TW202134438A - Xeroderma pigmentosum group f therapeutic agent - Google Patents

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 treatment drug

The present invention relates to a therapeutic drug for xeroderma pigmentosum group F, an antisense oligonucleotide of its active ingredient, or a pharmaceutically acceptable salt thereof, and the like.

Xeroderma pigmentosum (XP) is caused by a congenital abnormality of the following gene: it is involved in the transfer of nucleotides that are caused by ultraviolet light in the sun, such as photo-DNA damage, etc., removed from the gene body. In addition to repair mechanism (NER), or damage bypass (damage bypass) synthesis (TLS, transiesion synthesis (cross-damage synthesis)) genes. In addition to photosensitivity, abnormal pigmentation in the sun-exposed area, and a high incidence of cancer, XP patients have neurological symptoms. It is estimated that the prevalence of Japanese is about 1 in 25,000 births. So far, in terms of genes reported to be the cause of disease mutations, it is known that in addition to XPA to XPG genes, there are POLH genes encoding TLS polymerase. XP cases with their respective mutations are classified into the XP-A～XP-G group and the XP-V (variant) group complementary group. The proportion of each complementary group in XP cases varies according to race, but in the Japanese group, the abnormalities in the XP-A group are more common, followed by the abnormalities in the XP-V group.

Among the complementation groups of XP, in addition to the variant group that becomes TLS defect, the defect of NER is shown. NER is divided into whole-genome repair (GG-NER) and transcriptional conjugate repair (TC-NER) according to the recognition pattern of DNA damage. In the XP-C/E group, only the defect of GG-NER is displayed. In the XP-A/B/D/F/G group, the defect of both GG-NER and TC-NER is displayed. In XP cases, DNA damage accumulates in the gene body due to the defect of these NERs, which leads to the instability of the gene body, and skin cancer is more likely to occur in young people. Neurological symptoms vary depending on the complementary group or disease cause variation, but especially in cases with group A Japanese founder variation (IVS3-1G＞C), loss of NER due to loss of XPA protein expression All functions, and show severe neurological symptoms.

XP is a systemic genetic disease, and effective treatments have not yet been developed. Most of them started with severe sunburn in infants and went to the dermatologist to see XP, so they were diagnosed with XP. The approximate prognosis is also determined by genetic examination and so on. It can prevent skin cancer from being exposed to the sun, but there is no effective way to alleviate the neurological symptoms that have a major impact on the prognosis and QOL.

Regarding the diagnosis of XP, it is known to use a DNA repair activity assay of fibroblasts established from a patient's skin patch. Among them are: Unscheduled DNA synthesis (UDS) test that measures the activity of GG-NER by detecting repaired DNA synthesis after removal of light DNA damage, and by examining RNA in regions with high transcriptional activity after DNA damage A method for evaluating TC-NER activity by synthesizing recovery energy (RRS) (Non-Patent Documents 1 and 2). Evaluate UDS/RRS on patient-derived cells, and investigate the known XP genes in which these activities are restored, and the complementarity group can be specified (Non-Patent Documents 3 and 4).

Regarding genetic mutations maintained by patients in the XP-F group, in Non-Patent Document 5, mutations in the region outside the XPF gene (ERCC4) are reported as individual mutations in patients. However, there is no disclosure about the variation in the intron region of the XPF gene. [Prior Technical Literature] [Non-Patent Literature]

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

[The problem to be solved by the invention]

Regarding the XP-F group, there are cases of unspecified disease-causing mutations, and its treatment has not been possible so far. The purpose of the present invention is to provide a treatment method for this XP-F group. [Means to solve the problem]

The inventors of the present invention have identified two types of novel internal intron mutations that are the cause of disease in patients in the XP-F group. The first is a novel Japanese founder mutation, which exists deep in the intron (about 100 bp downstream from the exon 1-intron 1 boundary) and shows that it is caused by the low molecular weight ribonucleoprotein (U1 snRNP) in the U1 nucleus. ) The binding sequence is newly generated, which causes a decrease in gene expression caused by abnormal alternative splicing. The second type is a 1-base substitution mutation in the deep intron (hundreds of bp downstream from the exon 8-intron 8 boundary), which forms a pathological poly A additional sequence (cleaved and polyadenosine). Acidification factor binding sequence), and showed that the onset of disease caused by incomplete transcription termination and mRNA instability. The inventors of the present invention have further intensively studied and completed the present invention.

That is, the present invention includes the following inventions. [1] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which has a base sequence that can hybridize to a part of the intron region of the XPF gene, and has an abnormal post-transcriptional modification that inhibits the XPF gene The 5'end and/or 3'end of the active antisense oligonucleotide can be chemically modified. [2] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in [1], in the post-transcriptional modification of the XPF gene having the variant intron 1 sequence represented by SEQ ID NO: 63, Suppresses abnormal splicing using the 5'splice site on the 5'side of guanine No. 193 and the 3'splice site on the 3'side of guanine No. 1658 in the base sequence represented by SEQ ID NO: 63. [3] The antisense oligonucleotide as described in [1] or [2] or a pharmaceutically acceptable salt thereof, which can be combined with the 155th to 217th nucleotide sequence represented by SEQ ID NO: 63 The sequence of 15-30 nucleotides in the base sequence of the number hybridizes. [4] The antisense oligonucleotide according to any one of [1] to [3] or a pharmaceutically acceptable salt thereof, which contains the 155th nucleotide sequence represented by SEQ ID NO: 63 The sequence of 15-30 consecutive nucleotides in the base sequence No. to No. 217 is more than 90% complementary, preferably more than 95%, and is most suitable for a completely complementary sequence. [5] The antisense oligonucleotide according to any one of [1] to [4] or a pharmaceutically acceptable salt thereof, which comprises any one of the sequence identification numbers 65 to 82 and 101 to 108 The base sequence of (in the sequence, u can be substituted for t). [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 antisense oligonucleotide according to [6] or a pharmaceutically acceptable salt thereof, wherein the sugar-modified nucleoside comprises 4'-(CH ₂ ) _n -O-2' cross-linking (wherein, n is 1 or 2) or 2'-O-methylated nucleoside. [8] The antisense oligonucleotide according to any one of [1] to [7] or a pharmaceutically acceptable salt thereof, which comprises one or more modified internucleoside linkages. [9] The antisense oligonucleotide according to [8] or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is a phosphorothioate linkage. [10] The antisense oligonucleotide according to any one of [1] to [9] or a pharmaceutically acceptable salt thereof, which comprises any of the sequence identification numbers 1-18, 37-60 and 112-123 The base sequence represented by one. [11] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in [1], in the post-transcriptional modification of the XPF gene having the variant intron 8 sequence represented by SEQ ID NO: 64, Suppresses abnormal polyadenylation of the poly A additional sequence using the 324th to 329th base sequence represented by SEQ ID No. 64. [12] The antisense oligonucleotide according to [1] or [11] or a pharmaceutically acceptable salt thereof, which can be combined with the base sequence represented by SEQ ID NO: 309 to 343 The sequence of 15-30 nucleotides in the base sequence of the number hybridizes. [13] The antisense oligonucleotide according to any one of [1], [11], and [12], or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by SEQ ID NO: 64 The consecutive 15 to 30 nucleotide sequences in the base sequence of No. 309 to No. 343 have a sequence that is more than 90% complementary, preferably more than 95%, and is most suitable for a completely complementary sequence. [14] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of [1] and [11] to [13], which comprises any one of the sequence identification numbers represented by 83-100 The base sequence of (in the sequence, u can be substituted for t). [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 antisense oligonucleotide according to [15] or a pharmaceutically acceptable salt thereof, wherein the sugar-modified nucleoside comprises 4'-(CH ₂ ) _n -O-2' cross-linking (wherein, n is 1 or 2) and 2'-O-methylated nucleoside. [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 internucleoside linkages. [18] The antisense oligonucleotide according to [17] or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is a phosphorothioate linkage. [19] The antisense oligonucleotide according to any one of [1] and [11] to [18] or a pharmaceutically acceptable salt thereof, which comprises any one of the sequence identification numbers 19 to 36 The base sequence. [20] The antisense oligonucleotide according to any one of [1] to [19] or a pharmaceutically acceptable salt thereof, wherein the chemical modification is the addition of a molecular structure suitable for delivery of the oligonucleotide. [21] A medical composition for the treatment of Xeroderma F group, comprising the antisense oligonucleotide as described in any one of [1] to [20] or a pharmaceutically acceptable salt thereof. [22] A treatment method for group F of xeroderma chromosome, which comprises administering to a patient an effective amount of the antisense oligonucleotide as described in any one of [1] to [20] or its pharmaceutically acceptable Allowed salt. [23] An antisense oligonucleotide as described in any one of [1] to [20] or a pharmaceutically acceptable salt thereof for use in the treatment of Xeroderma pigmentosum F group. [24] A use of the antisense oligonucleotide as described in any one of [1] to [20] or a pharmaceutically acceptable salt thereof for the treatment of Xeroderma pigmentosum group F Pharmaceutical composition.

In addition, the present invention provides the following [A1] to [A20]. [A1] An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which has a base sequence that can hybridize to a part of the intron region of the XPF gene and has an abnormal post-transcriptional modification that inhibits the XPF gene active. [A2] The antisense oligonucleotide as described in [A1] or a pharmaceutically acceptable salt thereof in the post-transcriptional modification of the XPF gene having the variant intron 1 sequence represented by SEQ ID NO: 63, Suppresses abnormal splicing using the 5'splice site on the 5'side of guanine No. 193 and the 3'splice site on the 3'side of guanine No. 1658 in the base sequence represented by SEQ ID NO: 63. [A3] The antisense oligonucleotide as described in [A1] or [A2] or a pharmaceutically acceptable salt thereof, which can be combined with the 175th to the 217th base sequence represented by SEQ ID NO: 63 The sequence of 15-30 nucleotides in the base sequence of the number hybridizes. [A4] The antisense oligonucleotide according to any one of [A1] to [A3] or a pharmaceutically acceptable salt thereof, which contains the 175th nucleotide sequence in the base sequence represented by SEQ ID NO: 63 The sequence of 15-30 consecutive nucleotides in the base sequence No. to No. 217 is more than 90% complementary, preferably more than 95%, and is most suitable for a completely complementary sequence. [A5] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of [A1] to [A4], which is represented by any of the sequence identification numbers 65 to 82 and 101 to 108 The base sequence of (in the sequence, u can be substituted for t). [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 antisense oligonucleotide as described in [A6] or a pharmaceutically acceptable salt thereof, wherein the sugar-modified nucleoside comprises 4'-(CH ₂ ) _n -O-2' crosslinking (wherein, n It is 1 or 2) or 2'-O-methylated nucleoside. [A8] The antisense oligonucleotide according to any one of [A1] to [A7] or a pharmaceutically acceptable salt thereof, which comprises one or more modified internucleoside linkages. [A9] The antisense oligonucleotide as described in [A8] or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is a phosphorothioate linkage. [A10] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of [A1] to [A9], which is represented by any one of sequence identification numbers 1-18 and 37-60 The base sequence. [A11] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in [A1], in the post-transcriptional modification of the XPF gene having the variant intron 8 sequence represented by SEQ ID NO: 64, Suppresses abnormal polyadenylation of the poly A additional sequence using the 324th to 329th base sequence represented by SEQ ID No. 64. [A12] The antisense oligonucleotide as described in [A1] or [A11] or a pharmaceutically acceptable salt thereof, which can be combined with the 309th to the 343rd in the base sequence represented by SEQ ID No. 64 The sequence of 15-30 nucleotides in the base sequence of the number hybridizes. [A13] The antisense oligonucleotide as described in any one of [A1], [A11], and [A12] or a pharmaceutically acceptable salt thereof, which includes and includes the base sequence represented by SEQ ID NO: 64 The consecutive 15 to 30 nucleotide sequences in the base sequence of No. 309 to No. 343 have a sequence that is more than 90% complementary, preferably more than 95%, and is most suitable for a completely complementary sequence. [A14] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of [A1] and [A11] to [A13], which is represented by any one of the sequence identification numbers 83 to 100 The base sequence of (in the sequence, u can be substituted for t). [A15] The antisense oligonucleotide as described in any one of [A1] and [A11] to [A14] or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides. [A16] The antisense oligonucleotide as described in [A15] or a pharmaceutically acceptable salt thereof, wherein the sugar-modified nucleoside comprises 4'-(CH ₂ ) _n -O-2' cross-linking (wherein, n is 1 or 2) and 2'-O-methylated nucleoside. [A17] The antisense oligonucleotide as described in any one of [A1] and [A11] to [A16] or a pharmaceutically acceptable salt thereof, which comprises one or more modified internucleoside linkages. [A18] The antisense oligonucleotide according to [A17] or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is a phosphorothioate linkage. [A19] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of [A1] and [A11] to [A18], which is represented by any one of sequence identification numbers 19 to 36 The base sequence. [A20] A medical composition for the treatment of xeroderma group F, comprising the antisense oligonucleotide as described in any one of [A1] to [A19] or a pharmaceutically acceptable salt thereof. [Effects of Invention]

According to the present invention, it is possible to treat a specific XP-F group for which no treatment has been available.

[Form to implement the invention]

1. Definition In this manual, "Xeroderma pigmentosum F group" (also known as XP-F group) refers to one of the complementary groups of Xeroderma pigmentosum, showing both GG-NER and TC-NER The defect. The gene reported to be the cause of mutation is XPF gene.

In this specification, "XPF gene" refers to a human gene that encodes an endonuclease involved in the repair mechanism of nucleotide removal. XPF gene is also called ERCC4 gene, ERCC11 gene, FANCQ gene, RAD1 gene, or XFEPS gene. The sequence of XPF gene is known as Homo sapiens ERCC excision repair 4 (Homo sapiens ERCC excision repair 4), endonuclease catalytic subunit (ERCC4), RefSeqGene (LRG_463) on chromosome 16, for example (NCBI-GenBank accession number. NG_011442.1) and so on.

In this specification, the "variant intron 1 sequence" refers to the base sequence of intron 1 of the wild-type XPF gene (the base sequence of Nos. 5217 to 6874 in the aforementioned NG_011442.1). The sequence in which T mutated to A from the 5'end of sub 1 at No. 196 (No. 5412 in NG_011442.1) is the base sequence represented by SEQ ID NO: 63. This mutation site corresponds to the 196th base sequence represented by SEQ ID NO: 63.

In this specification, the "variant intron 8 sequence" refers to the base sequence of intron 8 of the wild-type XPF gene (in the aforementioned NG_011442.1, the base sequence of Nos. 20588-22609). The sequence in which C mutated to T from the 5'end of sub 8 at No. 326 (No. 20913 in NG_011442.1) is the base sequence represented by SEQ ID No. 64. This mutation site corresponds to the 326th base sequence represented by the sequence identification number 64.

In this specification, "post-transcriptional modification" refers to the modification in the process of mRNA precursor (pre-mRNA) becoming mature mRNA, including capping at the 5'end and polyadenylation at the 3'end , And editing.

In this specification, "abnormal post-transcriptional modification" refers to post-transcriptional modifications that are not usually seen in post-transcriptional modifications of wild-type genes, for example, abnormal splicing, abnormal polyadenylation, etc.

In this specification, "abnormal splicing" refers to splicing that is not usually seen in post-transcriptional modification of wild-type genes. The splicing of mRNA is the 5'splicing site, branch site, and 3'splicing site in introns by identification of the nuclear low-molecular-weight ribonucleoprotein (snRNP) as a splicing factor. In the post-transcriptional modification of the XPF gene with variant intron 1 sequence, the identification sequence of splicing factor U1 snRNP ( GTATGTAA), and the 5'side of guanine No. 193 becomes the 5'splicing site. As a result, an abnormality occurred using the 5'splice site on the 5'side of guanine No. 193 and the 3'splice site on the 3'side of guanine No. 1658 in the base sequence represented by SEQ ID NO: 63 Splicing.

In this specification, "abnormal polyadenylation" refers to polyadenylation that is not usually seen in post-transcriptional modification of wild-type genes. The polyadenylation of mRNA depends on the poly A additional sequence (also known as the severance/polyadenylation factor binding sequence or the polyadenylation message). The poly A additional sequence in humans usually contains AATAAA. In the post-transcriptional modification of the XPF gene with the variant intron 8 sequence, the poly A additional sequence (AATAAA) was newly generated by the mutation at No. 326 in the base sequence represented by SEQ ID No. 64. As a result, abnormal polyadenylation using the poly A addition sequence of No. 324 to No. 329 in the base sequence represented by SEQ ID No. 64 occurred.

In the present specification, the "antisense oligonucleotide" refers to a single-stranded oligonucleotide having the ability to hybridize to a nucleotide containing a target base sequence. "Hybridization" refers to the interaction between the bases (AG (adenine-guanine) and CT/U (cytosine-thymine/uracil)) to interact with the target nucleoside Acid can form double strands. The antisense oligonucleotide only needs to have sequence complementarity with the target sequence to the extent that it is hybridizable, and does not need to be a completely complementary sequence. In addition, antisense oligonucleotides can be DNA, RNA, and DNA/RNA chimeras. In addition, antisense oligonucleotides can include modifications such as modified nucleosides and modified internucleoside linkages.

"Performing hybridization" refers to hybridization under low stringency conditions, hybridization under moderate stringency conditions, and hybridization under high stringency conditions. The "low stringency conditions" may be, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formazan, 32° C.; or conditions equivalent thereto. "Medium stringent conditions" can be, for example, 5×SSC, 5×Danhart’s solution, 0.5% SDS, 50% formazan, 42°C, or 5×SSC, 1% SDS, 50 mM Tris-HCl( pH7.5), 50% formazan, 42℃; or its equivalent conditions. "Highly stringent conditions" can be, for example, 5×SSC, 5×Danhart’s solution, 0.5% SDS, 50% formazan, 50°C; or 0.2×SSC, 0.1% SDS, 65°C; or The same conditions. As far as the factors that affect the stringency of hybridization are concerned, it is conceivable that there are multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration. The same stringency can be achieved by appropriately selecting these elements. It is also possible to use commercially available hybridization reagents to achieve the above conditions. For example, "high stringency conditions" can be achieved by: performing ^{hybridization in a commercially available hybridization solution ExpressHyb TM} hybridization solution (manufactured by Clontech) at 68°C; or using a filter paper with immobilized DNA to perform hybridization at 0.7-1.0 After hybridization in the presence of M NaCl at 68°C, use 0.1-2 times the concentration of SSC solution (1 times the concentration of SSC contains 150mM NaCl, 15mM sodium citrate), and wash at 68°C.

In this specification, nucleosides include natural nucleosides and modified nucleosides. "Natural nucleoside" refers to 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, thymidine, 2 '-Deoxyuridine and other ribonucleosides such as 2'-deoxynucleosides, adenosine, guanosine, cytidine, 5-methylcytidine, and uridine. Among the nucleic acid bases, uracil (U) or (u) is interchangeable with thymine (T) or (t), and uracil (U) or (u) is interchangeable with thymine (T) or (t) Either one can be used to form a base pair with adenine (A) or (a) of the complementary chain.

In this specification, 2'-deoxyadenosine is sometimes expressed as A ^t , 2'-deoxyguanosine is expressed as G ^t , 2'-deoxycytidine is expressed as C ^t , and 2'- Deoxy-5-methylcytidine is represented as 5meC ^t , thymidine is represented as T ^t , and 2'-deoxyuridine is represented as U ^t . In addition, as the corresponding nucleotides, in this specification, 2'-deoxyadenosine nucleotides are sometimes expressed as ^Ap and 2'-deoxyguanosine nucleotides are expressed as G ^p , Denote 2'-deoxycytidine nucleotides as C ^p , 2'-deoxy-5-methyl cytidine nucleotides as 5meC ^p , and thymidine nucleotides as T ^p , and 2'-deoxyuridine nucleotides are denoted U ^p .

In this specification, "sugar-modified nucleoside" refers to a nucleoside in which the sugar portion of the nucleoside has been modified. The sugar-modified nucleoside includes all patterns of sugar modification known in the technical field of the present invention. 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 include halogen groups, allyl groups, amino groups, azido groups, O-allyl groups, OC ₁ -C ₁₀ alkyl groups, 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 of R _m and R _n is H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ alkyl group. Regarding 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methyluridine, commercially available reagents can be used. 2'-O-aminoethylguanosine, 2'-O-aminoethyladenosine, 2'-O-aminoethylcytidine, and 2'-O-aminoethyluridine can follow It is synthesized from literature (Blommers et al. Biochemistry (1998), 37, 17714-17725.). 2'-O-propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine, and 2'-O-propyluridine can be described in accordance with the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.). Regarding 2'-O-allylguanosine, 2'-O-allyladenosine, 2'-O-allylcytidine, and 2'-O-allyluridine, commercially available ones can be used The test drug. 2'-O-methoxyethylguanosine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethylcytidine, and 2'-O-methoxyethyl Uridine can be synthesized according to patent (US6261840) or literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504. 2'-O-butylguanosine, 2'-O-butyl Adenosine, 2'-O-butylcytidine, and 2'-O-butyluridine can be synthesized according to 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 according to the literature (Lesnik, EA et al. Biochemistry ( 1993), 32, 7832-7838.). About 2'-O-propargyl guanosine, 2'-O-propargyl adenosine, 2'-O-propargyl cytidine, and 2' -O-propargyluridine, commercially available reagents can be used.

Examples of 4'-sulfur modified nucleosides include β-D-ribonucleosides in which the 4'-oxygen atom is substituted with 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'-sulfur-2'-modified nucleosides include 4'-sulfur-2'-modified nucleosides that maintain 2'-H or 2'-O-methyl (Matsugami, et al. Nucleic Acids Res. 36, 1805 (2008)).

Examples of bicyclic sugar-modified nucleosides include nucleosides that maintain the second ring formed by cross-linking two atoms of the ribose ring. Examples of such nucleosides include 2',4'-BNA/LNA (bridged nucleic acids/locked nucleic acids) (Obika, S. et al. Tetrahedron Lett., 38, p.8735-(1997).; Obika, S. et al., Tetrahedron Lett., 39, p.5401-(1998).; AA Koshkin, AA et al. Tetrahedron, 54, p.3607(1998).; Obika, S. Bioorg. Med. Chem., 9, p.1001(2001).), to extend the methylene chain of 2',4'-BNA/LNA by one ENA (2'-O, 4'-C-ethylene-bridging nucleic acid) cross-linked by carbon ethylene chains (Morita, K. et al. Bioorg. Med. Chem. Lett., 12, p. 73 (2002).; Morita, K. et al. Bioorg. Med. Chem., 11, p. 2211 (2003).). The AmNA recorded in WO2014/109384 or the S-cEt(2', 4'-bound的ethyl).

In this specification, as a sugar-modified nucleoside containing 2'-O-methylation modification, for example, the one corresponding to A ^t is expressed as A ^m1t , the one corresponding to G ^t is expressed as G ^m1t , and the one corresponding to C ^t is expressed as C ^m1t , the one corresponding to 5meC ^t is expressed as 5meC ^m1t , the one corresponding to U ^t is expressed as U ^m1t and so on. Regarding the sugar-modified nucleotides that correspond to their 2'-O-methylation modifications, the one corresponding to A ^{p is sometimes} expressed as A m1p, the one corresponding to G ^p is expressed as G ^m1p , and the one corresponding to C ^p This is expressed as C ^m1p , the one corresponding to 5meC ^p is expressed as 5meC ^m1p , and the one corresponding to U ^p is expressed as U ^m1p .

In this specification, as a sugar-modified nucleoside containing a 2'-O-methoxyethylation modification, for example, the one corresponding to A ^t is expressed as A ^m2t , the one corresponding to G ^t is expressed as G ^m2t , and the corresponding 5meC ^t is expressed as 5meC ^m2t , and the corresponding T ^t is expressed as T ^m2t and so on. Regarding the corresponding sugar-modified nucleotides containing 2'-O-methoxyethylation modification, the corresponding A ^{p is sometimes} expressed as A m2p, the corresponding G ^{p is} expressed as G ^m2p , and the corresponding The case of 5meC ^p is represented as 5meC ^m2p , and the case corresponding to T ^p is represented as T ^m2p .

In this specification, as _{a sugar-modified nucleoside containing 4'-CH 2} -0-2' crosslinking, for example, the one corresponding to A ^t is expressed as A ^1t , the one corresponding to G ^t is expressed as G ^1t , and the corresponding The one with 5meC ^t is denoted as C ^1t , and the one corresponding to T ^t is denoted as T ^1t . Regarding the sugar-modified nucleotides corresponding to their 4'-CH ₂ -0-2' cross-links, the one corresponding to A ^{p is sometimes} expressed as A e1p, and the one corresponding to G ^p is expressed as G ^e1p , and The one corresponding to 5meC ^p is denoted as C ^e1p , and the one corresponding to T ^p is denoted as T ^e1p .

This specification, as comprising a _{4 '- (CH 2) 2} -0-2' sugar modified nucleoside crosslinked e.g. corresponding sometimes indicated as A ^t A ^2t, corresponding to those indicated as G ^t G ^2t, The one corresponding to 5meC ^t is denoted as C ^2t , and the one corresponding to T ^t is denoted as T ^2t . Regarding the sugar-modified nucleotides that correspond to their 4'-(CH ₂ ) ₂ -0-2' crosslinks, sometimes the one corresponding to A ^p is expressed as A ^e2p , and the one corresponding to G ^p is expressed as G ^e2p , the one corresponding to 5meC ^p is denoted as ^Ce2p , and the one corresponding to T ^p is denoted as T ^e2p .

In the present specification, "modified internucleoside linkage" refers to a bond in which a substitution or change is applied to a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage). That is, the antisense oligonucleotides containing modified internucleoside linkages include modification of the phosphate group of at least one nucleotide. With regard to modified internucleoside linkages, for example, phosphorothioate linkages, dithiophosphate linkages, alkyl phosphate linkages, boranophosphate linkages, amino phosphates ( phosphoramidate) bond and so on.

In this specification, regarding the ester of phosphorothioate that replaces the phosphate of nucleotide to become the ester of phosphorothioate, sometimes the one corresponding to ^Ap is expressed as A ^s , and the one corresponding to G ^p is expressed as G ^s , The one corresponding to C ^p is denoted as C ^s , the one corresponding to 5 meC ^p is denoted as 5 meC ^s , the one corresponding to T ^p is denoted as T ^s , and the one corresponding to U ^p is denoted as U ^s . Regarding sugar-modified nucleotides containing 2'-O-methylation modification and phosphorothioate, sometimes the one corresponding to A ^s is expressed as A ^m1s , the one corresponding to G ^s is expressed as G ^m1s , and the one corresponding to C ^s This is expressed as C ^m1s , the one corresponding to 5meC ^s is expressed as 5meC ^m1s , and the one corresponding to U ^s is expressed as U ^m1s . Regarding sugar-modified nucleotides containing 2'-O-methoxyethylation modification and phosphorothioate, the one corresponding to A ^{s is sometimes} expressed as A m2s, and the one corresponding to G ^s is expressed as G ^m2s , and The one corresponding to 5meC ^s is denoted as 5meC ^m2s , and the one corresponding to T ^s is denoted as T ^m2s . When also comprising 4'-CH ₂ -0-2 'phosphorothioate crosslinked and sugar modified nucleotide, as represented by the corresponding A ^{s A} e1s, expressed as the corresponding G ^{s G} e1s, expressed as the corresponding 5meC ^s C ^e1s , corresponding to T ^{s is} expressed as T ^e1s . When also comprising _{4 '- (CH 2) 2} -0-2' phosphorothioate crosslinked and sugar modified nucleotide, as represented by the corresponding A ^s A ^e2s, expressed as the corresponding G ^s G ^e2s, corresponding to 5meC ^{s is} expressed as C ^e2s , and the corresponding T ^{s is} expressed as T ^e2s .

In the following presentations A ^t , G ^t , 5meC ^t , C ^t , T ^t , U ^t , ^Ap , 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 , ^{meC 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 ^{The structure of m2s} , G ^m2s , 5meC ^m2s , and T ^m2s .

In this specification, the treatment of a disease or symptom includes the prevention, aggravation, or inhibition or hindrance of the onset of the disease, the reduction or aggravation or inhibition of more than one symptom presented by an individual suffering from the disease, two Treatment of secondary diseases, etc.

2. The pharmaceutical composition and antisense oligonucleotide of the present invention The present invention provides a medicinal composition for the treatment of xeroderma pigmentosum F group (XP-F group) (hereinafter referred to as the medicinal composition of the present invention), and antisense oligonucleotides or antisense oligonucleotides of its effective ingredients Its pharmaceutically acceptable salt.

The pharmaceutical composition of the present invention contains a post-transcriptional modification that inhibits abnormalities due to disease-causing mutations in the XP-F group, especially post-transcriptional modifications due to internal mutations in two types of novel introns. The modified abnormal antisense oligonucleotide serves as an effective ingredient, and inhibits the decrease in the expression level of XPF gene in the XP-F group with the mutation, and can treat the XP-F group with the mutation.

The two types of mutations that are the cause of diseases in the XP-F group newly identified by the inventors are derived from the base sequence of intron 1 of the wild-type XPF gene (the 5217 to 6874 in the aforementioned NG_011442.1 No. base sequence) in the 5'end of intron 1 from the 196th (No. 5412 in NG_011442.1) T mutated into A (in the base sequence represented by SEQ ID NO: 63, (Corresponding to No. 196) and the 5'end of intron 8 in the base sequence of intron 8 of the wild-type XPF gene (base sequence of Nos. 20588-22609 in the aforementioned NG_011442.1) The C from No. 326 (No. 20913 in NG_011442.1) was mutated into T (the base sequence represented by SEQ ID NO. 64 is equivalent to No. 326). Abnormal splicing of XPF gene occurs due to the former, and abnormal polyadenylation of XPF gene occurs due to the latter. As a result, it is believed that a decrease in the expression level of XPF gene occurs and causes symptoms in the XP-F group. Therefore, in one aspect, the applicable object 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. Furthermore, in the present invention, as long as the XPF gene contains the above-mentioned mutation, and thus the above-mentioned abnormal splicing or abnormal polyadenylation occurs, the XPF gene may further include a mutation (for example, deletion of 1 or several nucleotides, Replace, append or insert).

The antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof has a base sequence that can hybridize to 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 the antisense oligonucleotide of the present invention or its pharmaceutically acceptable salt can inhibit the abnormal post-transcriptional modification of XPF gene is based on the evaluation of the amount of XPF mRNA splicing products and the evaluation of protein expression as described in the following examples , Or the same evaluation method as the repair activity evaluation. It is confirmed by any evaluation method that the expression of the XPF gene that has been post-transcriptionally modified correctly or the recovery of the DNA repair activity is restored, and it is judged that the antisense oligonucleotide or its pharmaceutically acceptable salt inhibits the abnormality of the XPF gene Post-transcriptional modification.

In one aspect, the pharmaceutical composition of the present invention uses the above-mentioned mutation in intron 1 as the cause of disease as the applicable object of the XP-F group. The pharmaceutical composition of the present invention in this aspect is referred to as the pharmaceutical composition I of the present invention. The pharmaceutical composition I of the present invention includes: in the post-transcriptional modification of the XPF gene having the variant intron 1 sequence represented by the sequence identification number 63, inhibiting the use of the 193rd in the base sequence represented by the sequence identification number 63 No. 1 or more antisense oligonucleotides of abnormal splicing at the 5'splicing site on the 5'side of guanine and the 3'splicing site on the 3'side of guanine No. 1658. Furthermore, "abnormalities in the 5'splice site on the 5'side of guanine No. 193 and the 3'splice site on the 3'side of guanine No. 1658 in the base sequence represented by SEQ ID NO. 63 "Splicing" can also be changed to "Recognize guanine No. 1 to Adenine No. 192 in the base sequence represented by SEQ ID No. 63 as exons, and change guanine No. 193 to No. 1658. The number of guanine is recognized as an abnormal splicing of introns.”

Whether the antisense oligonucleotide in the pharmaceutical composition I of the present invention inhibits abnormal splicing in intron 1 is determined by evaluating the amount of XPF mRNA splicing products, evaluation of protein expression, or The repair activity evaluation is determined by the same evaluation method. It is confirmed by any evaluation method that the expression of the XPF gene that has been correctly spliced is increased or the DNA repair activity is restored, and it is judged that the antisense oligonucleotide inhibits the abnormal splicing in intron 1. In the evaluation of the amount of XPF mRNA splicing products, it was confirmed by droplet digital PCR (ddPCR) that cells derived from patients in the XP-F group who had the above-mentioned mutation in intron 1 as the cause of the disease In, whether there is an increase in XPF mRNA that has been correctly spliced by transfection of antisense oligonucleotides. ddPCR is a method of PCR amplification by dispersing critically diluted cDNA in micro-segments, and directly counting the number of micro-segments whose amplification information is positive, and absolutely measuring the concentration of the target in the sample . ddPCR can be performed using commercially available reagents and machines (for example, QX100 ^TM Droplet Digital ^TM PCR system (Bio-Rad Laboratories, Inc.), etc.). In the protein expression evaluation, Western blotting was used to confirm whether the cells from the XP-F group whose disease is caused by the above-mentioned mutation in intron 1 were transfected. An increase in XPF gene products stained with antisense oligonucleotides that have been correctly spliced. In addition, in the evaluation of repair activity, it was confirmed whether cells derived from patients in the XP-F group whose disease was caused by the above-mentioned mutation in intron 1 were repaired by transfection of antisense oligonucleotides. The activity of DNA damage caused by UV irradiation is restored.

The antisense oligonucleotide in the pharmaceutical composition I of the present invention is not limited to a specific one as long as it inhibits abnormal splicing in intron 1. However, for example, it may include the one represented by the sequence identification number 63 The 155th to 217th (preferably 175th to 217th, more preferably 179th to 213th, and still more preferably 183th to 203th) in the base sequence A sequence of 15 to 30 nucleotides (preferably 15 to 23 nucleotides, more preferably 15 to 18 nucleotides) within the sequence is the target. For example, the target base sequence of the antisense oligonucleotide in the pharmaceutical composition I of the present invention can be represented by any one selected from the group consisting of sequence identification numbers 65-82, 101-108, and 124-135 The sequence complementary to the base sequence of is preferably a sequence complementary to the base sequence represented by any one of the sequence identification numbers 65-82, 101, and 105-108, and more preferably may be a sequence selected from The sequence complementary to the base sequence represented by any one of the identification numbers 65 to 82 is more preferably a sequence complementary to the base sequence represented by any one selected from the group consisting of sequence identification numbers 75 to 78. In other words, the antisense oligonucleotide in the pharmaceutical composition I of the present invention can hybridize with the aforementioned sequence (ie, the target sequence). In a preferred aspect, the antisense oligonucleotide in the pharmaceutical composition I of the present invention can hybridize with the aforementioned target sequence under high stringency 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 without a tail sequence are most suitable.

After removing the base sequence of the part of the tail sequence of the antisense oligonucleotide in the pharmaceutical composition I of the present invention, as long as the activity of hybridizing with the aforementioned target sequence is maintained, the sequence complementarity with the target sequence can be 70% Above, it is preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more, and is most suitable for complete complementarity.

In a preferred aspect, the antisense oligonucleotides in the pharmaceutical composition I of the present invention include those selected from the group consisting of sequence identification numbers 65-82, 101-108, and 124-135 (preferably sequence identification number 65 ~82, 101, and 105 to 108, more preferably the base sequence represented by any of the sequence identification numbers 65 to 82, and still more preferably the sequence identification numbers 75 to 78). The base sequences represented by the sequence identification numbers 65 to 82, 101 to 108, and 124 to 135 are described as corresponding to the sequence identification numbers 1 to 18, 53 to 60, and 112 to 123 described in the examples described later. RNA sequence. In addition, the uridine residue in the antisense oligonucleotide may be substituted with a modified nucleotide based on the corresponding thymidine residue. Therefore, this aspect also includes the base sequence in which "u" is replaced with "t" in the base sequences represented by the sequence identification numbers 65 to 82, 101 to 108, and 124 to 135.

In addition, after removing the base sequence of the part of the tail sequence of the antisense oligonucleotide in the pharmaceutical composition I of the present invention, as long as it has the hybridization activity with the aforementioned target sequence, there can be 5 bases between the target sequence and the target sequence. The mismatch is less than 1 base (preferably 4 bases or less, more preferably 3 bases, 2 bases, or 1 base), but it is most suitable that there is no mismatch.

Here, it is considered that the antisense oligonucleotide in the pharmaceutical composition I of the present invention prevents U1snRNP from the newly generated 5'splicing site identification sequence GTAAGTAA (sequence identification number 63) of the variant intron 1 sequence. The 193-200th sequence in the base sequence shown) inhibits abnormal splicing. U1 snRNP has a sequence that is almost complementary to the recognition sequence of the 5'splicing site, and forms base pairs during the splicing process. Therefore, in a preferred aspect, the antisense oligonucleotide in the pharmaceutical composition I of the present invention can target the following sequence: including numbers 193 to 200 in the base sequence represented by SEQ ID No. 63 The sequence of at least 1 base, for example, a sequence of 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, or 8 bases.

The length of the antisense oligonucleotide in the pharmaceutical composition I of the present invention is not particularly limited as long as it inhibits abnormal splicing in intron 1, but may be, for example, 15-30 nucleotides, 15- 23 nucleotides, or 15-18 nucleotides.

In another aspect, the pharmaceutical composition of the present invention uses the above-mentioned mutation in intron 8 as the cause of disease as the applicable object of the XP-F group. The pharmaceutical composition of the present invention in this aspect is referred to as the pharmaceutical composition II of the present invention. The pharmaceutical composition II of the present invention includes: in the post-transcriptional modification of the XPF gene having the variant intron 8 sequence represented by the sequence identification number 64, the use of the 324th base sequence represented by the sequence identification number 64 is suppressed No.-No. 329, more than one antisense oligonucleotide with abnormal polyadenylation of poly A additional sequence.

Here, whether the antisense oligonucleotide in the pharmaceutical composition II of the present invention inhibits abnormal polyadenylation in intron 8 is based on the evaluation of protein expression or repair activity described in the following examples Determined by the same evaluation method. It is confirmed by any evaluation method that the expression of the XPF gene that has been correctly spliced is increased or the DNA repair activity is restored, and it is judged that the antisense oligonucleotide inhibits abnormal polyadenylation in intron 8. In the evaluation of protein expression level, it is confirmed whether the cells derived from the XP-F group whose disease is caused by the above-mentioned mutation in intron 8 are correct by transfection with antisense oligonucleotides. The position of the XPF gene product is increased by polyadenylation. In addition, in the evaluation of repair activity, it was confirmed whether cells derived from patients in the XP-F group whose disease was caused by the above-mentioned mutation in intron 8 were repaired by transfection of antisense oligonucleotides. The activity of DNA damage caused by UV irradiation is restored.

The antisense oligonucleotide in the pharmaceutical composition II of the present invention is not limited to a specific one as long as it inhibits abnormal polyadenylation in intron 8. For example, it may include the base represented by the sequence identification number 64 A sequence of 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides in the base sequence of No. 309 to No. 343 in the sequence is the target. In other words, the antisense oligonucleotide in the pharmaceutical composition II of the present invention can hybridize with the aforementioned sequence (ie, the target sequence). In a preferred aspect, the antisense oligonucleotide in the pharmaceutical composition II of the present invention can hybridize with the aforementioned target sequence under high stringency conditions.

The antisense oligonucleotide in the pharmaceutical composition II of the present invention may have a base sequence (tail sequence) that does not help hybridize 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 without a tail sequence are most suitable.

After removing the base sequence of the part of the tail sequence of the antisense oligonucleotide in the pharmaceutical composition II of the present invention, as long as the activity of hybridizing with the aforementioned target sequence is maintained, the sequence complementarity with the target sequence can be more than 70% , Preferably more than 80%, more preferably more than 90%, still more preferably more than 95%, most suitable for complete complementarity.

In a preferred aspect, the antisense oligonucleotide in the pharmaceutical composition II of the present invention includes the base sequence represented by the sequence identification number 83-100. The base sequences represented by the sequence identification numbers 83 to 100 are described as RNA sequences corresponding to the sequence identification numbers 19 to 36 described in the examples described later. In addition, the uridine residue in the antisense oligonucleotide may be substituted with a modified nucleotide based on the corresponding thymidine residue. Therefore, this aspect also includes the base sequence in which "u" is replaced with "t" in the base sequence represented by the sequence identification numbers 83-100.

Furthermore, after removing the base sequence of the part of the tail sequence of the antisense oligonucleotide in the pharmaceutical composition II of the present invention, as long as it has the hybridization activity with the aforementioned target sequence, there may be 5 bases between the target sequence and the target sequence. The mismatch is less than 1 base (preferably 4 bases or less, more preferably 3 bases, 2 bases, or 1 base), but it is most suitable that there is no mismatch.

Here, it is considered that the antisense oligonucleotide in the pharmaceutical composition II of the present invention prevents the addition of the sequence AATAAA (the base represented by the sequence identification number 64) to the newly generated poly A in the variant intron 8 sequence. In the sequence, the cleavage of the 324-329 sequence) and the binding of polyadenylation factor (CPSF) inhibit abnormal polyadenylation. Therefore, in a preferred aspect, the antisense oligonucleotide in the pharmaceutical composition II of the present invention can target the following sequence: including numbers 324 to 329 in the base sequence represented by SEQ ID No. 64 At least one base of the sequence, for example, a sequence containing 2 bases, 3 bases, 4 bases, 5 bases, or 6 bases.

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

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

The antisense oligonucleotide in the pharmaceutical composition of the present invention may contain more than one modification to increase nuclease resistance and improve stability in vivo. The modification includes, for example, the modification of the sugar moiety of nucleosides and the modification of the phosphate group of nucleotides. Examples of sugar-modified nucleosides and modified nucleoside linkages include those described in 1 above.

In a preferred aspect, the antisense oligonucleotide contains more than one sugar-modified nucleoside. The nucleosides contained in the antisense oligonucleotide may all be sugar modified nucleosides. The antisense oligonucleotide may include more than two types of sugar-modified nucleosides containing different sugar moieties. The sugar-modified nucleoside preferably contains 4'-(CH ₂ ) _n -O-2' cross-linking (where n is 1 or 2) or 2'-O-methylated nucleosides, more preferably 4'-(CH ₂ ) ₂ -O-2' cross-linked or 2'-O-methylated nucleosides. In antisense oligonucleotides, sugar-modified nucleosides containing 4'-(CH ₂ ) ₂ -O-2' cross-linking are included. The number is not particularly limited, but can be more than one. For example, 2 or more, 3 or more, 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 better aspect, all the nucleosides contained in the antisense oligonucleotides are sugar modified nucleosides, and the sugar modified nucleosides are sugar modifications that include 4'-(CH ₂ ) ₂ -O-2' cross-linking Nucleosides, sugar-modified nucleosides containing 2'-O-methylation, or combinations of these.

In a preferred aspect, the antisense oligonucleotide contains more than one modified internucleoside linkage. In antisense oligonucleotides, different modified internucleoside linkages can be included. In addition, the internucleoside linkages in antisense oligonucleotides can all be modified internucleoside linkages. In a more preferred aspect, the modified internucleoside linkages are phosphorothioate linkages. In a further preferred aspect, the internucleoside linkages in the antisense oligonucleotide are all modified internucleoside linkages, and the modified internucleoside linkages are phosphorothioate linkages.

In a preferred aspect, the antisense oligonucleotide in the pharmaceutical composition I of the present invention is selected from the group comprising the following oligonucleotides: comprising sequence identification numbers 1-18, 37-60 and 112~ Oligonucleotide of the base sequence represented by 123. The nucleosides contained in these oligonucleotides are _{sugar-modified nucleosides containing 4'-(CH 2} ) ₂ -O-2' cross-linking and sugar-modified nucleosides containing 2'-O-methylation Any of them. In addition, the internucleoside linkages in these oligonucleotides are all phosphorothioate linkages. As the antisense oligonucleotide in the pharmaceutical composition I of the present invention, more suitable ones 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 (serial identification number 11) Example 12 (XPF-int1-012): HO-C ^m1s -C ^{e2s-Um1s-Um1s} -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 (serial identification number 12) Example 13 ( XPF-int1-013): HO-C ^m1s -T ^e2s -U ^m1s -A ^m1s -C ^{e2s-Um1s-Um1s} -A ^e2s -C ^m1s -G ^m1s -T ^e2s -C ^m1s -U ^m1s -G ^e2s -U ^m1s -G ^m1s -T ^e2s -U ^m1t -H (serial identification number 13) Example 14 (XPF-int1-014): HO-U ^m1s -T ^e2s -A ^m1s -C ^m1s -T ^e2s -U ^m1s -A ^m1s -C ^e2s -G ^m1s -U ^m1s -C ^e2s -U ^m1s -G ^m1s -T ^e2s -G ^m1s -U ^m1s -T ^e2s -C ^m1t -H (serial identification number 14) Example 45 (XPF-int1- 013_1): HO-C ^m1s -T ^e2s -U ^m1s -A ^m1s -C ^{e2s-Um1s-Um1s} -A ^m1s -C ^m1s -G ^m1s -T ^e2s -C ^m1s -U ^m1s -G ^m1s -U ^m1s -G ^m1s -T ^e2s -U ^m1t -H (serial identification number 45) Example 46 (XPF-int1-013_2): HO-C ^m1s -T ^e2s -U ^m1s -A ^m1s -C ^{e2s-Um1s-Um1s} -A ^e2s -C ^m1s -G ^m1s -T ^e2s -C ^m1s -U ^m1s -G ^m1s -U ^m1s -G ^m1s -T ^e2s -U ^m1t -H (serial identification number 46) Example 62 (XPF-int1-013_5): HO-C ^m1s -T ^e2s -U ^m1s -A ^m1s -C ^{e2s-Um1s-Um1s} -A ^e2s -C ^m1s -G ^m1s -T ^e2s -C ^m1s -T ^e2s -G ^m1s -U ^m1s -G ^m1s -T ^e2s -U ^m1t -H (serial identification number 112) Example 63(XPF-int1-013_6): HO-C ^m1s -T ^e2s -U ^m1s -A ^m1s -C ^{e2s-Um1s-Um1s} -A ^e2s -C ^m1s -G ^m1s -T ^e2s -C ^m1s -U ^m1s -G ^m1s -T ^e2s -G ^m1s -T ^e2s -U ^m1t -H (serial identification number 113)

In a preferred aspect, the antisense oligonucleotide in the pharmaceutical composition II of the present invention is selected from the group comprising the following oligonucleotides: those containing the base sequence represented by the sequence identification numbers 19 to 36 Oligonucleotides. The nucleosides contained in these oligonucleotides are _{sugar-modified nucleosides containing 4'-(CH 2} ) ₂ -O-2' cross-linking and sugar-modified nucleosides containing 2'-O-methylation Any of them. In addition, the internucleoside linkages in these oligonucleotides are all phosphorothioate linkages.

The preparation method of antisense oligonucleotides is not particularly limited, but known chemical synthesis methods (phosphotriester method, phosphoramidite method, H-phosphonate method, etc.) can be used. Commercially available nucleic acid synthesizers can be used to synthesize using commercially available reagents for DNA/RNA synthesis. For example, by coupling the phosphoramidite reagent, sulfur, tetraethyl thiuram disulfide (TETD) (Applied Biosystems), Beaucage reagent (Glen Research), or hydroxanthin (xanthane hydride) and other reagent reactions, can synthesize antisense oligonucleotides with phosphorothioate bonds (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990) , PCT/WO98/54198).

The oligonucleotides (antisense oligonucleotides) of the present invention may also have the following groups at the 5'end and/or 3'end: they are expected to control the physical properties and in vivo dynamics of the oligonucleotide The base of the chemical structure. For example, an aminoalkyl group can be introduced into the 5'end and/or 3'end of the oligonucleotide (antisense oligonucleotide) of the present invention, and the desired chemical structure can be added through this group.

The introduction of an aminoalkyl group into the oligonucleotide can be carried out by a method known in the art, and it can also be carried out by using a commercially available reagent. For example, after the chain elongation of the oligonucleotide with the target sequence is completed, it is combined with 5'-Amino-Modifier C6 (Glen Research), 5'-TFA-Amino-Modifier C6-CE Phosphoramidite, 5'-TFA- Amino-Modifier-C5-CE Phosphoramidite (Link Technologies) and other amine modification reagents can be used to synthesize oligonucleotides with aminoalkyl phosphate groups bound to the 5'end. By reacting with 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG (Glen Research) and other amine modification reagents, oligomers with amino alkyl groups bound to the 3'end can be synthesized. Nucleotides.

With regard to the 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 possible to utilize the well-known controllable oligonucleotides in the technical field. The arbitrary chemical structure of physical properties and in vivo dynamics includes, for example, fatty acid, cholesterol, and GalNAc structure. For example, it is known that amidite compounds containing fatty acids that are thought to be useful for the migration of nucleic acids to muscle tissues can be used to synthesize antisense oligonucleotides and promote antisense oligonucleotides to muscle tissues. (For example, Link Technologies products such as Nucleic Acids Res. (2020) 47, 6029-6044, 5'-Palmitate-C6-CE Phosphoramidite, etc.). In addition, the effect of cholesterol-bound siRNA in the brain has been described in publicly known documents (for example, Mol. Cancer Ther. (2018) 17, 1251-1258). In addition, it is known that antisense oligonucleotides can be synthesized using galNAc compounds that are thought to be useful for the migration of nucleic acids to liver cells, and the antisense oligonucleotides can be specifically delivered to Liver cells (for example, Methods in Enzymology, 1999, Volume 313, pages 297-321, WO2009/073809, WO2014/076196, WO2014/179620, WO2015/006740, WO2015/105083, WO2016/055601, WO2017/023817, WO2017/ 084987, WO2017/131236, WO2019/172286, etc.).

啉鹽、葡萄糖胺鹽、苯基甘胺酸烷基酯鹽、乙二胺鹽、N-甲基還原葡糖胺鹽、胍鹽、二乙胺鹽、三乙胺鹽、二環己胺鹽、N,N’-二苄基乙二胺鹽、氯普魯卡因鹽、普魯卡因鹽、二乙醇胺鹽、N-苄基-苯乙胺鹽、哌

鹽、四甲基銨鹽、參(羥甲基)胺基甲烷鹽之有機鹽等之胺鹽；如氫氟酸鹽、鹽酸鹽、氫溴酸鹽、氫碘酸鹽的氫鹵酸鹽；如硝酸鹽、過氯酸鹽、硫酸鹽、磷酸鹽等之無機酸鹽；如甲磺酸鹽、三氟甲磺酸鹽、乙磺酸鹽之低級烷磺酸鹽；如苯磺酸鹽、對甲苯磺酸鹽之芳基磺酸鹽；乙酸鹽、蘋果酸鹽、反丁烯二酸鹽、琥珀酸鹽、檸檬酸鹽、酒石酸鹽、草酸鹽、順丁烯二酸鹽等之有機酸鹽；如甘胺酸鹽、離胺酸鹽、精胺酸鹽、鳥胺酸鹽、麩胺酸鹽、天冬胺酸鹽之胺基酸鹽等。此等之鹽可藉由公知方法製造。The antisense oligonucleotide in the pharmaceutical composition of the present invention can be in the form of a pharmaceutically acceptable salt. "The pharmaceutically acceptable salt thereof" refers to the salt of an oligonucleotide (antisense oligonucleotide). For such a salt, alkali metal salts such as sodium salt, potassium salt, and lithium salt; such as calcium Alkaline earth metal salts of salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc.; such as inorganic salt of ammonium salt; such as tertiary octylamine salt, dibenzyl Amine salt,

Salt, glucamine salt, alkyl phenylglycine salt, ethylenediamine salt, N-methyl reduced glucosamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt , N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine

Salt, tetramethylammonium salt, organic salt of ginseng (hydroxymethyl) aminomethane salt, etc. Amine salt; such as hydrofluoride, hydrochloride, hydrobromide, and hydrohalide of hydroiodide ; Inorganic acid salts such as nitrate, perchlorate, sulfate, phosphate, etc.; such as lower alkane sulfonate such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; such as benzenesulfonate , Aryl sulfonate of p-toluenesulfonate; among acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc. Organic acid salts; such as glycine, lysine, arginine, ornithine, glutamine, aspartate and the like. These salts can be produced by known methods.

In addition, the antisense oligonucleotides of the pharmaceutical composition of the present invention and pharmaceutically acceptable salts thereof may sometimes exist as solvates (for example, hydrates), and may also be such solvates.

The pharmaceutical composition of the present invention can be formulated by mixing antisense oligonucleotides and appropriate pharmaceutically acceptable additives. For example, the pharmaceutical composition of the present invention may be prepared as tablets, capsules, granules, etc., and administered orally, or prepared as injections, percutaneous absorption agents, etc., instead of being administered orally.

These preparations can use excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, diluents, solvents for injections, dissolution aids, suspending agents, isotonic agents, buffers, and analgesics Additives such as preservatives, antioxidants, etc., are manufactured by known methods.

Examples of excipients include organic excipients and inorganic excipients. For organic excipients, for example, sugar derivatives such as lactose and white sugar; starch derivatives such as corn starch and potato starch; cellulose derivatives such as crystalline cellulose; gum arabic and the like. As for the inorganic excipients, for example, sulfates of calcium sulfate can be cited.

As for the binding agent, for example, the above-mentioned excipients; gelatin; polyvinylpyrrolidone; polyethylene glycol and the like can be cited.

As for the disintegrant, for example, the aforementioned excipients; such as croscarmellose sodium, chemically modified starch or cellulose derivatives of sodium carboxymethyl starch; cross-linked polyvinylpyrrolidine Ketones and so on.

As for lubricants, for example, talc; stearic acid; colloidal silica; waxes such as beeswax and spermaceti; sulfates such as sodium sulfate; lauryl sulfates such as sodium lauryl sulfate; Starch derivatives in excipients, etc.

As for emulsifiers, for example, colloidal clays such as bentonite and VEEGUM; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; such as polyoxyethylene Non-ionic surfactants of ethyl alkyl ether, etc.

As for the stabilizer, for example, parabens such as methyl paraben and propyl paraben; alcohols such as chlorobutanol; phenols such as phenol and cresol.

As for the diluent, for example, water, ethanol, propylene glycol and the like can be cited.

Examples of solvents for injections include water, ethanol, and glycerin.

Examples of dissolution aids include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, and the like.

As for the suspending agent, for example, stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, monostearic acid Surfactants such as glycerin; such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, etc. Hydrophilic polymers, etc.

Examples of isotonic agents include sodium chloride, glycerin, D-mannitol, and the like.

As the buffer, for example, buffers such as phosphate, acetate, carbonate, citrate, etc. can be cited.

As an analgesic, benzyl alcohol etc. are mentioned, for example.

The preservatives include, for example, parabens, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like.

As the anti-acidifying agent, for example, sulfite, ascorbic acid, etc. can be mentioned.

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

Regarding the administration route of the pharmaceutical composition of the present invention, either oral administration or non-oral administration may be used, and a suitable administration route may be selected according to the target disease. In addition, the route of administration may be either systemic administration or local administration. For parenteral administration, for example, intravenous administration, intraarterial administration, intramedullary administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, and transdermal administration Administration, intraosseous administration, intra-articular administration, etc. For example, for skin symptoms, transdermal administration and subcutaneous administration can be selected, but for neurological symptoms, intramedullary administration can be selected.

The pharmaceutical composition of the present invention is administered to a subject in a therapeutically or preventively effective amount. "Therapeutically effective amount" means the amount that exerts a therapeutic effect on a specific disease, administration form, and route of administration, and is appropriately determined according to the type of subject, the type of disease, symptoms, gender, age, chronic disease, and other factors.

The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to the type of object, the type of disease, symptoms, sex, age, chronic disease, and other factors.

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

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

The "therapeutically effective amount" in the present invention means an amount that exerts a therapeutic effect on a specific disease or symptom, form of administration, and route of administration, depending on the type of subject, the type of disease or symptom, symptoms, gender, age, chronic disease, Other factors are appropriately decided. [Example]

Hereinafter, the present invention will be explained concretely with examples. In addition, these embodiments are used to illustrate the present invention, and are not intended to 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-Cm1s-Cm1s} -T ^e2s -U ^m1s- A ^m1s -C ^e2s -U ^m1t -H (XPF-int1-001) (Sequence ID 1) Using an automatic nucleic acid synthesizer (MerMade 192X manufactured by BioAutomation), and using the phosphoamid method (Nucleic Acids Research, 12, 4539) (1984) for synthesis. For reagents, activator solution-3 (0.25 mol/L 5-benzylthio-1H-tetrazole/acetonitrile solution, manufactured by Wako Pure Chemical Industries, Ltd., product number 013-20011) , CAP A for AKTA (1-methylimidazole/acetonitrile solution, manufactured by Sigma-Aldrich, product number L040050), Cap B1 for AKTA (acetic anhydride/acetonitrile solution, manufactured by Sigma-Aldrich, product number L050050), Cap B2 for AKTA (Pyridine/acetonitrile solution, manufactured by Sigma-Aldrich, product number L050150), DCA Deblock (dichloroacetic acid/toluene solution, manufactured by Sigma-Aldrich, product number L023050). As a sulfurization reagent for forming phosphorothioate bonds, Use acetonitrile (dehydration, Kanto Chemical Co., product number 01837-05), pyridine (dehydration, Kanto Chemical Co., product number 11339-05) 1:1 (v/v) solution to dissolve phenylacetonitrile in a way that becomes 0.2 M Disulfide (manufactured by CARBOSYNTH, product number FP07495). As for the imidamide reagent, the 2'-O-Me nucleoside phosphoramidite (adenosine product number ANP-5751, cytidine Product number ANP-5752, guanosine product number ANP-5753, uridine product number ANP-5754) are manufactured by ChemGenes. Non-natural phosphamidite is used in Example 14 of JP 2000-297097 ( 5'-O-Dimethoxytrityl-2'-O,4'-C-ethylene-6-N-benzyladenosine-3'-O-(2-cyanoethyl Group N,N-diisopropyl)phosphoramidite), Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-2- N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), Example 22 (5'-O-dimethoxytrityl Group-2'-O,4'-C-ethylene-4-N-benzyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N-diiso Propyl) Phosphosamide), implementation Example 9 (5'-O-Dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) compounds. Using Glen Unysupport FC 96-well format 0.2 μmol (manufactured by Glen Research) as a solid phase carrier, the compound of Example 1 was synthesized. However, the time required for the condensation of the amide form was set to about 9 minutes.

By treating the protected oligonucleotide analogue with the target sequence with 600 μL of concentrated ammonia water, the oligomer is cut out from the support while removing the protecting group cyanoethyl group and nucleic acid base on the phosphorus atom On the protecting base. The mixed solution of the oligomers was mixed with 300 μL of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex), and poured into a Clarity SPE 96-well plate (manufactured by Phenomenex). Sequentially add Clarity QSP DNA loading buffer: 1 mL of water = 1:1 solution, 3 mL of water, 3 mL of 3% dichloroacetic acid (DCA) aqueous solution, and 6 mL of water, and then collect the 20 mM Tris aqueous solution: Acetonitrile = 9:1 solution extracted components. After the solvent was distilled off, the target compound was obtained. This compound was subjected to reverse phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100A (2.1×50 mm)), A solution: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, B Solution: methanol, B%: 10% → 25% (4 minutes, linear gradient); 60°C; 0.5 mL/minute; 260nm) When analyzed, it was dissolved in 2.78 minutes. The compounds were identified by negative ion ESI mass analysis.

The base sequence of this compound is the mRNA (equivalent to 5001~ of NG_011442.1) encoded by Homo sapiens ERCC cleavage repair 4 endonuclease catalytic subunit (ERCC4) (NCBI-GenBank accession number. NG_011442.1) 37192), the 412th T mutated into the A sequence, the sequence complementary to the 412th to the 429th sequence.

(Examples 2～36) The compounds of Examples 2 to 36 were also synthesized in the same manner as the compound of Example 1. The information of the compounds of Examples 1 to 36 is described in Table 1.

[Table 1]

In the "sequence" in the table, uppercase letters indicate ENA, and lowercase letters indicate 2'-OMe RNA. The linkages between nucleosides are all phosphorothioate linkages. The "start" and "end" in the table indicate the nucleotide codes in the base sequence represented by SEQ ID No. 63 for Examples 1-18, and SEQ ID No. 64 for Examples 19 to 36. Nucleotide number in the base sequence shown. The "sequence" in the table, excluding Example 17, indicates a sequence complementary to the base sequence from "start" to "end". The "sequence" in Example 17 means a sequence with "c" appended at the 3'end of the sequence complementary to the base sequence from "start" to "end". The "molecular weight" in the table refers to the actual measured value caused by the negative ion ESI mass analysis.

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

[Table 2]

In the "sequence" in the table, uppercase letters indicate ENA, and lowercase letters indicate 2'-OMe RNA. The linkages between nucleosides are all phosphorothioate linkages. The "start" and "end" in the table indicate the nucleotide numbers of the base sequence represented by the sequence identification number 63, and the "sequence" in the table indicates the complement of the base sequence from "start" to "end" the sequence of. The "molecular weight" in the table refers to the actual measured value caused by the negative ion ESI mass analysis.

(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 phosphamidite method (Nucleic Acids Research, 12, 4539 (1984)). The LNA part used the one described in WO99/14226 It is synthesized by phosphamidite. Hereinafter, or in the figure, it may be expressed as "LNA".

This compound was used in reverse phase HPLC (tube column (X-Bridge C18 2.5 μm (4.6×75 mm)), A solution: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, B solution: methanol , B%: 5% → 30% (20 minutes, linear gradient); 60°C; 1 mL/minute; 260nm) When analyzed, it was dissolved in 10.23 minutes. The compound was identified by negative ion ESI mass analysis (actual value: 4972.26).

(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 is sometimes expressed as "control DNA".

This compound was used in reverse phase HPLC (tube column (X-Bridge C18 2.5 μm (4.6×75 mm)), A solution: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, B solution: methanol , B%: 5% → 30% (20 minutes, linear gradient); 60°C; 1 mL/minute; 260nm) When analyzed, it was dissolved in 10.46 minutes. The compound was identified by negative ion ESI mass analysis (actual value: 4971.16).

(Test Example 1) Analysis of the amount of XPF mRNA splicing product, XPF protein expression, and DNA damage repair activity caused by the compound of the example (1) 1-1. The amount of XPF mRNA splicing product was evaluated in a 6-well plate, per 1 Each well was seeded with 5× ^{10 4} patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7(6), 969-974). After 24 hours, LNA was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 40 nM. After 4 hours, exchange the new medium (DMEM 10% FBS). After 24 hours of transfection, the cells were recovered. The total RNA was extracted with Direct-zol ^TM RNA MiniPrep (ZYMO RESEARCH). CDNA was obtained from 100 ng of total RNA using SuperScript (registered trademark) IV (Thermo Fisher Scientific). The QX100 ^TM Droplet Digital ^TM PCR system (Bio-Rad Laboratories, Inc.) was used to perform droplet digital PCR (ddPCR), and the spliced products were quantified. For fibroblasts derived from healthy humans (48BR), the splicing products were also quantified in the same way.

The ddPCR system was performed in the following order. Add 10 μl of QX200 Eva Green ddPCR Supermix (Bio-Rad Laboratories, Inc.), and add forward and reverse primers to make the final concentration 100 nM. Add 5 μl of sample cDNA (adjust the total RNA to 100 ng) and an appropriate amount of water to make the total volume 20 μl. Put the sample and 70 μl of EvaGreen Droplet Generation oil into the DG8 box ^{for QX200 TM} /QX100 ^{TM Droplet Generator, and put it into QX100 TM} Droplet Generator. The turbid solution was placed in a 96-well plate (Bio-Rad Laboratories, Inc.), and sealed with a PX1 PCR plate sealer. Use QX100 ^TM Droplet Reader for analysis. In addition, for the quantification of the correct splicing product, the value of ddPCR with the primer combination of XPF ex1-F and XPF ex2-R is used, and for the quantification of the abnormal splicing product, the value of XPF int1-F and XPF ex2-R is used. The value of the primer combination for ddPCR. Forward primer XPF ex1-F: 5'-CTCCTCTACCACTTTCTCCAGCTG-3' (serial identification number 109) XPF int1-F: 5'-CGCGATGACACAGAGAAGGATG-3' (serial identification number 110) reverse primer XPF ex2-R: 5'- GAGGGAGGTGTTCAACTCCTTC-3' (serial identification number 111)

The results are shown in Figure 1A. As a result of analyzing the mRNA of patient-derived cells, there were fewer correctly spliced products. In the cells transfected with the compound of Example 61 (LNA-int1-001), an increase in correctly spliced products was observed.

1-2. The protein expression level was evaluated in a 6-well plate, and patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7(6), 969-974) were seeded in each well of 16×10 ⁴ indivual. After 24 hours, the compound of the example or the reference example was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 40 nM (the method of transfection was in accordance with the instructions of the product). Exchange with new medium after 4 hours. After 24 hours of transfection, the protein was recovered, and Western blotting (using XPF Ab-1 (Cat. #MS-1381, manufactured by Thermo Fisher Scientific) and β-actin antibody (sc-47778, manufactured by Santa Crus) ) Investigate protein expression.

The results are shown in Figure 1B. The result of analyzing the amount of XPF protein derived from patient-derived fibroblasts is less than that of fibroblasts derived from healthy humans (48BR). In the cells transfected with the compound of Example 61 (LNA-int1-001), an increase in the amount of XPF protein was observed. On the other hand, in cells transfected with the compound of Reference Example 1 (LNA-int1-001S, control DNA), no increase in the amount of XPF protein was observed. Also, as shown in Figure 2B, using the compounds of Examples 1 to 18, the patient-derived cells (XPF-int1-001～XPF-int1-018) were similarly transfected and the amount of XPF protein was analyzed As a result, in all the compounds of Example 1 to Example 18, an increase in the amount of XPF protein was observed. Especially in the case of using the compounds of Examples 11 to 14, the amount of increase is large.

1-3. Evaluation of repair activity in a 96-well plate, with patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7(6), 969-974), inoculating 1× ^{10 4 cells in each well} . Cells derived from the same patient were seeded into a total of 10 wells (5 wells x 2 groups). After 24 hours, the compound of the example or the reference example was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 40 nM (the method of transfection was in accordance with the method of the product specification). After 4 hours, exchange with new medium (DMEM 10% FBS). After 24 hours of transfection, the 5 wells x 1 group was irradiated with 20J/m ² UV-C (the remaining 5 wells x 1 group were not irradiated). Discard the medium, wash with PBS once. After discarding the PBS, perform UV irradiation without placing any liquid in the well. For the cells after UV irradiation, serum-free medium with 5-ethynyl-2'-deoxyuridine (EdU) added at a final concentration of 5 μM was added. After culturing in EdU-containing medium for 4 hours, wash the cells once with PBS, add fixative (300mM sucrose, 2% formalin, 0.5% TritonX-100 in PBS), and let stand at 4°C for 20 minutes. Fix the cells. The cells were washed 3 times with PBS. Add staining solution (50 mM Tris pH 7.3, ₄ mM CuSO 4, 10 mM L-sodium ascorbate, 10 μM fluorescent dye 488 azide, 0.03 mg/ml DAPI in water), then shading and standing at room temperature 1 hour. The cells were washed 3 times with PBS-T (0.05% tween20 in PBS). Add fixative solution (3.7% formalin in PBS) and let stand for 20 minutes. Discard the fixative and add PBS. Calculate the average fluorescence intensity of the 488 azide in the core. The fluorescence value indicates the repair activity. For fibroblasts (48BR) derived from healthy humans, the repair activity was also evaluated in the same way.

The results are shown in Figure 1C. The results of analyzing the repair activity of patient-derived cells are lower than those of healthy human-derived cells. Compared with the patient-derived cells transfected with the compound of Reference Example 1 (LNA-int1-001S, control DNA), the patient-derived cells transfected with the compound of Example 61 (LNA-int1-001) were observed Increased repair activity. Also, as shown in Figure 2A, the patient-derived cells were similarly transfected with the compounds of Example 1 to Example 18 (XPF-int1-001 to XPF-int1-018), and the results of the repair activity were analyzed. In all the compounds of Example 1 to Example 18, an increase in repair activity was observed.

(Examples 62-73) The compounds of Examples 62 to 73 were also synthesized in the same manner as in Example 1. About Examples 62 to 73, they are described in Table 3.

[table 3]

In the "sequence" in the table, uppercase letters indicate ENA, and lowercase letters indicate 2'-OMe RNA. The linkages between nucleosides are all phosphorothioate linkages. The "start" and "end" in the table indicate the RefSeqGene (LRG_463) (NCBI-GenBank accession number. NG_011442.1) on chromosome 16 of Homo sapiens ERCC splicing repair 4 endonuclease catalytic subunit (ERCC4) The T variant of No. 412 is the nucleotide number of the sequence of A. The "sequence" in the table indicates the sequence complementary to the nucleotide sequence from "start" to "end". The "molecular weight" in the table refers to the actual measured value caused by the negative ion ESI mass analysis.

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

[Table 4]

In the "sequence" in the table, uppercase letters indicate ENA, and lowercase letters indicate 2'-OMe RNA. The linkages between nucleosides are all phosphorothioate linkages. The sequence of the compound of Reference Example 2 is the same as that of the mouse (Mus musculus) strain mdx muscle dystrophin (dystrophin) gene, part of the nucleosides of cds (NCBI-GenBank accession number. AH007099.2) No. 223 to No. 240 A sequence that is complementary to an acid sequence.

(Test Example 2) Analysis of the amount of XPF mRNA splicing product, XPF protein expression, and DNA damage repair activity caused by the compound of the example (2) 2-1. Evaluation of protein expression The protein expression level evaluation of the compound of the example was performed in the same manner as in the test of 1-2. Transfection of patient-derived cells with the compounds of Examples 37, 38, 44 to 46, and 53 to 60 (XPF-int1-011_1, XPF-int1-011_2, XPF-int1-012_4, XPF-int1-013_1, XPF -int1-013_2, XPF-int1-0-1, XPF-int1-0-2, XPF-int1-0-3, XPF-int1-0-4, XPF-int1-19-22) and Example 11 and 14 compounds (XPF-int1-11 and XPF-int1-14) and analyze the amount of XPF protein. The compound of Reference Example 2 was used as a negative control. The results are shown in Figure 3B. The compounds of Examples 37, 38, 44 to 46, 53, 57 to 60, 11, and 14 were compared with the negative control (nega con. in the figure) and those without the compound of the embodiment (w/o in the figure) In comparison, an increase in the amount of XPF protein was observed. 2-2. Evaluation of repair activity The repair activity evaluation of the compound of the example was performed in the same manner as the test of 1-3. The patient-derived cells were transfected with the compounds of Example 37 to Example 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-int1-019～022) and the compounds of Examples 11-14 (XPF-int1-11-14) were analyzed for repair activity. The compound of Reference Example 2 was used as a negative control. The results are shown in Figure 3A. The compounds of Examples 37 to 53, 57 to 60, and 11 to 14 were compared with the negative control (nega con. in the figure) and those without the compound of the example (w/o in the figure), and restoration was observed Increased activity. [Possibility of Industrial Use]

With the medical composition of the present invention, it is possible to treat specific XP-F groups that have never existed for its treatment methods.

without. [Sequence list non-keyword text]

Sequence ID No. 1 to 61: The oligonucleotide sequence of Example Compound 1 to 61 Sequence ID No. 62: Reference Example The oligonucleotide sequence of Compound 1 Sequence ID No. 63: The DNA of variant intron 1 of XPF gene Sequence ID No. 64: The DNA sequence of the variant intron 8 of XPF gene. Sequence ID No. 65～100: RNA sequence corresponding to Sequence ID No. 1～36. Sequence ID No. 101～108: Corresponding to Sequence ID No. 53～60 RNA sequence ID 109～111: DNA sequence ID of primer used in ddPCR Sequence ID 112～123: Oligonucleotide sequence of Example compound 62～73 Sequence ID 124～135: Corresponding sequence ID 112～ The RNA sequence of 123 SEQ ID No. 136: Reference Example Compound 2 Oligonucleotide sequence ID No. 137: In the RNA sequence sequence table corresponding to SEQ ID No. 136, Am1s, Gm1s, Cm1s, Um1s, Ae2s, Ge2s, Ce2s, Te2s, Ae1s, Ge1s, Ce1s, Te1s, Cs, Ts, Am1t, Gm1t, Cm1t, Um1t, T2t, G1t, and C1t, respectively representing A ^m1s , G ^m1s , C ^m1s , U ^m1s , A ^e2s , G ^e2s , G e2s C ^e2s , T ^e2s , A ^e1s , G ^e1s , C ^e1s , T ^e1s , C ^s , T ^s , A ^m1t , G ^m1t , C ^m1t , U ^m1t , T ^2t , G ^1t , and C ^1t .

Figure 1A shows that the amount of XPF mRNA splicing products increased in XPF patient-derived cells transfected with the compound of Example 61. The black bar indicates the amount of correct splicing products, and the reverse white bar indicates the amount of abnormal splicing products. Figure 1B shows that XPF protein expression increased in XPF patient-derived cells transfected with the compound of Example 61. Figure 1C shows that in cells derived from XPF patients transfected with the compound of Example 61, the DNA repair activity increased after UV irradiation. Figure 2A shows that in cells derived from XPF patients transfected with the compounds of Examples 1-18, the DNA repair activity increased after UV irradiation. Figure 2B shows that XPF protein expression increased in XPF patient-derived cells transfected with the compounds of Examples 1-18. Figure 3A shows that in cells derived from XPF patients transfected with the compounds of Examples 11-14 and 37-60, the DNA repair activity increased after UV irradiation. The black bar indicates the amount of correct splicing products, and the white bar indicates the amount of abnormal splicing products. Fig. 3B shows that XPF protein expression increased in XPF patient-derived cells transfected with the compounds of Examples 11, 13, 37, 38, 44 to 46, and 53 to 60. The upper panel shows the expression level of XPF protein, and the lower panel shows the expression level of β-actin.

without.

Claims (24)

An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which has a base sequence that can hybridize to a part of the intron region of XPF gene and has the activity of inhibiting abnormal post-transcriptional modification of XPF gene The 5'end and/or 3'end of the antisense oligonucleotide can be chemically modified. Such as the antisense oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, which inhibits the use of sequence recognition in the post-transcriptional modification of the XPF gene having the variant intron 1 sequence represented by SEQ ID No. 63 In the base sequence represented by number 63, the 5'splice site on the 5'side of guanine number 193 and the 3'splice site on the 3'side of guanine number 1658 are abnormally spliced. For example, the antisense oligonucleotide of claim 1 or 2 or a pharmaceutically acceptable salt thereof can hybridize with the following sequence: including the 155th to the 217th of the base sequence represented by SEQ ID NO: 63 A sequence of 15-30 nucleotides in the base sequence of the number. The antisense oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, which comprises a sequence that is more than 90% complementary to the following sequence: comprising the base represented by the sequence identification number 63 A sequence of 15-30 consecutive nucleotides in the base sequence of No. 155 to No. 217 in the sequence. The antisense oligonucleotide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by any one of sequence identification numbers 65 to 82 and 101 to 108 (sequence Where u can be replaced with t). The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, which comprises one or more sugar-modified nucleosides. The antisense oligonucleotide of claim 6 or a pharmaceutically acceptable salt thereof, wherein the sugar-modified nucleoside comprises 4'-(CH ₂ ) _n -O-2' cross-linking (where n is 1 or 2) Or 2'-O-methylated nucleosides. The antisense oligonucleotide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, which comprises more than one modified internucleoside linkage. The antisense oligonucleotide according to claim 8 or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is a phosphorothioate linkage. The antisense oligonucleotide according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, which comprises a base represented by any one of sequence identification numbers 1-18, 37-60, and 112-123 Base sequence. For example, the antisense oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, which inhibits the use of sequence recognition in the post-transcriptional modification of the XPF gene having the variant intron 8 sequence represented by the sequence identification number 64 Abnormal polyadenylation of the poly A additional sequence of No. 324 to No. 329 in the base sequence represented by No. 64. For example, the antisense oligonucleotide of claim 1 or 11 or a pharmaceutically acceptable salt thereof can hybridize with the following sequence: including the 309th to the 343th of the base sequence represented by SEQ ID No. 64 A sequence of 15-30 nucleotides in the base sequence of the number. The antisense oligonucleotide of any one of 11 and 12 or a pharmaceutically acceptable salt thereof, which comprises a sequence that is more than 90% complementary to the following sequence: including the sequence of the base sequence represented by the sequence identification number 64 A sequence of 15 to 30 consecutive nucleotides in the base sequence of No. 309 to No. 343. The antisense oligonucleotide according to any one of claims 1 and 11 to 13, or a pharmaceutically acceptable salt thereof, which comprises a base sequence represented by any one of sequence identification numbers 83 to 100 (in the sequence, u can be replaced with t). The antisense oligonucleotide according to any one of claims 1 and 11 to 14 or a pharmaceutically acceptable salt thereof, which comprises more than one sugar-modified nucleoside. The antisense oligonucleotide of claim 15 or a pharmaceutically acceptable salt thereof, wherein the sugar-modified nucleoside comprises 4'-(CH ₂ ) _n -O-2' cross-linking (where n is 1 or 2) And 2'-O-methylated nucleosides. The antisense oligonucleotide according to any one of claims 1 and 11 to 16, or a pharmaceutically acceptable salt thereof, which comprises more than one modified internucleoside linkage. The antisense oligonucleotide according to claim 17 or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is a phosphorothioate linkage. The antisense oligonucleotide according to any one of claims 1 and 11 to 18 or a pharmaceutically acceptable salt thereof, which comprises a base sequence represented by any one of sequence identification numbers 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 delivery of the oligonucleotide. A medical composition for the treatment of xeroderma group F, which comprises the antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof. A treatment method for group F of xeroderma pigmentosum, 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. An 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 F group. A use of the antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof for the manufacture of a medical composition for the treatment of Xeroderma pigmentosum F group.

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