WO2021112106A1 - Agent thérapeutique contre le xeroderma pigmentosum de complémentation f - Google Patents

Agent thérapeutique contre le xeroderma pigmentosum de complémentation f Download PDF

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WO2021112106A1
WO2021112106A1 PCT/JP2020/044768 JP2020044768W WO2021112106A1 WO 2021112106 A1 WO2021112106 A1 WO 2021112106A1 JP 2020044768 W JP2020044768 W JP 2020044768W WO 2021112106 A1 WO2021112106 A1 WO 2021112106A1
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antisense oligonucleotide
pharmaceutically acceptable
acceptable salt
seq
xpf
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PCT/JP2020/044768
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English (en)
Japanese (ja)
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朋男 荻
由華 中沢
小泉　誠
貴生 小路
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第一三共株式会社
国立大学法人東海国立大学機構
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Publication of WO2021112106A1 publication Critical patent/WO2021112106A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a therapeutic agent for group F of xeroderma pigmentosum, an antisense oligonucleotide as an active ingredient thereof, or a pharmaceutically acceptable salt thereof.
  • Xeroderma pigmentosum is a congenital gene involved in nucleotide excision repair mechanism (NER) that removes photoDNA damage caused by ultraviolet rays in sunlight from the genome and damage bypass synthesis (TLS). It develops due to an abnormality.
  • NER nucleotide excision repair mechanism
  • TLS damage bypass synthesis
  • XP patients have photosensitivity, xeroderma pigmentosum in the sun-exposed area, high cancer incidence, and neurological symptoms, and the prevalence of Japanese is estimated to be about 1 in 25,000 births. ..
  • the POHL gene encoding TLS polymerase is known.
  • XP cases with each mutation are classified into the complementarity group of XP-A to XP-G group and XP-V (variant) group.
  • the proportion of each complementary group in XP cases varies depending on the race, but in the Japanese population, there are many abnormalities in the XP-A group and then in the XP-V group.
  • NER deficiency is shown.
  • NER is divided into whole genome repair (GG-NER) and transcriptional conjugated repair (TC-NER) according to the recognition form of DNA damage.
  • the XP-C / E group shows a deficiency of only GG-NER
  • the XP-A / B / D / F / G group shows a deficiency of both GG-NER and TC-NER.
  • DNA damage accumulates in the genome due to these NER deficiencies, leading to genome instability, and skin cancer occurs frequently at a young age.
  • Neurological symptoms differ in onset time depending on the complementary group and disease-causing mutation, but especially in cases with group A Japanese founder mutation (IVS3-1G> C), loss of XPA protein expression results in the loss of XPA protein expression and the full function of NER. And show severe neurological symptoms.
  • XP is a systemic hereditary disease, and no effective treatment has been developed at present. XP is often diagnosed by visiting a dermatologist after a severe sunburn in infancy. In addition, a rough prognosis can be determined by genetic testing. Although it is possible to avoid exposure to sunlight and try to prevent skin cancer, there is no effective way to alleviate the progression of neurological symptoms that have a significant effect on prognosis and quality of life.
  • a method for measuring DNA repair activity using fibroblasts established from a patient's skin patch is known. These include the Irregular DNA Synthesis (UDS) test, which measures GG-NER activity by detecting repair DNA synthesis after removal of photoDNA damage, and RNA synthesis in regions of high transcriptional activity after DNA damage.
  • UDS Irregular DNA Synthesis
  • GG-NER activity by detecting repair DNA synthesis after removal of photoDNA damage
  • RNA synthesis in regions of high transcriptional activity after DNA damage There is a method for evaluating TC-NER activity by examining resilience (RRS) (Non-Patent Documents 1 and 2).
  • RRS resilience
  • Complementarity groups can be identified by evaluating UDS / RRS in patient-derived cells and investigating which known XP genes restore these activities (Non-Patent Documents 3 and 4).
  • An object of the present invention is to provide a therapeutic means for such XP-F group.
  • the present inventors have identified two novel internal mutations in introns that are the cause of the disease in patients in the XP-F group.
  • the first is a novel Japanese founder mutation that exists deep in the intron (about 100 bp downstream from the exon 1-intron 1 boundary) and gives rise to a new U1 nuclear small ribonuclear protein (U1 snRNP) binding sequence. It was shown that this caused a decrease in gene expression due to abnormal alternative splicing.
  • the second is a single base substitution mutation in the deep intron (several hundred bp downstream from the exon 8-intron 8 boundary site), which forms a pathological polyA addition sequence (cleaved / polyadenylation factor binding sequence). It has been shown that incomplete transcription termination and mRNA destabilization lead to disease onset.
  • the present inventors have further studied and completed the present invention.
  • the present invention includes the following inventions.
  • the antisense oligonucleotide or pharmaceutically acceptable salt thereof according to [1], which suppresses abnormal splicing using the 3'splice site on the 3'side of the second guanine.
  • Antisense oligonucleotides or pharmaceutically acceptable salts thereof [4] 90% or more, preferably 95% or more, optimally completely complementary to the sequence consisting of 15 to 30 nucleotides in the 155th to 217th base sequences in the base sequence represented by SEQ ID NO: 63.
  • the sugar-modified nucleoside is a nucleoside containing 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) or 2'-O-methylation, [6] ]
  • the antisense oligonucleotide or a pharmaceutically acceptable salt thereof [8] The antisense oligonucleotide according to any one of [1] to [7] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
  • [12] The description in [1] or [11], which can hybridize with a sequence consisting of 15 to 30 nucleotides in a row in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64.
  • Antisense oligonucleotides or pharmaceutically acceptable salts thereof. [13] 90% or more, preferably 95% or more, optimally completely complementary to the sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64.
  • the antisense oligonucleotide according to any one of [1], [11] and [12] or a pharmaceutically acceptable salt thereof, which comprises a specific sequence.
  • the sugar-modified nucleoside is a nucleoside containing 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) and 2'-O-methylation, [15] ]
  • the antisense oligonucleotide or a pharmaceutically acceptable salt thereof [17] The antisense oligonucleotide according to any one of [1] and [11] to [16] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
  • a pharmaceutical composition for treating xeroderma pigmentosum F group which comprises the antisense oligonucleotide according to any one of [1] to [20] or a pharmaceutically acceptable salt thereof.
  • Xeroderma pigmentosum F which comprises administering to a patient an effective amount of the antisense oligonucleotide according to any one of [1] to [20] or a pharmaceutically acceptable salt thereof. How to treat the group.
  • the present invention also provides the following [A1] to [A20].
  • the antisense oligonucleotide or pharmaceutically acceptable salt thereof according to [A1], which suppresses abnormal splicing using the 3'splice site on the 3'side of the second guanine.
  • [A3] Described in [A1] or [A2], which can hybridize with a sequence consisting of 15 to 30 consecutive nucleotides in the 175th to 217th base sequences in the base sequence represented by SEQ ID NO: 63.
  • Antisense oligonucleotides or pharmaceutically acceptable salts thereof are examples of antisense oligonucleotides or pharmaceutically acceptable salts thereof.
  • 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.
  • the sugar-modified nucleoside is a nucleoside containing 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) or 2'-O-methylation, [A6].
  • the antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a pharmaceutically acceptable salt thereof.
  • [A8] The antisense oligonucleotide according to any one of [A1] to [A7] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
  • [A9] The antisense oligonucleotide according to [A8] or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
  • [A10] The antisense oligonucleotide according to any one of [A1] to [A9] or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by any of SEQ ID NOs: 1 to 18 and 37 to 60.
  • [A11] In post-transcriptional modification of the XPF gene having the mutant intron 8 sequence represented by SEQ ID NO: 64, abnormal polyadenylation using the 324th to 329th polyA addition sequence in the nucleotide sequence represented by SEQ ID NO: 64.
  • [A12] Described in [A1] or [A11], which can hybridize with a sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64.
  • Antisense oligonucleotides or pharmaceutically acceptable salts thereof are examples of antisense oligonucleotides or pharmaceutically acceptable salts thereof.
  • [A13] 90% or more, preferably 95% or more, optimally completely complementary to the sequence consisting of consecutive 15 to 30 nucleotides in the 309th to 343rd base sequences in the base sequence represented by SEQ ID NO: 64.
  • [A14] Described in any of [A1] and [A11] to [A13], which comprises a base sequence represented by any of SEQ ID NOs: 83 to 100 (u may be replaced with t in the sequence).
  • Antisense oligonucleotide or pharmaceutically acceptable salt thereof is described in any of [A1] and [A11] to [A13]
  • the antisense oligonucleotide according to any one of [A1] and [A11] to [A14] or a pharmaceutically acceptable salt thereof, which comprises one or more sugar-modified nucleosides.
  • the sugar-modified nucleoside is a nucleoside containing 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) and 2'-O-methylation, [A15].
  • the antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a nucleoside containing 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) and 2'-O-methylation, [A15].
  • [A17] The antisense oligonucleotide according to any one of [A1] and [A11] to [A16] or a pharmaceutically acceptable salt thereof, which comprises one or more modified nucleoside linkages.
  • [A18] The antisense oligonucleotide according to [A17] or a pharmaceutically acceptable salt thereof, wherein the modified nucleoside bond is a phosphorothioate bond.
  • [A19] The antisense oligonucleotide according to any one of [A1] and [A11] to [A18] or a pharmaceutically acceptable salt thereof, which comprises the base sequence represented by any of SEQ ID NOs: 19 to 36.
  • a therapeutic pharmaceutical composition for group F of xeroderma pigmentosum which comprises the antisense oligonucleotide according to any one of [A1] to [A19] or a pharmaceutically acceptable salt thereof.
  • FIG. 1A shows an increase in the amount of XPF mRNA splicing product in cells derived from an XPF patient transfected with the compound of Example 61. Black bars represent the correct amount of splicing products and white bars represent the amount of abnormal splicing products.
  • FIG. 1B shows an increase in XPF protein expression in cells derived from XPF patients transfected with the compound of Example 61.
  • FIG. 1C shows that DNA repair activity after UV irradiation is increased in cells derived from XPF patients transfected with the compound of Example 61.
  • FIG. 2A shows that DNA repair activity after UV irradiation is increased in cells derived from XPF patients transfected with the compounds of Examples 1-18.
  • FIG. 1A shows an increase in the amount of XPF mRNA splicing product in cells derived from an XPF patient transfected with the compound of Example 61. Black bars represent the correct amount of splicing products and white bars represent the amount of abnormal splicing products
  • FIG. 2B shows an increase in XPF protein expression in cells derived from XPF patients transfected with the compounds of Examples 1-18.
  • FIG. 3A shows that DNA repair activity after UV irradiation is increased in cells derived from XPF patients transfected with the compounds of Examples 11-14 and 37-60. Black bars represent the correct amount of splicing products and white bars represent the amount of abnormal splicing products.
  • FIG. 3B shows increased XPF protein expression levels in cells derived from XPF patients transfected with the compounds of Examples 11, 13, 37, 38, 44-46, and 53-60. The upper panel shows the expression level of XPF protein, and the lower panel shows the expression level of ⁇ -actin.
  • xeroderma pigmentosum F group (also referred to as XP-F group) is one of the complementary groups of xeroderma pigmentosum, and both GG-NER and TC-NER. Shows a defect.
  • the gene for which the causative mutation has been reported is the XPF gene.
  • the "XPF gene” is a human gene encoding an endonuclease involved in the nucleotide excision and repair mechanism.
  • the XPF gene is also referred to as the ERCC4 gene, ERCC11 gene, FANCQ gene, RAD1 gene, or XFEPS gene.
  • the sequence of the XPF gene is known as, for example, Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4), RefSeqGene (LRG_463) on chromosome 16 (NCBI-GenBank accession No. NG_011442.1).
  • the "mutant intron 1 sequence” is the base sequence of intron 1 of the wild-type XPF gene (base sequence 5217 to 6874 in the above NG_011442.1), which is 196th from the 5'end of intron 1 (the base sequence of intron 1).
  • T of 5412 is a sequence mutated to A, and is a base sequence represented by SEQ ID NO: 63.
  • the mutation site corresponds to the 196th position in the base sequence represented by SEQ ID NO: 63.
  • the "mutant intron 8 sequence” refers to the 326th from the 5'end of the intron 8 in the base sequence of the wild-type XPF gene intron 8 (the base sequence of 20588 to 22609 in the above NG_011442.1).
  • C is the sequence mutated to T (20913th), and is the base sequence represented by SEQ ID NO: 64.
  • the mutation site corresponds to the 326th position in the base sequence represented by SEQ ID NO: 64.
  • post-transcriptional modification refers to the modification that pre-mRNA undergoes in the process of becoming a mature mRNA, which includes cap addition to the 5'end, polyadenylation at the 3'end, and splicing. included.
  • abnormal post-transcriptional modification refers to post-transcriptional modification that is not normally observed in post-transcriptional modification of wild-type genes, and includes, for example, abnormal splicing and abnormal polyadenylation.
  • abnormal splicing refers to splicing that is not normally found in post-transcriptional modification of wild-type genes.
  • mRNA splicing proceeds by recognizing the 5'splice site, branch site, and 3'splice site in the intron by the splicing factor, the small nuclear ribonuclear protein (snRNP).
  • the mutation at position 196 in the nucleotide sequence represented by SEQ ID NO: 63 gives rise to a new recognition sequence for the splicing factor U1 snRNP (GTATGTAA), which is number 193.
  • GTATGTAA splicing factor
  • the 5'side of guanine is the 5'splice site.
  • abnormal splicing occurs using the 5'splice site on the 5'side of the 193rd guanine and the 3'splice site on the 3'side of the 1658th guanine in the nucleotide sequence represented by SEQ ID NO: 63.
  • abnormal polyadenylation refers to polyadenylation that is not normally observed in post-transcriptional modification of wild-type genes.
  • Polyadenylation of mRNA occurs depending on the polyA addition sequence (also referred to as cleavage / polyadenylation factor binding sequence or polyadenylation signal).
  • Poly-A addition sequences in humans usually include AATAAA.
  • AATAAA poly A addition sequence
  • SEQ ID NO: 64 is generated by the 326th mutation in the nucleotide sequence represented by SEQ ID NO: 64.
  • abnormal polyadenylation using the 324th to 329th polyA addition sequences in the nucleotide sequence represented by SEQ ID NO: 64 occurs.
  • the "antisense oligonucleotide” is a single-stranded oligonucleotide having an ability to hybridize to a nucleotide containing a target base sequence. “Able to hybridize” means double-stranded with a target nucleotide by interaction between bases (AG (adenine-guanine) and CT / U (cytosine-thymine / uracil)). Means that can be formed. The antisense oligonucleotide need only have sequence complementarity with the target sequence to the extent that it can hybridize, and does not have to be a completely complementary sequence.
  • the antisense oligonucleotide can be a DNA, RNA, and DNA / RNA chimera.
  • the antisense oligonucleotide may also contain modifications such as modified nucleosides and bonds between modified nucleosides.
  • Hybridizing includes hybridizing under low stringent conditions, hybridizing under medium stringent conditions, and hybridizing under high stringent conditions.
  • Low stringent conditions can be, for example, 5 x SSC, 5 x Denhardt solution, 0.5% SDS, 50% formamide, 32 ° C., or equivalent.
  • Medium stringent conditions are, for example, 5 x SSC, 5 x Denhardt solution, 0.5% SDS, 50% formamide, 42 ° C, or 5 x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5). , 50% formamide, 42 ° C., or equivalent.
  • “High stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 50% formamide, 50 ° C, or 0.2 ⁇ SSC, 0.1% SDS, 65 ° C, or equivalent conditions. Can be. Multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art will appropriately select these factors. By doing so, it is possible to achieve similar stringency. The above conditions can also be realized by using a commercially available hybridization reagent.
  • high stringent conditions can be defined as 0.7 in a commercially available hybridization solution ExpressHyb TM hybridization solution (manufactured by Clontech) by hybridizing at 68 ° C. or by using a filter on which DNA is fixed. After hybridization at 68 ° C. in the presence of ⁇ 1.0 M NaCl, a 0.1-2 times concentration SSC solution (1 time concentration SSC consists of 150 mM NaCl and 15 mM sodium citrate) was used at 68 ° C. It can be realized by cleaning with.
  • nucleosides include natural nucleosides and modified nucleosides.
  • Natural nucleosides are 2'-deoxy such as 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, thymidine, 2'-deoxyuridine, etc.
  • Ribonucleosides such as nucleosides, adenosine, guanosine, thymidine, 5-methylcytidine, and uridine.
  • uracil (U) or (u) and thymine (T) or (t) are compatible, and either uracil (U) or (u) and thymine (T) or (t) It can be used for base pairing with the complementary strand adenine (A) or (a).
  • 2'-deoxy adenosine A t, 2'-deoxyguanosine and G t, 2'-deoxycytidine and C t, 2'-deoxy-5-methylcytidine 5meC t, thymidine T t, 2'-deoxyuridine may represent a U t.
  • nucleotides corresponding thereto in the present specification, 2'-deoxyadenosine nucleotide A p, 2'-deoxyguanosine nucleotides G p, 2'-deoxycytidine nucleotides C p, 2'-deoxy - 5meC 5-methyl cytidine nucleotides p, the thymidine nucleotides T p, sometimes a 2'-deoxyuridine nucleotide represented as U p.
  • sugar-modified nucleoside means a nucleoside in which the sugar portion of the nucleoside is modified.
  • Sugar-modified nucleosides include all forms of sugar modification known in the art to which the present invention belongs.
  • Sugar-modified nucleosides include, for example, 2'-modified nucleosides, 4'-thio-modified nucleosides, 4'-thio-2'-modified nucleosides and bicyclic sugar-modified nucleosides.
  • Examples of 2'-modified nucleotides are halo, allyl, amino, azide, O-allyl , OC 1- C 10 alkyl, OCF 3 , O- (CH 2 ) 2- O-CH 3 , 2'-.
  • O (CH 2 ) 2 SCH 3 , O- (CH 2 ) 2 -ON (R m ) (R n ), or O-CH 2- C ( O) -N (R m ) (R n )
  • Each R m and R n is H, an amino protecting group, or a substituted or unsubstituted C 1- C 10 alkyl individually.
  • 2'-O-methylguanosine 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methyluridine.
  • 2'-O-aminoethylguanosine, 2'-O-aminoethyladenosine, 2'-O-aminoethylcytidine, and 2'-O-aminoethyluridine are described in the literature (Blommers et al. Biochemistry (1998), 37). , 17714-17725.).
  • 2'-O-propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine, and 2'-O-propyluridine are described in the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832). -7838.) Can be synthesized according to. Commercially available reagents can be used for 2'-O-allyl guanosine, 2'-O-allyl adenosine, 2'-O-allyl cytidine, and 2'-O-allyl uridine.
  • 2'-O-Methoxyethyl guanosine, 2'-O-Methoxyethyl adenosine, 2'-O-Methoxyethyl cytidine, and 2'-O-Methoxyethyl uridine are available in the patent (US6261840) or in the literature (Martin, P. et al. Synthesized according to Helv. Chim. Acta. (1995) 78, 486-504.
  • 2'-O-butylguanosine, 2'-O-butyladenosine, 2'-O-butylcytidine, and 2'-O-butyl Uridine can be synthesized according to the literature (Lesnik, EA et al.
  • Examples of 4'-thio-modified nucleosides include ⁇ -D-ribonucleosides in which the 4'-oxygen atom is replaced by a sulfur atom (Hoshika, S. et al. FEBS Lett. 579, p. 3115). -3118, (2005); Dande, P. et al. J. Med. Chem. 49, p. 1624-1634 (2006); Hoshika, S. et al. ChemBioChem. 8, p. 2133-2138, (2007). )).
  • 4'-thio-2'-modified nucleosides examples include 2'-H or 4'-thio-2'-modified nucleosides carrying 2'-O-methyl (Matsugami, et.). al. Nucleic Acids Res. 36, 1805 (2008)).
  • bicyclic sugar-modified nucleosides include nucleosides that retain a second ring formed by cross-linking two atoms of the ribose ring, and examples of such nucleosides are 2'-. 2', 4'-BNA / LNA (bridged nucleoside acids / locked nucleoside acids) (Obika, S. et al. Tetrahedron Lett., 38, p. 8735-) in which an oxygen atom and a 4'-carbon atom are crosslinked with a methylene chain. (1997) .; Obika, S. et al., Tetrahedron Lett., 39, p.5401- (1998) .; A.A.
  • a sugar-modified nucleosides that include a 2'-O- methylation modification for example, those corresponding to A t A m1t, G m1t those corresponding to G t, those corresponding to the C t C M1T, may 5meC those corresponding to 5meC t M1T, those corresponding to U t is expressed as like U M1T.
  • sugar modified nucleotides containing 2'-O- methylation modification corresponding to them those corresponding to A p A m1p, G m1p those corresponding to G p, those corresponding to the C p C m1p , may represent 5meC m1p those corresponding to 5meC p, those corresponding to U p and U m1p.
  • a sugar-modified nucleosides that include a 2'-O- methoxyethyl of modifications, for example, those corresponding to A t A m2t, G m2t those corresponding to G t, those corresponding to 5meC t 5meC m2t, those corresponding to T t may be represented by such T m2t.
  • the sugar modified nucleosides containing 4'-CH 2 -0-2 'bridge correspond to one corresponding to A t A 1t, those corresponding to the G t in G 1t, 5meC t sometimes represent things C 1t, those corresponding to T t and T 1t.
  • the sugar-modified nucleotides comprising a 4'-CH 2 -0-2 'bridge their corresponding, which corresponds to that corresponding to A p A E1p, those corresponding to the G p G E1p, the 5meC p certain C E1p, also those corresponding to T p represents a T E1p.
  • 4 - as a sugar-modified nucleosides that include a '(CH 2) 2 -0-2' bridge for example, those corresponding to A t A 2t, those corresponding to the G t G 2t, 5meC t
  • the one corresponding to T t may be expressed as C 2t
  • the one corresponding to T t may be expressed as T 2t.
  • the "modified nucleoside bond” refers to a bond in which a naturally occurring nucleoside bond (that is, a phosphodiester nucleoside bond) is replaced or changed. That is, the antisense oligonucleotide containing the modified nucleoside linkage comprises the modification of the phosphate group of at least one nucleotide.
  • the modified nucleoside bond include a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a boranophosphate bond, a phosphoramidate bond and the like.
  • phosphorothioate esters has a phosphorothioate ester in place of phosphoric acid esters of nucleotides, the one corresponding to A p A s, a G s which corresponds to G p, those corresponding to the C p some C s, 5meC those corresponding to 5meC p s, the T s corresponds to T p, also those corresponding to U p expressed as U s.
  • T For 2'-O- methoxyethyl of modified and sugar-modified nucleotides comprising a phosphorothioate, those corresponding to A s A m2s, those corresponding to the G s G m2s, 5meC those corresponding to 5meC s m2s, T The one corresponding to s may be expressed as T m2s.
  • the treatment of a disease or symptom includes prevention of the onset of the disease, suppression or inhibition of exacerbation or progression, alleviation or exacerbation or suppression of progression of one or more symptoms exhibited by an individual suffering from the disease. Includes treatment of secondary illnesses.
  • the pharmaceutical composition of the present invention and the antisense oligonucleotide relates to a pharmaceutical composition for treating xeroderma pigmentosum F group (XP-F group) (hereinafter referred to as the pharmaceutical composition of the present invention), and Provided is an antisense oligonucleotide which is an active ingredient thereof or a pharmaceutically acceptable salt thereof.
  • XP-F group xeroderma pigmentosum F group
  • the pharmaceutical composition of the present invention contains an antisense oligonucleotide that suppresses abnormal post-transcriptional modifications caused by disease-causing mutations in the XP-F group, particularly abnormal post-transcriptional modifications caused by two types of novel intron internal mutations.
  • an antisense oligonucleotide that suppresses abnormal post-transcriptional modifications caused by disease-causing mutations in the XP-F group, particularly abnormal post-transcriptional modifications caused by two types of novel intron internal mutations.
  • the two mutations that are the cause of the disease in the XP-F group newly identified by the present inventors are the introns in the base sequence of intron 1 of the wild XPF gene (base sequence 5217 to 6874 in the above NG_011442.1).
  • the application target of the pharmaceutical composition of the present invention is the XP-F group in which at least one of these two types of mutations is the cause of the disease.
  • the XPF gene contains the above-mentioned mutation, and as long as the above-mentioned abnormal splicing or abnormal polyadenylation can occur, the XPF gene is further mutated (for example, deletion or substitution of one or several nucleotides). Addition or insertion) may be included.
  • the antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof has a base sequence capable of hybridizing with a part of the intron region of the XPF gene. As long as the abnormal post-transcriptional modification of the XPF gene can be suppressed, a part of the intron region, that is, the range of the target sequence is not particularly limited. Whether or not the antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress abnormal post-transcriptional modification of the XPF gene is described in the following Examples, Evaluation of XPF mRNA Splicing Product Amount, Protein. It is determined by the same evaluation method as the expression level evaluation or the repair activity evaluation.
  • the antisense oligonucleotide or its pharmaceutically acceptable salt is abnormally transcribed of the XPF gene. It is judged to suppress post-modification.
  • the pharmaceutical composition of the present invention applies to the XP-F group whose disease cause is the above-mentioned mutation in intron 1.
  • the pharmaceutical composition of the present invention in this embodiment is referred to as the pharmaceutical composition I of the present invention.
  • the pharmaceutical composition I of the present invention is located on the 5'side of the 193rd guanine in the nucleotide sequence represented by SEQ ID NO: 63. It contains one or more antisense oligonucleotides that suppress abnormal splicing using the 5'splice site and the 3'splice site on the 3'side of position 1658 guanine.
  • SEQ ID NO: 63 "abnormal splicing using the 5'splice site on the 5'side of the 193rd guanine and the 3'splice site on the 3'side of the 1658th guanine in the nucleotide sequence represented by SEQ ID NO: 63" is referred to as "SEQ ID NO: It can also be rephrased as "abnormal splicing in which the 1st to 192nd adenine in the base sequence represented by 63 is recognized as an exon and the 1658th guanine from the 193rd guanine is recognized as an intron".
  • Whether or not the antisense oligonucleotide in the pharmaceutical composition I of the present invention suppresses abnormal splicing in intron 1 is determined by the XPF mRNA splicing product amount evaluation, protein expression level evaluation, or repair activity evaluation described in the following Examples. It is determined by a similar evaluation method. If an increase in the expression of the correctly spliced XPF gene or a recovery in the DNA repair activity is confirmed by any of the evaluation methods, it is judged that the antisense oligonucleotide suppresses the abnormal splicing in the intron 1.
  • ddPCR Droplet digital PCR
  • ddPCR can be performed using commercially available reagents and equipment (eg, QX100 TM Droplet Digital TM PCR system (Bio-Rad Laboratories, Inc.), etc.).
  • protein expression assessment whether or not transfection of antisense oligonucleotides increases correctly spliced XPF gene products in cells derived from patients in the XP-F group whose disease is caused by the above mutation in intron 1. Is confirmed by Western blotting.
  • the repair activity evaluation in cells derived from patients in the XP-F group caused by the above mutation in intron 1, the activity of repairing DNA damage caused by UV irradiation by transfecting an antisense oligonucleotide was found. Check if it recovers.
  • the antisense oligonucleotide in the pharmaceutical composition I of the present invention is not limited to a specific one as long as it suppresses abnormal splicing in intron 1, but for example, it is located at positions 155 to 217 (preferably) in the base sequence represented by SEQ ID NO: 63. Is a contiguous 15-30 nucleotides (preferably 15-23 nucleotides, more preferably 15) in the 175th-217th, more preferably 179th-213rd, even more preferably 183rd-203rd base sequence. A sequence consisting of ( ⁇ 18 nucleotides) can be targeted.
  • the base sequence targeted by the antisense oligonucleotide in the pharmaceutical composition I of the present invention is a base sequence represented by any one selected from SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135.
  • the antisense oligonucleotide in the pharmaceutical composition I of the present invention can hybridize with the sequence (ie, the target sequence).
  • the antisense oligonucleotide in the pharmaceutical composition I of the present invention can hybridize with the target sequence under highly stringent conditions.
  • the antisense oligonucleotide in the pharmaceutical composition I of the present invention may have a base sequence (tail sequence) that does not contribute to hybridization with the target sequence at its 5'end and / or 3'end.
  • the number of bases contained in each tail sequence is 5 or less (preferably 4, 3, 2 or 1), and those having no tail sequence are optimal.
  • the base sequence of the portion of the pharmaceutical composition I of the present invention excluding the tail sequence of the antisense oligonucleotide has a sequence complementarity of 70% or more with the target sequence as long as it retains the hybridization activity with the target sequence. It is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and is optimally completely complementary.
  • the antisense oligonucleotides in pharmaceutical composition I of the present invention are SEQ ID NOs: 65-82, 101-108, and 124-135 (preferably SEQ ID NOs: 65-82, 101, and 105-108, more preferably. Includes a base sequence represented by any one selected from SEQ ID NOs: 65-82, and even more preferably SEQ ID NOs: 75-78).
  • the nucleotide sequences represented by SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135 are RNA sequences corresponding to SEQ ID NOs: 1 to 18, 53 to 60, and 112 to 123, respectively, which are described in Examples described later. It is described as.
  • the uridine residue in the antisense oligonucleotide may be replaced with a modified nucleotide based on the corresponding thymidine residue. Therefore, the base sequence in which "u” is replaced with "t” in the base sequences represented by SEQ ID NOs: 65 to 82, 101 to 108, and 124 to 135 is also included in this embodiment.
  • the base sequence of the portion of the pharmaceutical composition I of the present invention excluding the tail sequence of the antisense oligonucleotide is 5 bases or less (preferably) with the target sequence as long as it has hybrid activity with the target sequence. There may be a mismatch of 4 bases or less, more preferably 3 bases, 2 bases or 1 base), but optimally there is no mismatch.
  • the antisense oligonucleotide in the pharmaceutical composition I of the present invention is the recognition sequence GTAAGTAA of the 5'splice site newly generated in the mutant intron 1 sequence (No. 193 to 200 in the base sequence represented by SEQ ID NO: 63). It is considered that abnormal splicing is suppressed by inhibiting the binding of U1snRNP to (sequence).
  • U1 snRNP has a sequence that is almost complementary to the recognition sequence at the 5'splice site, and base pairs are formed during the splicing process.
  • the antisense oligonucleotide in the pharmaceutical composition I of the present invention is at least one base of the 193 to 200th sequence in the base sequence represented by SEQ ID NO: 63, for example, 2 bases, 3 bases, 4 bases. Sequences containing 5, 6 bases, 7 bases, or 8 bases can be targeted.
  • the length of the antisense oligonucleotide in the pharmaceutical composition I of the present invention is not particularly limited as long as it suppresses abnormal splicing in intron 1, and is, for example, 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides. obtain.
  • the pharmaceutical composition of the present invention applies to the XP-F group whose disease cause is the above-mentioned mutation in intron 8.
  • the pharmaceutical composition of the present invention in this embodiment is referred to as the pharmaceutical composition II of the present invention.
  • the pharmaceutical composition II of the present invention adds polyA at positions 324 to 329 in the base sequence represented by SEQ ID NO: 64. Includes one or more antisense oligonucleotides that suppress aberrant polyadenylation using sequences.
  • whether or not the antisense oligonucleotide in the pharmaceutical composition II of the present invention suppresses abnormal polyadenylation in intron 8 is determined by the same evaluation method as the protein expression level evaluation or repair activity evaluation described in the following Examples. It is determined. If an increase in the expression of the correctly spliced XPF gene or a recovery in the DNA repair activity is confirmed by any of the evaluation methods, it is judged that the antisense oligonucleotide suppresses the abnormal polyadenylation in the intron 8.
  • transfection of antisense oligonucleotides increases the polyadenylated XPF gene product at the correct location in cells from patients in the XP-F group whose disease is caused by the mutation in intron 8. Check if it is.
  • repair activity cells derived from patients in the XP-F group caused by the above mutation in intron 8 were found to have the activity of repairing DNA damage caused by UV irradiation by transfecting antisense oligonucleotides. Check if it recovers.
  • the antisense oligonucleotide in the pharmaceutical composition II of the present invention is not limited to a specific one as long as it suppresses abnormal polyadenylation in intron 8, but for example, the 309th to 343rd nucleotides in the nucleotide sequence represented by SEQ ID NO: 64.
  • a sequence consisting of 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides in a sequence within a base sequence can be targeted.
  • the antisense oligonucleotide in the pharmaceutical composition II of the present invention can hybridize with the above sequence (ie, the target sequence).
  • the antisense oligonucleotide in the pharmaceutical composition II of the present invention can hybridize with the target sequence under highly stringent conditions.
  • the antisense oligonucleotide in the pharmaceutical composition II of the present invention may have a base sequence (tail sequence) that does not contribute to hybridization with the target sequence at its 5'end and / or 3'end.
  • the number of bases contained in each tail sequence is 5 or less (preferably 4, 3, 2 or 1), and those having no tail sequence are optimal.
  • the base sequence of the portion of the pharmaceutical composition II of the present invention excluding the tail sequence of the antisense oligonucleotide has a sequence complementarity of 70% or more with the target sequence as long as it retains the hybridization activity with the target sequence. It is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and is optimally completely complementary.
  • the antisense oligonucleotide in the pharmaceutical composition II of the present invention comprises the base sequence represented by SEQ ID NOs: 83-100.
  • the base sequences represented by SEQ ID NOs: 83 to 100 are described as RNA sequences corresponding to SEQ ID NOs: 19 to 36 described in Examples described later.
  • the uridine residue in the antisense oligonucleotide may be replaced with a modified nucleotide based on the corresponding thymidine residue. Therefore, the base sequence in which "u" is replaced with "t” in the base sequence represented by SEQ ID NOs: 83 to 100 is also included in this embodiment.
  • the base sequence of the portion of the pharmaceutical composition II of the present invention excluding the tail sequence of the antisense oligonucleotide is 5 bases or less (preferably) with the target sequence as long as it has hybrid activity with the target sequence. There may be a mismatch of 4 bases or less, more preferably 3 bases, 2 bases or 1 base), but optimally there is no mismatch.
  • the antisense oligonucleotide in the pharmaceutical composition II of the present invention is a polyA addition sequence AATAAA newly generated in the mutant intron 8 sequence (sequences 324 to 329 in the base sequence represented by SEQ ID NO: 64). It is thought that abnormal polyadenylation is suppressed by inhibiting cleavage and binding of polyadenylation factor (CPSF) to. Therefore, in a preferred embodiment, the antisense oligonucleotide in the pharmaceutical composition II of the present invention is at least one base of the 324 to 329th sequence in the base sequence represented by SEQ ID NO: 64, for example, 2 bases, 3 bases, 4 bases. Sequences containing 5, or 6 bases can be targeted.
  • the length of the antisense oligonucleotide in the pharmaceutical composition II of the present invention is not particularly limited as long as it suppresses abnormal polyadenylation in intron 1, but is, for example, 15 to 30 nucleotides, 15 to 23 nucleotides, or 15 to 18 nucleotides. possible.
  • the antisense oligonucleotide in the pharmaceutical composition of the present invention may be any of DNA, RNA, and DNA / RNA chimera. By making it a DNA / RNA chimera, the nuclease resistance of the antisense oligonucleotide can be increased and the stability in vivo can be enhanced.
  • the antisense oligonucleotide in the pharmaceutical composition of the present invention may contain one or more modifications to increase nuclease resistance and enhance in vivo stability.
  • Modifications include, for example, modification of the sugar moiety of the nucleoside and modification of the phosphate group of the nucleotide. Examples of the bond between the sugar-modified nucleoside and the modified nucleoside include those described in 1 above.
  • the antisense oligonucleotide comprises one or more sugar modified nucleosides. All of the nucleosides contained in the antisense oligonucleotide may be sugar-modified nucleosides.
  • the antisense oligonucleotide may contain two or more sugar-modified nucleosides containing modifications of different sugar moieties.
  • the sugar-modified nucleoside is preferably a nucleoside containing 4'-(CH 2 ) n- O-2'cross-linking (where n is 1 or 2 in the formula) or 2'-O-methylation.
  • the antisense oligonucleotide contains a sugar-modified nucleoside containing a 4'-(CH 2 ) 2- O-2'crosslink
  • the number is not particularly limited, but one or more, for example, two or more, or three. It can be 4 or more, 5 or more, or 6 or more, 12 or less, for example, 11 or less, 10 or less, or 9 or less.
  • all of the nucleosides contained in the antisense oligonucleotide are sugar-modified nucleosides, where the sugar-modified nucleoside is a sugar-modified nucleoside containing a 4'-(CH 2 ) 2- O-2'bridge, 2'-.
  • the antisense oligonucleotide comprises one or more modified nucleoside linkages. Different modified nucleoside linkages may be included in the antisense oligonucleotide. Further, all the nucleoside linkages in the antisense oligonucleotide may be modified nucleoside linkages. In a more preferred embodiment, the modified nucleoside bond is a phosphorothioate bond. In an even more preferred embodiment, all nucleoside linkages in the antisense oligonucleotide are modified nucleoside linkages and the modified nucleoside linkages are phosphorothioate linkages.
  • the antisense oligonucleotide in the pharmaceutical composition I of the present invention is selected from the group consisting of oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 1-18, 37-60 and 112-123.
  • the nucleosides contained in these oligonucleotides are either sugar-modified nucleosides containing 4'-(CH 2 ) 2 -O-2'crosslinks or sugar-modified nucleosides containing 2'-O-methylation.
  • all the nucleoside-to-nucleoside bonds in these oligonucleotides are phosphorothioate bonds.
  • More suitable antisense oligonucleotides in the pharmaceutical composition I of the present invention are the oligonucleotides shown below.
  • Example 11 (XPF-int1-011): HO -C m1s -C e2s -C m1s -U m1s -T e2s -A m1s -C m1s -T e2s -U m1s -A m1s -C e2s -G m1s -U m1s- C e2s- U m1s- G m1s- T e2s- G m1t- H (SEQ ID NO: 11)
  • Example 12 (XPF-int1-012): HO -C m1s -C e2s -U m1s -U m1s -A e2s -C m1s -U m1s -T e2s -A m1s -C m1s -G e2s -U m1s -C m1s- T
  • the antisense oligonucleotide in the pharmaceutical composition II of the present invention is selected from the group consisting of oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 19 to 36.
  • the nucleosides contained in these oligonucleotides are either sugar-modified nucleosides containing 4'-(CH 2 ) 2 -O-2'crosslinks or sugar-modified nucleosides containing 2'-O-methylation.
  • all the nucleoside-to-nucleoside bonds in these oligonucleotides are phosphorothioate bonds.
  • the method for preparing the antisense oligonucleotide is not particularly limited, but a known chemical synthesis method (phosphoric acid triester method, phosphoramidite method, H-phosphonate method, etc.) can be used. It may be synthesized by using a commercially available nucleic acid synthesizer and using a commercially available reagent used for DNA / RNA synthesis. For example, after coupling a phosphoramidite reagent, a reagent such as sulfur, tetraethylthiura disulfide (TETD, Applied Biosystem), Beaucage reagent (Glen Research), or xanthan hydride is reacted to have a phosphorothioate bond.
  • TETD tetraethylthiura disulfide
  • Beaucage reagent Glen Research
  • xanthan hydride xanthan hydride
  • Antisense oligonucleotides can be synthesized (T
  • the oligonucleotide (antisense oligonucleotide) of the present invention may have a group having a desired chemical structure at the 5'end and / or 3'end to control the physical properties and pharmacokinetics of the oligonucleotide.
  • an aminoalkyl group can be introduced at the 5'end and / or 3'end of the oligonucleotide (antisense oligonucleotide) of the present invention, through which the desired chemical structure can be added.
  • the introduction of the aminoalkyl group into the oligonucleotide may be carried out by a method known in the art, or may be carried out using a commercially available reagent.
  • 5'-Amino-Modifier C6 Glen Research
  • 5'-TFA-Amino-Modifier C6-CE Phosphoramidite 5'-TFA-Amino-Modifier-
  • an amino modification reagent such as C5-CE Phosphoramidite (Link Technologies)
  • an oligonucleotide in which an aminoalkylphosphate group is bonded to the 5'end can be synthesized.
  • an oligonucleotide in which an aminoalkyl group is bonded to the 3'end can be synthesized.
  • oligonucleotide As a chemical structure that can be introduced into the 5'end and / or 3'end of the oligonucleotide (antisense oligonucleotide) of the present invention, it is known in the art that the physical properties and pharmacokinetics of the oligonucleotide can be controlled. Any chemical structure can be utilized, including, for example, fatty acids, cholesterol, GalNAc structures and the like.
  • antisense oligonucleotides can be synthesized using amidite compounds containing fatty acids that are useful for the transfer of nucleic acids to muscle tissue, and the transfer of antisense oligonucleotides to muscle tissue can be promoted.
  • amidite compounds containing fatty acids that are useful for the transfer of nucleic acids to muscle tissue, and the transfer of antisense oligonucleotides to muscle tissue can be promoted.
  • Known eg, Link Technologies products such as Nucleic Acids Res.
  • antisense oligonucleotides can be synthesized using amidite compounds containing a GalNAc structure, which is useful for the transfer of nucleic acids to hepatocytes, and that antisense oligonucleotides can be specifically delivered to hepatocytes.
  • the antisense oligonucleotide in the pharmaceutical composition of the present invention may be in the form of a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt means a salt of an oligonucleotide (antisense oligonucleotide), and examples of such a salt include alkali metal salts such as sodium salt, potassium salt and lithium salt, calcium salt and magnesium.
  • Alkaline earth metal salts such as salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts and other metal salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts , Morphorine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocine salt, Amine salts such as prokine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylammonium salts, organic salts such as tris (hydroxymethyl) aminomethane salts; hydrofluorates, hydrochlorides, bromide Hydroch
  • the antisense oligonucleotide of the pharmaceutical composition of the present invention and a pharmaceutically acceptable salt thereof may also exist as a solvate (for example, a hydrate), and even such a solvate may exist. Good.
  • the pharmaceutical composition of the present invention can be formulated by mixing an antisense oligonucleotide and an appropriate pharmaceutically acceptable additive.
  • the pharmaceutical composition of the present invention can be administered orally as a preparation such as tablets, capsules and granules, or parenterally as a preparation such as an injection and a transdermal absorbent.
  • formulations are excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, diluents, solvents for injections, solubilizers, suspending agents, isotonic agents, buffers, painless It can be produced by a well-known method using additives such as an agent, a preservative, and an antioxidant.
  • excipients examples include organic excipients and inorganic excipients.
  • examples of the organic excipient include sugar derivatives such as lactose and sucrose; starch derivatives such as corn starch and potato starch; cellulose derivatives such as crystalline cellulose; and gum arabic.
  • examples of the inorganic excipient include sulfates such as calcium sulfate.
  • binder examples include the above-mentioned excipients; gelatin; polyvinylpyrrolidone; polyethylene glycol and the like.
  • disintegrant examples include the above-mentioned excipients; chemically modified starch or cellulose derivatives such as croscarmellose sodium and sodium carboxymethyl starch; and crosslinked polyvinylpyrrolidone.
  • Lubricants include, for example, talc; stearic acid; colloidal silica; bead wax, waxes such as gay wax; sulfates such as sodium sulfate; lauryl sulfates such as sodium lauryl sulfate; in the above excipients.
  • examples include starch derivatives.
  • the emulsifiers include, for example, colloidal clays such as bentonite, beagum; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ethers. Examples include ionic surfactants.
  • stabilizers examples include parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol; phenols and phenols such as cresol.
  • diluent examples include water, ethanol, propylene glycol and the like.
  • Examples of the solvent for injection include water, ethanol, glycerin and the like.
  • solubilizing agent examples include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
  • suspending agent for example, surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, carboxy Examples thereof include hydrophilic polymers such as sodium methylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.
  • tonicity agent examples include sodium chloride, glycerin, D-mannitol and the like.
  • the buffer examples include a buffer solution such as phosphate, acetate, carbonate, and citrate.
  • Examples of the pain-relieving agent include benzyl alcohol and the like.
  • preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • antioxidants examples include sulfites and ascorbic acid.
  • the subject to which the pharmaceutical composition of the present invention is administered is a human.
  • the route of administration of the pharmaceutical composition of the present invention may be either oral administration or parenteral administration, and a suitable administration route may be selected according to the target symptomatology.
  • the route of administration may be either systemic administration or topical administration.
  • parenteral administration include intravenous administration, intraarterial administration, intrathecal administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intra-articular administration. be able to.
  • transdermal and subcutaneous administration may be selected for cutaneous symptoms
  • intrathecal administration may be selected for neurological symptoms.
  • the pharmaceutical composition of the present invention is administered to a subject in a therapeutically effective amount.
  • “Therapeutic effective amount” means the amount that exerts a therapeutic effect for a specific disease, administration form and administration route, depending on the target species, type of disease, symptom, gender, age, chronic disease, and other factors. Will be decided as appropriate.
  • the dose of the pharmaceutical composition of the present invention can be appropriately determined according to the target species, the type of disease, symptoms, gender, age, chronic disease, and other factors.
  • the pharmaceutical composition of the present invention may be used in combination with at least one known therapeutic agent or method.
  • the present invention also provides a method for treating xeroderma pigmentosum F group, which comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.
  • the "therapeutic effective amount” in the present invention means an amount that exerts a therapeutic effect for a specific disease or symptom, administration form and administration route, and refers to a target species, disease or symptom type, symptom, sex, age, chronic disease, etc. It is determined as appropriate according to other factors.
  • the reagent was activator solution-3 (0.25 mol / L). 5-benzylthio-1H-Tetrazole / acetonitrile solution, manufactured by Wako Pure Chemical Industries, Ltd., product No. 013-20011), CAP A for AKTA (1-methylimidazole / acetonitrile solution, manufactured by Sigma-Aldrich, product No. L040050), Cap B1 for AKTA (anhydrous acetic acid / acetonitrile solution, manufactured by Sigma-Aldrich, product No. L050050), Cap B2 for AKTA (pyridine / acetonitrile solution, manufactured by Sigma-Aldrich, product No.
  • the one manufactured by ChemGenes was used.
  • the non-natural phosphoramidite used was Example 14 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-6-N-) of JP-A-2000-297097.
  • Example 27 Benzoyladenosine-3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite
  • Example 27 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-2-N -Isobutyryl guanosine-3'-O- (2-Cyanoe) Phosphoramidite)
  • Example 22 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'- O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite)
  • Example 9 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-5-methyluridine-3'-O -(2-Cyanoethyl N, N-diisopropyl) phosphoramidite), compound was used.
  • the oligomer was excised from the support by treating the protected oligonucleotide analog having the target sequence with 600 ⁇ L of concentrated aqueous ammonia, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed.
  • a mixed solution of oligomers was mixed with 300 ⁇ L of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex) and charged onto a Clarity SPE 96 well plate (manufactured by Phenomenex).
  • the components extracted in the above were collected. After distilling off the solvent, the target compound was obtained.
  • This compound is a reverse phase HPLC (column (Phenomenex, Clarity 2.6 ⁇ m Oligo-MS 100A (2.1 ⁇ 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), aqueous solution 8 mM triethylamine, solution B: methanol, B. %: 10% ⁇ 25% (4min, linear gradient); 60 ° C; 0.5mL / min; 260nm), elution was performed at 2.78 minutes.
  • Compounds were identified by negative ion ESI mass spectrometry.
  • the base sequence of this compound is the 412th in the mRNA encoded by Homo sapiens ERCC excision repair 4, endonuclease catalytic subunit (ERCC4) (NCBI-GenBank accession No. NG_011442.1).
  • T is a sequence complementary to the 412-429th sequence of the sequence in which T is mutated to A.
  • Example 2 to 36 The compounds of Examples 2 to 36 were also synthesized in the same manner as the compounds of Example 1. Information on the compounds of Examples 1 to 36 is given in Table 1.
  • Examples 37 to 60 The compounds of Examples 37 to 60 were also synthesized in the same manner as in Example 1. Information on the compounds of Examples 37-60 is shown in Table 2.
  • sequence In the "sequence” in the table, uppercase letters indicate ENA and lowercase letters indicate 2'-OMeRNA. All bonds between nucleosides are phosphorothioate bonds. “Start” and “end” in the table indicate the nucleotide number of the base sequence represented by SEQ ID NO: 63, and “sequence” in the table is complementary to the base sequence from “start” to “end”. Shows the sequence. "Molecular weight” in the table indicates the measured value by negative ion ESI mass spectrometry.
  • Example 61 HO-C e1s -C s -T e1s -T s -A e1s -C s -T e1s -T s -A e1s -C s -G e1s -T s -C e1s -T s -G 1t -H (LNA) -int1-001) (SEQ ID NO: 61)
  • the compound of Example 61 was synthesized using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984).
  • the LNA moiety was synthesized using the phosphoramidite compound described in WO99 / 14226. , Or, in the figure, it may be expressed as "LNA”.
  • This compound is a reverse phase HPLC (column (X-Bridge C18 2.5 ⁇ m (4.6 ⁇ 75 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), aqueous solution of 8 mM triethylamine, solution B: methanol, B%: 5 When analyzed at% ⁇ 30% (20 min, linear gradient); 60 ° C; 1 mL / min; 260 nm), elution was performed at 10.23 minutes. The compound was identified by negative ion ESI mass spectrometry (measured value: 4927.26).
  • This compound is a reverse phase HPLC (column (X-Bridge C18 2.5 ⁇ m (4.6 ⁇ 75 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), aqueous solution of 8 mM triethylamine, solution B: methanol, B%: 5 When analyzed at% ⁇ 30% (20 min, linear gradient); 60 ° C; 1 mL / min; 260 nm), elution was performed at 10.46 minutes. The compound was identified by negative ion ESI mass spectrometry (measured value: 4791.16).
  • Example 1 Analysis of XPF mRNA splicing product amount, XPF protein expression level, and DNA damage repair activity by Example compounds (1) 1-1. Evaluation of XPF mRNA splicing product amount Patient-derived cells (Matsumura et al. Hum. Mol. Genet. 1998, 7 (6), 969-974) were seeded in 5x10 4 cells per well on a 6-well plate. After 24 hours, LNA was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 40 nM. After 4 hours, the medium was replaced with new medium (DMEM 10% FBS). Cells were harvested 24 hours after transfection. Total RNA was extracted with Direct-zol TM RNA MiniPrep (ZYMO RESEARCH).
  • CDNA was obtained from 100 ng of total RNA using SuperScript® IV (Thermo Fisher Scientific). Droplet digital PCR (ddPCR) was performed on the QX100 TM Droplet Digital TM PCR system (Bio-Rad Laboratories, Inc.) to quantify splicing products. Similarly, splicing products were quantified for healthy human-derived fibroblasts (48BR).
  • ddPCR Droplet digital PCR
  • ddPCR was performed according to the following procedure.
  • QX200 Eva Green ddPCR Supermix Bio-Rad Laboratories, Inc.
  • Forward primer and Reverse primer were added to a final concentration of 100 nM, respectively.
  • Sample cDNA total RNA adjusted to 100 ng
  • Sample cDNA total RNA adjusted to 100 ng
  • QX200 TM / QX100 put the sample and 70 ⁇ l of Droplet Generation oil for EvaGreen to DG8 cartridge for TM Droplet Generator was subjected to QX100 TM Droplet Generator.
  • the turbid solution was placed in a 96-well plate (Bio-Rad Laboratories, Inc.) and sealed using a PX1 PCR plate sealer. Analysis was performed using the QX100 TM Droplet Reader.
  • the value obtained by ddPCR using a combination of primers of XPF ex1-F and XPF ex2-R was used, and for the quantification of abnormal splicing products, XPF int1-F and XPF ex2-
  • the value obtained by ddPCR using the combination of R primers was used.
  • Fixative (3.7% formalin PBS solution) was added and allowed to stand for 20 minutes. The fixative was discarded and PBS was added. The average fluorescence intensity of 488 azide in the nucleus was calculated. The fluorescence value indicates the repair activity.
  • the repair activity of healthy human-derived fibroblasts (48BR) was also evaluated in the same manner.
  • Fig. 1C The result is shown in Fig. 1C.
  • the patient-derived cells transfected with the compound of Example 61 (LNA-int1-001) had a repair activity as compared with the patient-derived cells transfected with the compound of Reference Example 1 (LNA-int1-001S, control DNA).
  • LNA-int1-001S the compound of Reference Example 1
  • FIG. 2A the compounds of Examples 1 to 18 (XPF-int1-001 to XPF-int1-018) were similarly transfected into patient-derived cells and the repair activity was analyzed. An increase in repair activity was observed in all the compounds of Examples 1 to 18.
  • Example 62 to 73 The compounds of Examples 62 to 73 were also synthesized in the same manner as in Example 1. Examples 62 to 73 are shown in Table 3.
  • Reference example 2 The compound of Reference Example 2 was also synthesized in the same manner as in Example 1. Reference example 2 is shown in Table 4.
  • sequence of the compound of Reference Example 2 is a sequence complementary to the nucleotide sequence from the 223rd to the 240th of Musmusculus strain mdx dystrophin genes, partial cds (NCBI-GenBank accession No. AH007099.2).
  • Example 2 Analysis of XPF mRNA splicing product amount, XPF protein expression level, and DNA damage repair activity by Example compounds (2) 2-1. Evaluation of protein expression level The protein expression level of the example compound was evaluated in the same manner as in the test 1-2.
  • the pharmaceutical composition of the present invention can treat a specific XP-F group for which there has been no conventional therapeutic means.
  • SEQ ID NO: 1 to 61 oligonucleotide sequence of Examples Compounds 1 to 61 SEQ ID NO: 62: oligonucleotide sequence of Reference Example Compound 1 SEQ ID NO: 63: RNA sequence of XPF gene variant Intron 1 SEQ ID NO: 64: Mutation of XPF gene DNA sequence of type intron 8 SEQ ID NO: 65-100: RNA sequence corresponding to SEQ ID NO: 1-36 SEQ ID NO: 101-108: RNA sequence corresponding to SEQ ID NO: 53-60 SEQ ID NO: 109-111: Primer used for ddPCR DNA Sequence SEQ ID NOs: 112 to 123: Oligonucleotide Sequence of Examples Compounds 62 to 73 SEQ ID NOs: 124 to 135: RNA Sequence Corresponding to SEQ ID NOs: 112 to 123 SEQ ID NO: 136: Oligonucleotide Sequence of Reference Example Compound 2 SEQ ID NO: 137: RNA sequence corresponding

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Abstract

La présente invention fournit un moyen de traitement contre le Xeroderma pigmentosum de complémentation F, pour lequel aucune mutation provoquant une maladie n'a été identifiée à ce jour.　La présente invention concerne : un oligonucléotide antisens ayant une séquence de bases capable de s'hybrider avec une partie de la région d'intron d'un gène XPF et ayant une activité pour inhiber une modification post-transcriptionnelle anormale du gène XPF, ou un sel pharmaceutiquement acceptable dudit oligonucléotide antisens ; et un agent thérapeutique ou une composition thérapeutique contre le Xeroderma pigmentosum de complémentation F contenant ledit oligonucléotide antisens ou son sel pharmaceutiquement acceptable en tant que principe actif.
PCT/JP2020/044768 2019-12-02 2020-12-02 Agent thérapeutique contre le xeroderma pigmentosum de complémentation f WO2021112106A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08510130A (ja) * 1993-05-11 1996-10-29 ザ・ユニヴァーシティ・オヴ・ノース・キャロライナ・アト・チャペル・ヒル 異常スプライシングを阻害するアンチセンスオリゴヌクレオチドと同物質の利用法
JP2017519766A (ja) * 2014-06-16 2017-07-20 ユニバーシティ・オブ・サザンプトン イントロン保持の減少

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08510130A (ja) * 1993-05-11 1996-10-29 ザ・ユニヴァーシティ・オヴ・ノース・キャロライナ・アト・チャペル・ヒル 異常スプライシングを阻害するアンチセンスオリゴヌクレオチドと同物質の利用法
JP2017519766A (ja) * 2014-06-16 2017-07-20 ユニバーシティ・オブ・サザンプトン イントロン保持の減少

Non-Patent Citations (3)

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Title
MARÍN MARIA, RAMÍREZ MARÍA JOSÉ, CARMONA MIRIAM AZA, JIA NAN, OGI TOMOO, BOGLIOLO MASSIMO, SURRALLÉS JORDI: "Functional Comparison of XPF Missense Mutations Associated to Multiple DNA Repair Disorders", GENES, vol. 10, no. 1, 17 January 2019 (2019-01-17), pages 1 - 15, XP055832162 *
PARK, J. E. ET AL.: "In Vitro Modulation of Endogenous Alternative Splicing Using Splice- Switching Antisense Oligonucleotides", METHODS IN MOLECULAR BIOLOGY, vol. 1648, 2017, pages 39 - 52 *
SENJU, CHIKAKO ET AL.: "A Novel Gene Mutation Of Japanese Xeroderma Pigmentosum Group F Patients", CONSORTIUM OF BIOLOGICAL SCIENCES, 2017 *

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