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 (SEQ ID NO: 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 (SEQ ID NO: 113)

In a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition II of the present invention is selected from the group consisting of oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 19 to 36. The nucleosides contained in these oligonucleotides are _{either sugar-modified nucleosides containing 4'-(CH 2} ) ₂ -O-2'crosslinks or sugar-modified nucleosides containing 2'-O-methylation. Moreover, all the nucleoside-to-nucleoside bonds in these oligonucleotides are phosphorothioate bonds.

The method for preparing the antisense oligonucleotide is not particularly limited, but a known chemical synthesis method (phosphoric acid triester method, phosphoramidite method, H-phosphonate method, etc.) can be used. It may be synthesized by using a commercially available nucleic acid synthesizer and using a commercially available reagent used for DNA / RNA synthesis. For example, after coupling a phosphoramidite reagent, a reagent such as sulfur, tetraethylthiura disulfide (TETD, Applied Biosystem), Beaucage reagent (Glen Research), or xanthan hydride is reacted to have a phosphorothioate bond. Antisense oligonucleotides can be synthesized (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

The oligonucleotide (antisense oligonucleotide) of the present invention may have a group having a desired chemical structure at the 5'end and / or 3'end to control the physical properties and pharmacokinetics of the oligonucleotide. For example, an aminoalkyl group can be introduced at the 5'end and / or 3'end of the oligonucleotide (antisense oligonucleotide) of the present invention, through which the desired chemical structure can be added.

The introduction of the aminoalkyl group into the oligonucleotide may be carried out by a method known in the art, or may be carried out using a commercially available reagent. For example, after the strand extension of the oligonucleotide having the target sequence is completed, 5'-Amino-Modifier C6 (Glen Research), 5'-TFA-Amino-Modifier C6-CE Phosphoramidite, 5'-TFA-Amino-Modifier- By reacting with an amino modification reagent such as C5-CE Phosphoramidite (Link Technologies), an oligonucleotide in which an aminoalkylphosphate group is bonded to the 5'end can be synthesized. Further, by reacting with an amino modification reagent such as 3'-amino-Modifier C3 CPG and 3'-amino-Modifier C7 CPG (Glen Research), an oligonucleotide in which an aminoalkyl group is bonded to the 3'end can be synthesized.

As a chemical structure that can be introduced into the 5'end and / or 3'end of the oligonucleotide (antisense oligonucleotide) of the present invention, it is known in the art that the physical properties and pharmacokinetics of the oligonucleotide can be controlled. Any chemical structure can be utilized, including, for example, fatty acids, cholesterol, GalNAc structures and the like. For example, antisense oligonucleotides can be synthesized using amidite compounds containing fatty acids that are useful for the transfer of nucleic acids to muscle tissue, and the transfer of antisense oligonucleotides to muscle tissue can be promoted. Known (eg, Link Technologies products such as Nucleic Acids Res. (2020) 47, 6029-6044, 5'-Palmitate-C6-CE Phosphoramidite). In addition, it is described in publicly known literature (for example, Mol. Cancer Ther. (2018) 17, 1251-1258) that cholesterol-bound siRNA acts in the brain. 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 to hepatocytes, and that antisense oligonucleotides can be specifically delivered to hepatocytes. (For example, Methods in Enzymology, 1999, 313, 297-321, WO2009 / 073809, WO2014 / 076196, WO2014 / 179620, WO2015 / 006740, WO2015 / 105083, WO2016 / 055601, WO2017 / 023817, WO2017 / 084987, WO2017 / 131236, WO2019 / 172286, etc.).

The antisense oligonucleotide in the pharmaceutical composition of the present invention may be in the form of a pharmaceutically acceptable salt thereof. "The pharmaceutically acceptable salt" means a salt of an oligonucleotide (antisense oligonucleotide), and examples of such a salt include alkali metal salts such as sodium salt, potassium salt and lithium salt, calcium salt and magnesium. Alkaline earth metal salts such as salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts and other metal salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts , Morphorine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocine salt, Amine salts such as prokine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylammonium salts, organic salts such as tris (hydroxymethyl) aminomethane salts; hydrofluorates, hydrochlorides, bromide Hydrochloride, halogenated hydrorates such as hydroiodide, inorganic acid salts such as nitrates, perchlorates, sulfates, phosphates; methanesulfonates, trifluoromethanesulfonates, ethanesulfonic acid Lower alkane sulfonates such as salts, benzene sulfonates, aryl sulfonates such as p-toluene sulfonates, acetates, apple salts, fumarates, succinates, citrates, tartrates , Organic salts such as oxalate, maleate; amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, asparaginate and the like. These salts can be produced by known methods.

In addition, the antisense oligonucleotide of the pharmaceutical composition of the present invention and a pharmaceutically acceptable salt thereof may also exist as a solvate (for example, a hydrate), and even such a solvate may exist. Good.

The pharmaceutical composition of the present invention can be formulated by mixing an antisense oligonucleotide and an appropriate pharmaceutically acceptable additive. For example, the pharmaceutical composition of the present invention can be administered orally as a preparation such as tablets, capsules and granules, or parenterally as a preparation such as an injection and a transdermal absorbent.

These formulations are excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, diluents, solvents for injections, solubilizers, suspending agents, isotonic agents, buffers, painless It can be produced by a well-known method using additives such as an agent, a preservative, and an antioxidant.

Examples of excipients include organic excipients and inorganic excipients. Examples of the organic excipient 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 the inorganic excipient include sulfates such as calcium sulfate.

Examples of the binder include the above-mentioned excipients; gelatin; polyvinylpyrrolidone; polyethylene glycol and the like.

Examples of the disintegrant include the above-mentioned excipients; chemically modified starch or cellulose derivatives such as croscarmellose sodium and sodium carboxymethyl starch; and crosslinked polyvinylpyrrolidone.

Lubricants include, for example, talc; stearic acid; colloidal silica; bead wax, waxes such as gay wax; sulfates such as sodium sulfate; lauryl sulfates such as sodium lauryl sulfate; in the above excipients. Examples include starch derivatives.

The emulsifiers include, for example, colloidal clays such as bentonite, beagum; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ethers. Examples include ionic surfactants.

Examples of stabilizers include parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol; phenols and phenols such as cresol.

Examples of the diluent include water, ethanol, propylene glycol and the like.

Examples of the solvent for injection include water, ethanol, glycerin and the like.

Examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

As the suspending agent, for example, surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, carboxy Examples thereof include hydrophilic polymers such as sodium methylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

Examples of the tonicity agent include sodium chloride, glycerin, D-mannitol and the like.

Examples of the buffer include a buffer solution such as phosphate, acetate, carbonate, and citrate.

Examples of the pain-relieving agent include benzyl alcohol and the like.

Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

Examples of antioxidants include sulfites and ascorbic acid.

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

The route of administration of the pharmaceutical composition of the present invention may be either oral administration or parenteral administration, and a suitable administration route may be selected according to the target symptomatology. The route of administration may be either systemic administration or topical administration. Examples of parenteral administration include intravenous administration, intraarterial administration, intrathecal administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intra-articular administration. be able to. For example, transdermal and subcutaneous administration may be selected for cutaneous symptoms, and intrathecal administration may be selected for neurological symptoms.

The pharmaceutical composition of the present invention is administered to a subject in a therapeutically effective amount. "Therapeutic effective amount" means the amount that exerts a therapeutic effect for a specific disease, administration form and administration route, depending on the target species, type of disease, symptom, gender, age, chronic disease, and other factors. Will be decided as appropriate.

The dose of the pharmaceutical composition of the present invention can be appropriately determined according to the target species, the type of disease, symptoms, gender, age, chronic disease, and other factors.

The pharmaceutical composition of the present invention may be used in combination with at least one known therapeutic agent or method.

The present invention also provides a method for treating xeroderma pigmentosum F group, which comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.

The "therapeutic effective amount" in the present invention means an amount that exerts a therapeutic effect for a specific disease or symptom, administration form and administration route, and refers to a target species, disease or symptom type, symptom, sex, age, chronic disease, etc. It is determined as appropriate according to other factors.

Hereinafter, the present invention will be specifically described with reference to Examples. It should be noted that these examples are for explaining the present invention and do not limit the scope of the present invention.

(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 performed using an automatic nucleic acid synthesizer (MerMade 192X manufactured by BioAutomation) and a phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984). The reagent was activator solution-3 (0.25 mol / L). 5-benzylthio-1H-Tetrazole / acetonitrile solution, manufactured by Wako Pure Chemical Industries, Ltd., product No. 013-20011), CAP A for AKTA (1-methylimidazole / acetonitrile solution, manufactured by Sigma-Aldrich, product No. L040050), Cap B1 for AKTA (anhydrous acetic acid / acetonitrile solution, manufactured by Sigma-Aldrich, product No. L050050), Cap B2 for AKTA (pyridine / acetonitrile solution, manufactured by Sigma-Aldrich, product No. L050150), DCA Deblock (dichloroacetic acid / toluene solution) , Sigma-Aldrich, product No. L023050) was used. As a thiolation reagent for forming a phosphorothioate bond, phenylacetyldisulfide (CARBOSYNTH, product No. FP07495) was dehydrated to 0.2 M. , Kanto Kagaku, product No. 01837-05), pyridine (dehydration, Kanto Kagaku, product No. 11339-05) 1: 1 (v / v) solution was used to dissolve and use. , 2'-O-Me nucleoside phosphoramidite (adenosine product No. ANP-5751, citidine product No. ANP-5752, guanosine product No. ANP-5753, uridine product No. ANP-5754) The one manufactured by ChemGenes was used. The non-natural phosphoramidite used was Example 14 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-6-N-) of JP-A-2000-297097. Benzoyladenosine-3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite), Example 27 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-2-N -Isobutyryl guanosine-3'-O- (2-Cyanoe) Phosphoramidite), Example 22 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'- O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite), Example 9 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-5-methyluridine-3'-O -(2-Cyanoethyl N, N-diisopropyl) phosphoramidite), compound was used. The compound of Example 1 was synthesized using Glen Unysupport FC 96-well format 0.2 μmol (manufactured by Glen Research) as a solid phase carrier. However, the time required for condensation of the amidite compound was about 9 minutes.

The oligomer was excised from the support by treating the protected oligonucleotide analog having the target sequence with 600 μL of concentrated aqueous ammonia, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed. A mixed solution of oligomers was mixed with 300 μL of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex) and charged onto a Clarity SPE 96 well plate (manufactured by Phenomenex). Clarity QSP DNA Loading Buffer: Water = 1: 1 solution 1 mL, water 3 mL, 3% dichloroacetic acid (DCA) aqueous solution 3 mL, water 6 mL in that order, then 20 mM Tris aqueous solution: acetonitrile = 9: 1 solution The components extracted in the above were collected. After distilling off the solvent, the target compound was obtained. This compound is a reverse phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100A (2.1 × 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), aqueous solution 8 mM triethylamine, solution B: methanol, B. %: 10% → 25% (4min, linear gradient); 60 ° C; 0.5mL / min; 260nm), elution was performed at 2.78 minutes. Compounds were identified by negative ion ESI mass spectrometry.

The base sequence of this compound is the 412th in the mRNA encoded by Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4) (NCBI-GenBank accession No. NG_011442.1). T is a sequence complementary to the 412-429th sequence of the sequence in which T is mutated to A.

(Examples 2 to 36)
The compounds of Examples 2 to 36 were also synthesized in the same manner as the compounds of Example 1. Information on the compounds of Examples 1 to 36 is given in Table 1.

In the "sequence" in the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMeRNA. All bonds between nucleosides 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" of Example 17 indicates a sequence in which "c" is added to the 3'end of a sequence complementary to the base sequence from "start" to "end". "Molecular weight" in the table indicates the measured value by negative ion ESI mass spectrometry.

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

In the "sequence" in the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMeRNA. All bonds between nucleosides are phosphorothioate bonds. “Start” and “end” in the table indicate the nucleotide number of the base sequence represented by SEQ ID NO: 63, and “sequence” in the table is complementary to the base sequence from “start” to “end”. Shows the sequence. "Molecular weight" in the table indicates the measured value by negative ion ESI mass spectrometry.

(Example 61)
HO-C ^e1s -C ^s -T ^e1s -T ^s -A ^e1s -C ^s -T ^e1s -T ^s -A ^e1s -C ^s -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 compound described in WO99 / 14226. , Or, in the figure, it may be expressed as "LNA".

This compound is a reverse phase HPLC (column (X-Bridge C18 2.5 μm (4.6 × 75 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), aqueous solution of 8 mM triethylamine, solution B: methanol, B%: 5 When analyzed at% → 30% (20 min, linear gradient); 60 ° C; 1 mL / min; 260 nm), elution was performed at 10.23 minutes. The compound was identified by negative ion ESI mass spectrometry (measured value: 4927.26).

(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 figure, it may be expressed as "control DNA".

This compound is a reverse phase HPLC (column (X-Bridge C18 2.5 μm (4.6 × 75 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), aqueous solution of 8 mM triethylamine, solution B: methanol, B%: 5 When analyzed at% → 30% (20 min, linear gradient); 60 ° C; 1 mL / min; 260 nm), elution was performed at 10.46 minutes. The compound was identified by negative ion ESI mass spectrometry (measured value: 4791.16).

(Test Example 1) Analysis of XPF mRNA splicing product amount, XPF protein expression level, and DNA damage repair activity by Example compounds (1)
1-1. Evaluation of XPF mRNA splicing product amount Patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7 (6), 969-974) were seeded in ^{5x10 4 cells per well on a 6-well plate.} After 24 hours, LNA was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 40 nM. After 4 hours, the medium was replaced with new medium (DMEM 10% FBS). Cells were harvested 24 hours after transfection. Total RNA was extracted with Direct-zol ^TM RNA MiniPrep (ZYMO RESEARCH). CDNA was obtained from 100 ng of total RNA using SuperScript® IV (Thermo Fisher Scientific). Droplet ^{digital PCR (ddPCR) was performed on the QX100 TM} Droplet Digital ^TM PCR system (Bio-Rad Laboratories, Inc.) to quantify splicing products. Similarly, splicing products were quantified for healthy human-derived fibroblasts (48BR).

ddPCR was performed according to the following procedure. QX200 Eva Green ddPCR Supermix (Bio-Rad Laboratories, Inc.) was added to 10 μl, and Forward primer and Reverse primer were added to a final concentration of 100 nM, respectively. Sample cDNA (total RNA adjusted to 100 ng) was added to 5 μl, and an appropriate amount of water was added to bring the total volume to 20 μl. QX200 ^TM / QX100 put the sample and 70 μl of Droplet Generation oil for EvaGreen to DG8 cartridge for ^TM Droplet Generator was subjected to QX100 ^TM Droplet Generator. The turbid solution was placed in a 96-well plate (Bio-Rad Laboratories, Inc.) and sealed using a PX1 PCR plate sealer. Analysis was performed using the QX100 ^{TM Droplet Reader.} For the correct quantification of splicing products, the value obtained by ddPCR using a combination of primers of XPF ex1-F and XPF ex2-R was used, and for the quantification of abnormal splicing products, XPF int1-F and XPF ex2- The value obtained by ddPCR using the combination of R primers was used.
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)

The results are shown in Fig. 1A. As a result of analyzing the mRNA of patient-derived cells, the number of correctly spliced products was small, whereas in the cells transfected with the compound of Example 61 (LNA-int1-001), the number of correctly spliced products increased. Admitted.

1-2. Protein expression evaluation Patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7 (6), 969-974) were seeded on ^{6-well plates at a rate of 16x10 4 cells per well.} After 24 hours, the Example compound or Reference Example compound was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 40 nM (the transfection method was as described in the product instructions). After 4 hours, the medium was replaced with new medium. Twenty-four hours after transfection, protein was recovered and Western blotting (using XPF Ab-1 (Cat. # MS-1381, Thermo Fisher Scientific) and β-Actin antibody (sc-47778, Santa Crus)). The protein expression level was examined in.

The results are shown in FIG. 1B. As a result of analyzing the amount of XPF protein in patient-derived fibroblasts, it was lower than that in healthy human-derived fibroblasts (48BR). An increase in the amount of XPF protein was observed in cells transfected with the compound of Example 61 (LNA-int1-001). On the other hand, no increase in the amount of XPF protein was observed in the cells transfected with the compound of Reference Example 1 (LNA-int1-001S, control DNA). Further, as shown in FIG. 2B, (XPF-int1-001 to XPF-int1-018) were similarly transfected into patient-derived cells using the compounds of Examples 1 to 18 to determine the amount of XPF protein. As a result of the analysis, an increase in the amount of XPF protein was observed in all the compounds of Examples 1 to 18. In particular, when the compounds of Examples 11 to 14 were used, the amount of increase was large.

1-3. Evaluation of repair activity 96 patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7 (6), 969-974) were seeded in ^{1x10 4 cells per well.} Cells derived from the same patient were seeded in a total of 10 wells (5 wells x 2 sets). After 24 hours, the Example compound or Reference Example compound was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 40 nM (the transfection method was as described in the product instructions). After 4 hours, the medium was replaced with new medium (DMEM 10% FBS). Twenty-four hours after transfection, 5 well x 1 set was ^{irradiated with 20 J / m 2} of UV-C (the remaining 5 well x 1 set was unirradiated). The medium was discarded, washed once with PBS, the PBS was discarded, and then UV irradiation was performed with no liquid in the well. Serum-free medium containing 5-ethynyl-2'-deoxyuridine (EdU) at a final concentration of 5 μM was added to the cells after UV irradiation. After culturing in medium containing EdU for 4 hours, the cells were washed once with PBS, fixed solution (300 mM sucrose, 2% formalin, 0.5% TritonX-100 PBS solution) was added, and the cells were allowed to stand at 4 degrees for 20 minutes. The cells were fixed. The cells were washed 3 times with PBS. Add stain (50 mM Tris pH 7.3, ₄ mM CuSO 4, 10 mM Sodium L-ascorbate, 10 μM Fluorescent dye 488 azide, 0.03 mg / ml DAPI aqueous solution), and leave at room temperature for 1 hour. did. Cells were washed 3 times with PBS-T (0.05% tween 20 PBS solution). Fixative (3.7% formalin PBS solution) was added and allowed to stand for 20 minutes. The fixative was discarded and PBS was added. The average fluorescence intensity of 488 azide in the nucleus was calculated. The fluorescence value indicates the repair activity. The repair activity of healthy human-derived fibroblasts (48BR) was also evaluated in the same manner.

The result is shown in Fig. 1C. As a result of analyzing the repair activity of patient-derived cells, it was lower than that of healthy human-derived cells. The patient-derived cells transfected with the compound of Example 61 (LNA-int1-001) had a repair activity as compared with the patient-derived cells transfected with the compound of Reference Example 1 (LNA-int1-001S, control DNA). Was observed to increase. Further, as shown in FIG. 2A, the compounds of Examples 1 to 18 (XPF-int1-001 to XPF-int1-018) were similarly transfected into patient-derived cells and the repair activity was analyzed. An increase in repair activity was observed in all the compounds of Examples 1 to 18.

(Examples 62 to 73)
The compounds of Examples 62 to 73 were also synthesized in the same manner as in Example 1. Examples 62 to 73 are shown in Table 3.

In the "sequence" in the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMeRNA. All bonds between nucleosides are phosphorothioate bonds. "Start" and "End" in the table are the 412th T of Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4), RefSeqGene (LRG_463) on chromosome 16 (NCBI-GenBank accession No. NG_011442.1). The nucleotide number of the sequence mutated to A is shown, and the "sequence" in the table indicates a sequence complementary to the nucleotide sequence from "start" to "end". "Molecular weight" in the table indicates the measured value by negative ion ESI mass spectrometry.

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

In the "sequence" in the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMeRNA. All bonds between nucleosides are phosphorothioate bonds. The sequence of the compound of Reference Example 2 is a sequence complementary to the nucleotide sequence from the 223rd to the 240th of Musmusculus strain mdx dystrophin genes, partial cds (NCBI-GenBank accession No. AH007099.2).

(Test Example 2) Analysis of XPF mRNA splicing product amount, XPF protein expression level, and DNA damage repair activity by Example compounds (2)
2-1. Evaluation of protein expression level The protein expression level of the example compound was evaluated in the same manner as in the test 1-2. Compounds of Examples 37, 38, 44-46, and 53-60 (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, XPF-int1-19-22) and the compounds of Examples 11 and 14 (XPF-int1- 11 and XPF-int1-14) were transfected into patient-derived cells and the amount of XPF protein was analyzed. As a negative control, the compound of Reference Example 2 was used. The results are shown in FIG. 3B. The compounds of Examples 37, 38, 44 to 46, 53, 57 to 60, 11, and 14 did not contain the negative control (nega con. In the figure) and the compound of Example (w / in the figure). An increase in the amount of XPF protein was observed compared to o).
2-2. Evaluation of repair activity The repair activity of the example compound was evaluated in the same manner as in the tests 1-3. Compounds of Examples 37 to 60 (XPF-int1-011_1-4, XPF-int1-012_1-4, XPF-int1-013_1-4, XPF-int1-014_1-4, XPF-int1-0-1- 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 their repair activity was analyzed. As a negative control, the compound of Reference Example 2 was used. The results are shown in FIG. 3A. The compounds of Examples 37 to 53, 57 to 60, and 11 to 14 were compared with those to which the negative control (nega con. In the figure) and the compound of Example were not added (w / o in the figure). An increase in repair activity was observed.

The pharmaceutical composition of the present invention can treat a specific XP-F group for which there has been no conventional therapeutic means.

SEQ ID NO: 1 to 61: oligonucleotide sequence of Examples Compounds 1 to 61 SEQ ID NO: 62: oligonucleotide sequence of Reference Example Compound 1 SEQ ID NO: 63: RNA sequence of XPF gene variant Intron 1 SEQ ID NO: 64: Mutation of XPF gene DNA sequence of type intron 8 SEQ ID NO: 65-100: RNA sequence corresponding to SEQ ID NO: 1-36 SEQ ID NO: 101-108: RNA sequence corresponding to SEQ ID NO: 53-60 SEQ ID NO: 109-111: Primer used for ddPCR DNA Sequence SEQ ID NOs: 112 to 123: Oligonucleotide Sequence of Examples Compounds 62 to 73 SEQ ID NOs: 124 to 135: RNA Sequence 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 Am1s, Gm1s, Cm1s, Um1s, Ae2s, Ge2s, Ce2s, Te2s, Ae1s, Ge1s, Ce1s, Te1s, Cs, Ts, Am1t, Gm1t, Cm1t, Um1t G1t and C1t are A ^m1s , G ^m1s , C ^m1s , U ^m1s , A ^e2s , G ^e2s , C ^e2s , ^Te2s , A ^e1s , G ^e1s , C ^e1s , ^Te1s , C ^s , T ^s , A, respectively. ^{Represents m1t} , G ^m1t , C ^m1t , U ^m1t , T ^2t , G ^1t , and C ^1t .

Claims

An antisense oligonucleotide having a base sequence capable of hybridizing with a part of the intron region of the XPF gene and having an activity of suppressing abnormal post-transcriptional modification of the XPF gene, at the 5'end and / or An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which may be chemically modified at the 3'end.
In the post-transcription modification of the XPF gene having the mutant intron 1 sequence represented by SEQ ID NO: 63, the 5'splice site on the 5'side of the 193rd guanine and the 1658th guanine in the nucleotide sequence represented by SEQ ID NO: 63. The antisense oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, which suppresses abnormal splicing using the 3'splice site on the 3'side of.
The antisense oligonucleotide according to claim 1 or 2, which can hybridize with a sequence consisting of consecutive 15 to 30 nucleotides in the 155th to 217th base sequences in the base sequence represented by SEQ ID NO: 63. Or its pharmaceutically acceptable salt.
Any one of claims 1 to 3, which comprises a sequence consisting of 90% or more complementary to a sequence consisting of consecutive 15 to 30 nucleotides in the 155th to 217th base sequences in the base sequence represented by SEQ ID NO: 63. The antisense oligonucleotide described in the section or a pharmaceutically acceptable salt thereof.
The antisense according to any one of claims 1 to 4, which comprises the base sequence represented by any of SEQ ID NOs: 65 to 82 and 101 to 108 (u may be replaced with t in the sequence). Oligonucleotides or pharmaceutically acceptable salts thereof.
The antisense oligonucleotide according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides.
6. The sugar-modified nucleoside is a nucleoside comprising 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) or 2'-O-methylation. Antisense oligonucleotide or pharmaceutically acceptable salt thereof.
The antisense oligonucleotide according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
The antisense oligonucleotide of claim 8 or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
The antisense oligonucleotide according to any one of claims 1 to 9, which comprises the base sequence represented by any of SEQ ID NOs: 1 to 18, 37 to 60 and 112 to 123, or a pharmaceutically acceptable salt thereof. ..
Post-transcriptional modification of the XPF gene having the mutant intron 8 sequence represented by SEQ ID NO: 64 suppresses abnormal polyadenylation using the 324th to 329th polyA addition sequences in the nucleotide sequence represented by SEQ ID NO: 64. , The antisense oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof.
The antisense oligonucleotide according to claim 1 or 11, which can hybridize with a sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64. Or its pharmaceutically acceptable salt.
Any of claims 1, 11 and 12, which comprises a sequence consisting of 90% or more complementary to a sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to the above item.
The antisense oligonucleotide according to any one of claims 1 and 11 to 13, which comprises the base sequence represented by any of SEQ ID NOs: 83 to 100 (u may be replaced with t in the sequence). Or its pharmaceutically acceptable salt.
The antisense oligonucleotide according to any one of claims 1 and 11 to 14, or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides.
15. The sugar-modified nucleoside is a nucleoside comprising 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) and 2'-O-methylation. Antisense oligonucleotide or pharmaceutically acceptable salt thereof.
The antisense oligonucleotide according to any one of claims 1 and 11 to 16, or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
The antisense oligonucleotide of claim 17, or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
The antisense oligonucleotide according to any one of claims 1 and 11 to 18, or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by any of SEQ ID NOs: 19 to 36.
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 transporting the oligonucleotide.
A pharmaceutical composition for treating xeroderma pigmentosum F group, which comprises the antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof.
A method for treating xeroderma pigmentosum F group, which comprises administering to a patient an effective amount of the antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof.
The antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof for use in the treatment of xeroderma pigmentosum group F.
Use of the antisense oligonucleotide according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, for producing a therapeutic pharmaceutical composition for xeroderma pigmentosum group F.