[go: up one dir, main page]

WO2021002359A1 - Médicament d'acide nucléique et son utilisation - Google Patents

Médicament d'acide nucléique et son utilisation Download PDF

Info

Publication number
WO2021002359A1
WO2021002359A1 PCT/JP2020/025676 JP2020025676W WO2021002359A1 WO 2021002359 A1 WO2021002359 A1 WO 2021002359A1 JP 2020025676 W JP2020025676 W JP 2020025676W WO 2021002359 A1 WO2021002359 A1 WO 2021002359A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
aso
oligonucleotide
composition according
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2020/025676
Other languages
English (en)
Japanese (ja)
Inventor
慎吾 中村
玲 金
孝 上村
遼平 ▲高▼田
正之 梨本
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Veritas In Silico Inc
Original Assignee
Veritas In Silico Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Veritas In Silico Inc filed Critical Veritas In Silico Inc
Priority to JP2021500250A priority Critical patent/JP6934695B2/ja
Publication of WO2021002359A1 publication Critical patent/WO2021002359A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • 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 the regulation of gene expression using oligonucleotides. More specifically, it relates to a pharmaceutical composition containing a relatively short oligonucleotide for regulating gene expression and treatment of a disease using the same. More specifically, the present invention relates to a pharmaceutical composition containing an antisense oligonucleotide targeting the p53 gene and a method for preventing or treating a disease using the same.
  • nucleic acid drugs are expected as a new modality (drug discovery method).
  • Non-Patent Document 1 As basic research, research on antisense nucleic acids and RNAi (RNA interference) has been conducted (Non-Patent Document 1), drug development based on that technology has been carried out, and some products have begun to be put on the market. Furthermore, the function of nucleic acids that do not encode proteins is being clarified, and application to drug discovery as a gene expression regulation technique as miRNA (microRNA) is being promoted (Non-Patent Document 2).
  • miRNA miRNA
  • Acute renal failure (also called acute renal failure (AKI)) is a condition in which the homeostasis of body fluid cannot be maintained due to a rapid decline in renal function.
  • ARF is a clinical syndrome characterized by rapid deterioration of renal function within a few days and is generally based on a rapid rise in serum creatinine levels or a rapid rise in serum creatinine and BUN.
  • Acute renal failure (acute renal injury) is diagnosed. In general, 170-200 severe ARF cases per million people occur each year.
  • Acute renal failure due to renal tubular necrosis caused by nephrotoxic substances such as ischemia due to shock, cisplatin, aminoglycoside, and contrast media may develop in connection with medical practice such as surgery, contrast examination, and administration of anticancer drugs.
  • Acute kidney injury often develops as a result of renal ischemia-reperfusion injury (IRE) in patients who have undergone major surgery such as heart surgery.
  • IRE renal ischemia-reperfusion injury
  • Acute renal failure due to surgery, contrast examination, administration of antineoplastic drugs or antibiotics is likely to occur when the extracellular fluid volume is low, and it can be caused by sufficient fluid replacement in advance. It is known that the frequency is reduced and the degree of renal failure can be reduced.
  • there is no specific treatment method for ARF there is no specific treatment method for ARF, and further improvement is required for the prevention and treatment method of acute renal failure.
  • U.S. Pat. No. 9,334,499 provides a method for treating patients at risk of developing acute renal failure or those who develop acute renal failure, which down-regulates the expression of the p53 gene. Methods are disclosed that include administering to a patient a double-stranded siRNA compound for an amount effective for p53 gene. The same document also discloses that down-regulation of p53 gene expression can be effective against alopecia caused by cancer chemotherapy and radiation therapy.
  • US Pat. No. 7,910,566 describes acute renal failure after renal ischemia-reperfusion, characterized in that a double-stranded siRNA compound having a specific nucleotide sequence targeting the p53 gene is used at a specific time. Treatment methods for patients at risk are disclosed. This document shows that in a rat ischemia-reperfusion-induced ARF model, p53-targeted siRNA can protect renal tissue from the effects of ischemia-reperfusion injury and reduce the severity of ARF.
  • the present invention comprises a pharmaceutical composition containing a relatively short oligonucleotide (antisense oligonucleotide; hereinafter also referred to as ASO) for regulating the expression of the p53 gene, and a method for preventing or treating a disease or condition using the pharmaceutical composition.
  • ASO antisense oligonucleotide
  • One of the purposes is to provide.
  • Another object of the present invention is to provide ASO having high knockdown efficiency for expression of p53 gene and high efficiency of uptake into cells.
  • the present inventors have identified an ASO that acts on a transcript of the p53 gene for the purpose of regulating the expression of the p53 gene. It is considered that p53-related diseases such as acute renal failure can be treated and prevented by suppressing the translation of the p53 gene from the transcript using these ASOs.
  • the present invention is based on these nucleic acid medicines developed by the present inventors and includes the following aspects:
  • a pharmaceutical composition for inhibiting the expression of the p53 gene in cells wherein an oligonucleotide containing a complementary region substantially complementary to at least a part of mRNA encoding the p53 gene is used as an active ingredient.
  • Aspect 2 The pharmaceutical composition according to Aspect 1, wherein the p53 gene is a human p53 gene.
  • Aspect 3 The pharmaceutical composition according to Aspect 1 or 2, wherein the p53 gene has the sequence of SEQ ID NO: 132.
  • Aspect 4 The pharmaceutical composition according to any one of aspects 1 to 3, wherein the oligonucleotide is essentially a single-stranded molecule.
  • Aspect 5 The pharmaceutical composition according to any one of aspects 1 to 4, wherein the oligonucleotide is an antisense oligonucleotide (ASO).
  • Aspect 6 The pharmaceutical composition according to Aspect 5, wherein the antisense oligonucleotide (ASO) is a gapmer.
  • Aspect 7 The pharmaceutical composition according to any one of aspects 1 to 6, wherein the oligonucleotide has a length of 12 to 18 bases.
  • Aspect 8 The pharmaceutical composition according to Aspect 7, wherein the oligonucleotide is 14 bases long.
  • Aspect 9 The pharmaceutical composition according to Aspect 8, wherein the oligonucleotide is a 2-10-2 gapmer.
  • Aspect 10 The pharmaceutical composition according to any one of aspects 1 to 9, wherein the base in the oligonucleotide is 80% or more complementary to the p53 gene.
  • Aspect 11 The pharmaceutical composition according to any one of aspects 1 to 10, wherein the base in the oligonucleotide is 100% complementary to the p53 gene.
  • Aspect 12 The pharmaceutical composition according to any one of aspects 1 to 11, wherein the oligonucleotide contains a modified nucleoside and / or a bond between modified nucleosides.
  • Aspect 13 The pharmaceutical composition according to Aspect 12, wherein the modified nucleoside is a bridged nucleic acid.
  • oligonucleotide is an oligonucleotide containing any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 125.
  • oligonucleotide is an oligonucleotide consisting of any one sequence of SEQ ID NO: 1 to SEQ ID NO: 125.
  • the oligonucleotide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 125.
  • the disease or symptom is ischemia-reperfusion disorder, hearing loss, hearing disorder, balance disorder, hearing loss, chemotherapy-induced alopecia, radiation therapy-induced alopecia, acute renal failure, acute renal disorder, Chronic kidney disease (CKD), side effects associated with anticancer drug therapy, delayed transplant function (DGF) in patients with bone marrow transplantation, spinal cord injury, brain injury, stroke, stroke, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, tumor, burn , Wound, hyperthermia, hypoxia, ischemia, organ transplantation, bone marrow transplantation (BMT), myocardial infarction / heart attack, cardiotoxicity, p53-positive cancer, and acute liver failure, selected from the group, Aspect 26 or 27.
  • DGF delayed transplant function
  • a pharmaceutical composition for use in the treatment or prevention of a disease or symptom in a subject which comprises a complementary region that is substantially complementary to at least a portion of the mRNA encoding the p53 gene. It contains a single-stranded oligonucleotide as an active ingredient, the oligonucleotide is a 14-base long gapmer, and the wing regions on the 5'side and 3'side of the gapmer each consist of 2 bases of LNA.
  • a pharmaceutical composition for use in the treatment or prevention of a disease or condition in a subject p53-ASO-15, p53-ASO-15-11, p53-ASO-15-12, and p53-ASO.
  • FIG. 1 shows the nucleotide sequence of the human p53 gene (NM_000546.5) (SEQ ID NO: 132).
  • FIG. 2 is a graph showing that the addition of p53-ASO-01-23 with a transfection reagent using HeLa cells reduces the mRNA level of p53. The vertical axis shows the relative value of the mRNA level of p53.
  • FIG. 3 is a graph showing the evaluation result of ASO in which the target site is shifted back and forth. The vertical axis shows the relative value of the mRNA level of p53. The left side shows the result 1 day after transfection, and the right side shows the result 2 days after transfection.
  • FIG. 4 is a graph showing the evaluation results of ASO shortened to 13 base lengths.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the left side shows the result 1 day after transfection, and the right side shows the result 2 days after transfection.
  • FIG. 5 is a graph showing the evaluation results of ASO having a long chain length of 15 bases.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the left side shows the result 1 day after transfection, and the right side shows the result 2 days after transfection.
  • FIG. 6 is a graph showing the evaluation results of ASO having a longer chain length of 16 bases.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the left side shows the result 1 day after transfection, and the right side shows the result 2 days after transfection.
  • FIG. 5 is a graph showing the evaluation results of ASO having a long chain length of 15 bases.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the left side shows the result 1
  • FIG. 7 is a graph showing the evaluation results of ASO having a longer chain length of 17 bases.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the left side shows the result 1 day after transfection, and the right side shows the result 2 days after transfection.
  • FIG. 8 is a graph showing the evaluation results of ASO having a longer chain length of 18 bases.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the left side shows the result 1 day after transfection, and the right side shows the result 2 days after transfection.
  • FIG. 9 is a graph showing the evaluation result of ASO in which the wing region is modified.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • FIG. 10 is a graph showing the evaluation results of ASO with various modifications.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the results one day after transfection are shown on the left side, and the results two days after transfection are shown side by side on the right side.
  • FIG. 11 is a graph showing the evaluation results of ASO with added cholesterol.
  • the vertical axis shows the relative value of the mRNA level of p53. The results one day after transfection are shown on the left side, and the results two days after transfection are shown side by side on the right side.
  • FIG. 10 is a graph showing the evaluation results of ASO with various modifications.
  • the vertical axis shows the relative value of the mRNA level of p53.
  • the results one day after transfection are shown on the left side, and the results two days after transfection are shown side by side on the right side.
  • FIG. 12 is a graph showing the results of measuring the amount of p53 protein one day after transfection by a luciferase assay using a reporter plasmid with a p53 response element.
  • the results using p53-ASO-15 are shown on the left side, and the results using QPI1002 are shown on the right side.
  • FIG. 13 is a graph showing the results of measuring the amount of p53 protein 2 days after transfection by a luciferase assay using a reporter plasmid having a p53 response element.
  • the results using p53-ASO-15 are shown on the left side, and the results using QPI1002 are shown on the right side.
  • FIG. 14 is a graph showing the results of evaluating the expression-suppressing effect of p53-ASO-15 and Quark's siRNA QP1002 by qPCR. The upper row shows the results after 1 day, and the lower row shows the results after 2 days.
  • FIG. 15 shows an experimental protocol for evaluation by Western blotting.
  • FIG. 16 shows the results of evaluating the effects of ASO-15, 15-16 and QPI-1002 by Western blotting.
  • the present inventors have developed a method for efficiently regulating the expression of the p53 gene in cells using a relatively short oligonucleotide (ASO).
  • ASO oligonucleotide
  • p53 gene The p53 gene is involved in DNA repair in cells, arrest of cell growth, suppression of cell growth cycle, etc., and is a gene that is said to cause apoptosis when cells become cancerous.
  • p53 is one of the so-called tumor suppressor genes, and it is thought that cancer occurs when the function of this gene is impaired. It is thought that changes in multiple oncogenes and tumor suppressor genes are necessary for cells to become cancerous, but p53 is the gene with the highest frequency of abnormalities in malignant tumors.
  • the p53 polypeptide responds to cell stress by converting a variety of different stimuli, such as DNA damage conditions such as gamma irradiation, transcription or replication dysregulation, and transformation by oncogenes, into cell growth arrest or apoptosis. It plays an important role in the mechanism.
  • the p53 polypeptide induces apoptosis, or programmed cell death, in response to such stimuli.
  • p53 has important roles such as maintaining cell homeostasis and inducing apoptosis.
  • Most anticancer therapies also damage normal cells with p53, causing serious side effects associated with damage or death of healthy cells.
  • One aspect of the invention relates to a pharmaceutical composition for use in the treatment or prevention of a disease or condition associated with the expression of the p53 gene in a subject.
  • the targeted disease or condition is ischemic-reperfusion injury, hearing loss, hearing impairment, balance disorder, hearing loss, chemotherapy-induced alopecia, radiation therapy-induced alopecia, acute renal failure.
  • DGF Delayed transplantation function
  • the base sequence of the p53 gene and its mRNA is known and can be easily obtained from a database such as GenBank.
  • GenBank a database such as GenBank.
  • the sequence of NCBI accession number NM_000546.5 (FIG. 1; SEQ ID NO: 132) can be used.
  • Antisense oligonucleotide (also called ASOs) Antisense oligonucleotides (also called ASOs) have a sequence that is substantially complementary to at least a portion of the target nucleobase and is Watson-Crick, Hoogsteen, or reverse hoo between the corresponding nucleobases. Refers to a single-stranded oligonucleotide that hybridizes by a Gusteen-type hydrogen bond. Antisense oligonucleotides can exhibit detectable or measurable antisense activity due to hybridization to their target nucleic acid.
  • antisense activity is a reduction in the amount or expression of a target nucleic acid, or a reduction in the amount or expression of a protein encoded by such a target nucleic acid.
  • antisense activity in certain embodiments, is antisense inhibition by degradation (cleave) of the target, which reduces the level of the target nucleic acid in the presence of antisense oligonucleotides complementary to the target nucleic acid. means.
  • antisense oligonucleotides containing at least a part of continuous DNA of 4 bases or more hybridize with the target RNA and become a substrate for intracellular RNase H, which induces specific degradation (cleavage) of the target RNA.
  • antisense activity is protein binding inhibition due to steric hindrance due to target occupation, resulting in translational repression and splicing regulation (eg, exon skipping).
  • Antisense oligonucleotides are single-stranded oligomers mainly composed of deoxyribonucleosides (DNA), ribonucleosides (RNAs), modified nucleosides, and nucleoside mimics (morpholinonucleic acids, peptide nucleic acids, etc.). It has an outer region having one or more nucleosides (eg, sugar-modified nucleosides such as LNA) on both sides or one side of an internal region having multiple nucleosides (eg, deoxyribonucleosides of 4 or more consecutive bases) that induce RNase H cleavage. Chimeric antisense oligonucleotides are called gapmers.
  • nucleosides eg, sugar-modified nucleosides such as LNA
  • an external region consisting entirely of LNA is called an LNA gapmer.
  • Chimeric antisense oligonucleotides that have an external region on only one side are also called hemigapmers, in particular.
  • the nucleosides contained in the inner region are chemically different from the nucleosides contained in the outer region.
  • the inner area is sometimes called the "gap” and the outer area is sometimes called the "wing".
  • a 14-base long gapmer having a wing region of 3 bases on the 5'side and a gap region of 8 bases, respectively, is sometimes called a 3-8-3 gapmer.
  • the antisense oligonucleotides are 2-10-2 gapmers, 2-9-3 gapmers, 3-9-2 gapmers, 3-8-3 gapmers, 3-7-4 gaps. It can be any of Mar, 4-7-3 Gap Mar, and 4-6-4 Gap Mar. In certain embodiments, the antisense oligonucleotides are 2-10-2 LNA gapmer, 2-9-3 LNA gapmer, 3-9-2 LNA gapmer, 3-8-3 LNA gapmer, 3-7-4 LNA gapmer. It can be any of Mar, 4-7-3 LNA Gap Mar, and 4-6-4 LNA Gap Mar.
  • the number of bases in the internal region can be 1 or more, for example, 2,3,4,5,6,7,8,9, or 10 bases, but is not limited thereto.
  • the number of bases in the external region can be 0 bases or more, for example, 1, 2, 3, 4, 5, or 6 bases independently on the 5'side and 3'side, but the number is limited to these. Not done.
  • the 5'side and 3'side wing regions may have different numbers of bases.
  • the wing region may be composed of the same or different sugar-modified nucleosides, and the sugar-modified nucleoside in the wing region can be, for example, LNA, but is not limited thereto.
  • Antisense oligonucleotides can, in certain embodiments, include modified nucleoside and / or modified nucleoside interlinks. Further, in a specific embodiment, the antisense oligonucleotide may be modified with one or both of the terminal hydroxyl groups of the oligonucleotide. For example, a phosphate group is added to one or both of the terminal hydroxyl groups of the oligonucleotide. You may be. In certain embodiments, the antisense oligonucleotide is, for example, at least 8 bases long or longer, eg, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. It can be base length, but is not limited to these. In some embodiments, an ASO of about 14 bases is preferably used.
  • the antisense oligonucleotides according to the present invention are p53-ASO-15, p53-ASO-15-11, p53-ASO-15-12, and p53-ASO- disclosed in Examples. It can be any of 15-16.
  • p53-ASO-15 and p53-ASO-15-16 have a sequence complementary to the 189th to 202nd bases of SEQ ID NO: 132.
  • p53-ASO-15-11 and p53-ASO-15-12 have sequences complementary to the 185th to 202nd bases and the 183rd to 202nd bases of SEQ ID NO: 132, respectively. There is.
  • the antisense oligonucleotide according to the present invention has a sequence complementary to the 189th to 202nd bases of SEQ ID NO: 132, the 185th to 202nd bases, and the 183rd to 202nd bases. It can be an oligonucleotide. Such complementary oligonucleotides can include the modifications described herein. Also, the antisense oligonucleotides according to the invention are in the form of pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. May be good. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • the ASO according to the present invention can be produced by a method using known chemical synthesis, an enzymatic transcription method, or the like.
  • methods using known chemical synthesis include a phosphoramidite method, a phosphorothioate method, a phosphotriester method, and the like.
  • ABI3900 high-throughput nucleic acid synthesizer manufactured by Applied Biosystems
  • NTS H-6 nucleic acid synthesis It can be synthesized by a machine (manufactured by Nihon Techno Service Co., Ltd.) and an Oligoilot10 nucleic acid synthesizer (manufactured by GE Healthcare).
  • Examples of the enzymatic transcription method include transcription using an RNA polymerase such as T7, T3, and SP6 RNA polymerase using a plasmid or DNA having the desired base sequence as a template.
  • the ASO produced by the synthetic method or the transcription method is then purified by HPLC or the like.
  • HPLC purification ASO is eluted from the column using triethylammonium acetate (TEAA) or a mixed solution of hexalylammonium acetate (HAA) and acetonitrile. Then, the elution solution is dialyzed against 1000 times the elution volume of distilled water for 10 hours, the dialysis solution is freeze-dried, and then stored frozen until use. At the time of use, for example, it is dissolved in distilled water so that the final concentration is about 100 ⁇ M.
  • TEAA triethylammonium acetate
  • HAA hexalylammonium acetate
  • the nucleic acid used in the ASO according to the present invention may be any nucleoside or a molecule having a function equivalent to that of the nucleoside, which is polymerized via an internucleoside bond.
  • Nucleoside is a type of compound in which a base (nucleobase) and a sugar are bound.
  • the bases include purine bases such as adenine and guanine, pyrimidine bases such as thymine, cytosine and uracil, nicotinamide and dimethylisoaroxazine.
  • Typical nucleosides are adenosine, thymidine, guanosine, cytidine, and uridine.
  • Nucleotides are substances in which a phosphate group is bound to a nucleoside.
  • oligonucleotides also referred to as polynucleotides
  • RNA which is a polymer of ribonucleotides
  • DNA which is a polymer of deoxyribonucleotides
  • nucleotide polymers containing modified nucleosides Be done. Natural DNA and RNA have phosphodiester bonds as internucleoside bonds.
  • the nucleic acid used for ASO according to the present invention may contain modifications.
  • Nucleic acid modification positions include sugar moieties, backbone (linkage) moieties, nucleobase (base) moieties, and 3'or 5'terminal moieties.
  • the ASO used in the present invention may contain a morpholino nucleic acid and a peptide nucleic acid.
  • Modified nucleosides include, for example, ribo to improve or stabilize nuclease resistance, to increase affinity with complementary-stranded nucleic acids, to increase cell permeability, or to visualize, as compared to RNA or DNA.
  • Examples include nucleosides, deoxyribonucleosides, RNA or DNA-modified molecules, such as sugar-modified nucleosides such as 2'-MOE, LNA, and ENA.
  • the ASO of the present invention may contain, for example, the modified nucleic acid molecule disclosed in Khvorova & Watts (Nature Biotechnology 35, 238-248 (2017) doi: 10.1038 / nbt.3765).
  • Modified sugar refers to sugars that have substitutions and / or arbitrary changes from the natural sugar moiety (ie, the sugar moiety found in DNA (2'-H) or RNA (2'-OH)).
  • a modified nucleoside refers to a modified nucleoside containing a modified sugar.
  • the sugar-modified nucleoside may be any one obtained by adding or substituting an arbitrary chemical structural substance to a part or all of the chemical structure of the sugar of the nucleoside, for example, substituting with 2'-O-methylribose.
  • Modified nucleosides substituted with 2'-O-propylribose Modified nucleosides substituted with 2'-methoxyethoxyribose, Modified nucleosides substituted with 2'-O-methoxyethylribose, 2'- Modified nucleoside substituted with O- [2- (guanidium) ethyl] ribose, modified nucleoside substituted with 2'-O-fluororibose, bridge structure having two cyclic structures by introducing a bridge structure into the sugar moiety.
  • Type artificial nucleic acid (Bridged Nucleic Acid) (BNA), more specifically, Locked Nucleic Acid (LNA) in which an oxygen atom at the 2'position and a carbon atom at the 4'position are crosslinked via methylene, ethylene.
  • Cross-linked artificial nucleic acids (Ethylene bridged nucleic acid: ENA) [Nucleic Acid Research, 32, e175 (2004)], etc., and peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)) ], Oxypeptide Nucleic Acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and Peptide Ribonucleic Acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] Etc. can be mentioned.
  • 2'-O-methyl (2'-OMe) modification (2'-OMe-RNA) of RNA is a naturally occurring modification that improves the binding affinity and nuclease resistance of modified oligonucleotides and is immune. Reduces irritation.
  • nuclease resistance is further increased over 2'-OMe modification, and the binding affinity ( ⁇ Tm) of modified nucleotides is also significantly increased.
  • 2'-fluoro (2'-F) modification (2'-F-RNA) of RNA can also be used to increase the affinity of oligonucleotides.
  • examples of other 2'modified nucleic acids include 2'-F-ANA and Sekine et al.'S 2'-modified derivative (Patent No. 5194256, JP-A-2015-02994).
  • LNA Locked Nucleic Acid
  • LNA Locked Nucleic Acid
  • the 2'oxygen and 4'carbon of the ribose sugar of RNA are fixed in the ring structure. This modification increases specificity, affinity, and half-life, allowing effective delivery to the tissue of interest with lower toxicity.
  • oligomers longer than about 8 nucleotides fully modified with LNA are known to tend to aggregate and are commonly used in admixture with DNA and other sugar-modified nucleic acids.
  • CEt which is a methylation analog of LNA, is as useful as LNA.
  • Tricyclo-DNA tkCD is a constrained nucleotide based on a tricyclic skeleton.
  • modified nucleosides include atoms (eg, hydrogen atoms, oxygen atoms) or functional groups (eg, hydroxyl groups, amino groups) in the base portion of the nucleic acid, other atoms (eg, hydrogen atoms, sulfur atoms), functional groups.
  • nucleoside for example, lipid, phospholipid, phenazine, forate , Phenantridin, anthraquinone, aclysine, fluorescein, rhodamine, coumarin, dye, and other molecules to which another chemical substance is added may be used.
  • Modified nucleobases include any nucleobase except adenine, cytosine, guanine, timine, or uracil, including, for example, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-. Iodocytosine, N4-methylcytosine, 5-fluorouracil, 5-bromouracil, 5-iodouracil, 2-thiothymine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, 8-bromoguanine, and inosine Can be mentioned.
  • At least one cytosine may be replaced with 5-methylcytosine, and in some embodiments, all cytosines are replaced with 5-methylcytosine. It may have been.
  • Nucleoside bond Natural DNA and RNA have a phosphodiester bond as a nucleoside bond.
  • the internucleoside bond may include modification.
  • a modified nucleoside bond is a nucleoside bond that has a substitution or arbitrary change from a naturally occurring nucleoside bond (that is, a phosphodiester bond), and a modified nucleoside bond is a nucleoside bond containing a phosphorus atom.
  • nucleoside bonds that do not contain phosphorus atoms are included.
  • the modified nucleoside bond may be one in which an arbitrary chemical substance is added or substituted to a part or all of the chemical structure of the phosphate diester bond of the nucleotide.
  • a modified nucleoside bond substituted with a phosphorothioate bond examples thereof include a modified nucleoside bond substituted with an N3'-P5'phosphoamidate bond.
  • Other modified nucleoside linkages include ( SC5'R p ) - ⁇ , ⁇ -CNA, PMO and the like.
  • Typical phosphorus-containing nucleoside bonds include, for example, phosphodiester bonds, phosphorothioate bonds (also referred to as thiophosphate bonds), phosphorodithioate bonds, phosphotriester bonds, and methylphosphonate bonds, methylthiophosphonate bonds, and borane phosphates. Examples include binding and phosphodiester bonding.
  • PS phosphorothioate
  • Phosphorothioate (PS) modifications were originally incorporated into oligonucleotides to confer nuclease resistance, but these modifications also have a significant impact on oligonucleotide transport and uptake.
  • PS increases the binding of receptor sites and plasma proteins by altering the charge of ASO, increasing the amount of ASO reaching the target tissue.
  • Heparin-binding proteins are one of the most compatible targets for phosphorothioate-modified oligonucleotides. Proper binding by plasma proteins suppresses rapid elimination from the blood by the renal system and promotes optimal delivery.
  • the ASO of the invention comprises at least one modified internucleotide bond, eg, 20% or more, 30% or more, 40% or more, 50% or more, 60% of the total number of internucleotide bonds. More than 70%, more than 80%, more than 90%, or more than 95% may be modified internucleotide bonds. In one embodiment of the invention, ASO is used in which all internucleotide bonds are modified internucleotide bonds (eg, phosphorothioate bonds).
  • Phosphorothioate linkages has a stereogenic phosphorus atom part
  • fully modified oligonucleotide is typically a mixture of 2 n-1 diastereomers (e.g., phosphorothioate oligonucleotides 14mer is 2 thirteen It becomes a mixture of diastereomers).
  • Sp and R p diastereomeric bonds are known to exhibit different properties.
  • R p diastereomers are less nuclease resistance than S p diastereomers, it joins with a complementary strand with a higher affinity.
  • chiral control may be performed during the synthesis of the modified nucleoside bond, and the synthesis may be controlled so that the specific phosphorothioate bond becomes a specific diastereomer.
  • Examples of molecules obtained by adding another chemical substance to an oligonucleotide / terminal-modified oligonucleotide nucleic acid in which a ligand or the like is linked include, for example, a 5'-polyamine addition derivative, a cholesterol addition derivative, a steroid addition derivative, a bile acid addition derivative, and a vitamin addition derivative. , Cy5 Derivatives, Cy3 Derivatives, 6-FAM Derivatives, Biotin Derivatives, etc. and Derivatives of Kitade et al. (PCT / JP2007 / 00877, PCT / JP2016 / 59398).
  • the site to which the ligand or the like is added may be the terminal (5'end or 3'end) of the oligonucleotide and / or the inside of the oligonucleotide.
  • the ligand or the like may be indirectly bound via hybridization with an oligonucleotide complementary to the ASO to which the ligand or the like is added (WO2013 / 089283A1).
  • ASO and cholesterol can be linked via triethylene glycol (TEG), for example, as shown below.
  • TOG triethylene glycol
  • GalNAc-linked oligonucleotides and PUFA-linked oligonucleotides are known as examples of terminal modification.
  • a ligand such as GalNAc may be directly or indirectly linked to the ASO used in the present invention.
  • Short oligonucleotides tend to be delivered more to the kidneys and long oligomers tend to be delivered more to the liver. Short oligonucleotides are less likely to bind to plasma proteins and, as a result, tend to have shorter half-lives in plasma, but multimers can be constructed using cleavable linkers and the like.
  • the ASO used in the present invention may be linked to another ASO using a cleavable linker or the like.
  • the ASO according to the present invention may have a phosphate group added to the 5'end and / or the 3'end.
  • Other terminal modifications include E-VP, methylphosphonate, phosphorothioate, C-methyl analog, etc., which are known to enhance the stability of oligonucleotides.
  • the ASO used in the present invention may include these terminal modifications.
  • Sequence design of ASO The sequence of ASO can be designed based on the base sequence of the target gene.
  • a method for designing an ASO sequence is known to those skilled in the art, and a large number of ASOs have been designed so far and their activities have been evaluated.
  • examples of antisense oligonucleotides targeting the p53 gene include EP1012267B1, EP1889911A2, WO98 / 33904, EP1598420A2, WO95 / 09916, WO20 / 248885, WO98 / 22142, WO1993 / 003770, US Pat. No. 5,654,415, US Pat. No. 5641754, US Pat. No. 5087617, Japanese Patent No.
  • ASO may be determined in consideration of the secondary or tertiary structure of the target RNA.
  • the present inventors use a unique algorithm named MobyDick TM for sequencing.
  • nucleic acids For the structure prediction of nucleic acids, the following references may be referred to: --Markham, N.R. & Zuker, M. (2005) DINAMelt web server for nucleic acid acid melting prediction. Nucleic Acids Res., 33, W577-W581; --Markham, NR & Zuker, M. (2008) UNAFold: software for nucleic acid folding and hybridization. In Keith, JM, editor, Bioinformatics, Volume II. Structure, Function and Applications, number 453 in Methods in Molecular Biology, chapter 1 , pages 3-31. Humana Press, Totowa, NJ. ISBN 978-1-60327-428-9.).
  • the antisense oligonucleotide (ASO) according to the present invention can be substantially identical to any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 125.
  • substantially the same means that the oligonucleotide does not have to be completely (100%) identical to the target sequence and has 80% or more identity.
  • the oligonucleotide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of any one of the sequences of SEQ ID NOs: 1 to 125. , Or an oligonucleotide having 100% identity.
  • One aspect of the invention is a pharmaceutical composition for inhibiting the expression of the p53 gene in a cell, an oligonucleotide containing a complementary region that is substantially complementary to at least a portion of the mRNA encoding the p53 gene.
  • the oligonucleotide is an antisense oligonucleotide having a sequence substantially the same as that of any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 125.
  • substantially complementary means that the oligonucleotide does not have to be completely (100%) complementary to the target sequence and is 80% or more, for example 85%, 90%, 95%, 98% or It means having 99% complement.
  • This oligonucleotide contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences of SEQ ID NOs: 1 to 125. It can be an oligonucleotide consisting of sequences having the same identity. In certain embodiments, the oligonucleotide consists of a sequence that is 100% identical to any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 125. In certain embodiments, the oligonucleotide may have additional sequences on the 5'and / or 3'side of the complementary region.
  • the antisense oligonucleotide is essentially a single-stranded molecule.
  • being essentially single-stranded means that in the process of delivering or formulating an oligonucleotide, a double-strand may be formed with a separate nucleic acid that is temporarily complementary.
  • oligonucleotide When an oligonucleotide hybridizes to a target RNA and exerts an antisense effect, it acts in the form of a single-stranded oligonucleotide, and finally a double strand is formed by the target RNA and ASO. ..
  • oligonucleotides having the sequences of SEQ ID NOs: 1 to 125 can also be used as part of siRNA.
  • Hybrid ASO International Publication No. 2013/089283 and Nishina et al., Nature Communications volume 6, Article number: 7969 (2015) describe double-stranded oligonucleotides (also called HDOs) containing RNA oligonucleotides complementary to ASO. It is described that the target gene is efficiently delivered and accumulated in the liver and the expression of the target gene in the liver is suppressed.
  • International Publication No. 2015/105083 describes ASO in which a GalNAc derivative is bound to HDO via a linker, and such antisense oligonucleotides are more efficient than tocopherol (Toc) modified products. It is described that the expression of the target gene is suppressed. Furthermore, in International Publication No.
  • single-stranded oligonucleotides in which ASO-complementary oligonucleotides are linked to form double strands in the molecule are equivalent to double-stranded oligonucleotides. It is described that it exhibits the above antisense effect. Therefore, in some aspects of the invention, the ASO may form a double strand with a nucleic acid strand complementary thereto. Further, in some aspects of the present invention, the ASO may be linked to a nucleic acid strand complementary thereto, and a double-stranded portion may be formed by intramolecular self-annealing.
  • ligands such as tocopherol (Toc) and GalNAc may be linked to the nucleic acid region complementary to ASO.
  • ligands such as tocopherol (Toc) and GalNAc may be linked to the nucleic acid region complementary to ASO.
  • the oligonucleotides of the present disclosure may be used as part of a siRNA duplex.
  • the pharmaceutical composition antisense nucleic acid can be formulated by itself, but is usually mixed with one or more pharmacologically acceptable carriers and any of the well-known technical fields of pharmaceutics. It is desirable to administer it as a pharmaceutical preparation produced by the above method.
  • the pharmaceutical composition may contain a mixture of multiple ASOs having different sequences.
  • the administration target includes human or non-human animals, for example, non-human mammals. It is desirable to use the most effective route of administration for treatment, including oral administration or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, intravenous and transdermal administration. It can be done, preferably intravenously.
  • preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
  • Liquid preparations such as emulsions and syrups include water, sucrose, sorbitol, sugars such as fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, and p-hydroxybenzoic acid. It can be produced by using preservatives such as esters, flavors such as strawberry flavor and peppermint as additives. Capsules, tablets, powders, granules, etc.
  • excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol, hydroxy. It can be produced by using a binder such as propyl cellulose and gelatin, a surfactant such as a fatty acid ester, and a plastic agent such as glycerin as additives.
  • Injections, suppositories, sprays, etc. are examples of preparations suitable for parenteral administration.
  • the injection is prepared by using a carrier consisting of a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids.
  • the spray agent is prepared using a carrier or the like that does not irritate the oral cavity and airway mucosa of the recipient and disperses the active ingredient as fine particles to facilitate absorption.
  • the carrier include lactose, glycerin, liposomes, and nanomicelles.
  • preparations such as aerosols and dry powders are possible. Further, also in these parenteral preparations, the components exemplified as additives in the oral preparation can be added.
  • the dose or frequency of administration varies depending on the target therapeutic effect, administration method, treatment period, age, body weight, etc., but is, for example, 10 ⁇ g / kg to 100 mg / kg per day for adults.
  • One aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising about 14-20 mer oligonucleotides (ASOs) for use in the treatment or prevention of a disease or condition in a patient.
  • the patient can be a human or non-human animal.
  • the term "about 14 mer” is understood to include at least the range of 1 base before and after that, that is, 13 mer, 14 mer, and 15 mer, and the term "about 20 mer” means 19 mer, 20 mer, and It is understood to include 21 mer.
  • a 14 mer ASO is preferably used.
  • the disease or condition is ischemic-reperfusion injury, hearing loss, hearing impairment, balance impairment, hearing loss, chemotherapy-induced alopecia, radiation therapy-induced alopecia, acute renal failure, acute renal injury.
  • Chronic kidney disease (CKD) side effects associated with anticancer drug therapy, late transplant function (DGF) in kidney transplant patients, spinal cord injury, brain injury, stroke, stroke, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, tumor, It can be any of burns, wounds, hyperthermia, hypoxia, ischemia, organ transplantation, bone marrow transplantation (BMT), myocardial infarction / heart attack, cardiotoxicity, p53-positive cancer, and acute liver failure.
  • BMT bone marrow transplantation
  • One embodiment of the present invention is a pharmaceutical composition for treating or preventing a disease or symptom, or a pharmaceutical composition for use in treating or preventing a disease or symptom, any sequence selected from the following.
  • pharmaceutical compositions comprising, or comprising an ASO consisting of any of the sequences: p53-ASO-15, p53-ASO-15-11, p53-ASO-15-12, and p53-ASO-15-16.
  • One aspect of the present invention relates to the use of oligonucleotides (ASOs) of about 14-20 mer in the manufacture of medicines for use in the treatment or prevention of diseases or disorders in patients.
  • ASOs oligonucleotides
  • a 14 mer ASO is preferably used.
  • one aspect of the present invention is a method for treating or preventing a disease or disorder in a patient, which comprises a step of administering about 14 to 20 mer of oligonucleotide (ASO) to the patient.
  • ASO oligonucleotide
  • a 14 mer ASO is preferably used.
  • one aspect of the present invention is a method for treating or preventing acute renal failure (acute kidney injury) in a patient, which comprises or consists of any sequence selected from the following. Consistent with methods comprising administering to a patient an oligonucleotide consisting of: p53-ASO-15, p53-ASO-15-11, p53-ASO-15-12, and p53-ASO-15-16.
  • Acute Kidney Insufficiency occurs, for example, in patients undergoing major cardiovascular surgery after reduced local blood flow to the kidney during surgery and recovery of blood flow. Due to reperfusion injury, it may develop within hours to days after surgery, and the mortality rate 30 days after onset exceeds 50%.
  • ASOs according to the invention can be administered to patients to prevent, reduce their severity, or treat acute renal failure. Administration of ASO is, for example, from before the start of surgery (eg, 2 hours, 4 hours, or 6 hours) to within 8 hours after surgery (eg, 3 hours, 4 hours, or 5 hours after surgery). It can be done at any time. Administration can be single or multiple doses. Administration can be, for example, by intravenous injection.
  • Example 1 Design of antisense (ASO) for p53 , synthesis A sequence targeting p53 was designed for ASO and synthesized.
  • the human transcript NM_000546.5 (SEQ ID NO: 132, FIG. 1) registered in the NCBI Refseq collection was used for the design. The synthesis was outsourced to GeneDesign Co., Ltd.
  • the synthesized nucleotide sequences are shown below. Uppercase letters indicate RNA or sugar-modified nucleic acid, and lowercase letters indicate DNA. "5" represents 5-methylcytidine.
  • the parentheses in each nucleotide indicate the modification of the 2'position of ribose, and L indicates LNA.
  • G (L) represents LNA type guanosine.
  • the acute accent flex " ⁇ " between bases indicates that the nucleoside bond is a thiophosphate bond (phosphorothioate bond).
  • P53-ASO-01-23 shown below are 14-base long 2-10-2 gapmers with 2 bases 5'wing region and 3'wing region, respectively. No phosphate group was added to the 5'end and 3'end of these ASOs.
  • p53-ASO-01 to 23 which had been previously refolded (85 ° C., 5 min, then rapidly cooled on ice), were adjusted to a final concentration of 125 nM, respectively, and the non-cationic lipid carrier TransIT-TKO ( Mirus Cat no. MIR2150) Add 2.5 ⁇ L per well to each of the above serum-free mediums, mix well by pipetting to a final 50 ⁇ L, and incubate for 15 minutes at room temperature. The mixture was slowly added to each well for transfection. Cells were cultured for either 24 or 48 hours.
  • Total RNA was prepared using RNAiso Plus (total RNA extraction reagent) (Cat No. 9109 manufactured by TakaRa) or RNA extraction and purification kit Zymo-Spin IC Columns (ZYMO Research R2062).
  • the expression change of p53 mRNA was examined using RT-qPCR.
  • the Revertra Ace qPCR RT kit (Code No.FSQ-101) was used.
  • a reverse transcription reaction was performed using 150 to 300 ng of total RNA as a template according to the recommended conditions of the kit. Specifically, 150 to 300 ng / 8 ⁇ L of total RNA was prepared, heated at 65 ° C. for 5 minutes, and then 2 ⁇ L of 5xRT Master Mix included in the kit was added to each sample at 37 ° C.
  • a reaction solution for PCR was prepared as follows using TB Green Premix ExTaq II (TaKaRa Code: RR820). Each well was set to a final volume of 25 ⁇ L containing 400 nM of forward and reverse primers, 12.5 ⁇ L of TB Green Premix EX Tag II, and 2 ⁇ L of reverse transcriptase.
  • the PCR reaction was carried out on a 96-well plate using Thermal Cycler Dice Real Time Sysytem (manufactured by TakaRa).
  • the housekeeping gene GAPDH was used as an endogenous control. We also used the housekeeping gene UBC as a control.
  • the following primer sequences were used for each of the three genes.
  • the "Rel.Qty” value (relative amount) was calculated based on the PCR analysis results of Thermal Cycler Dice Real Time Sysytem (TaKaRa).
  • the "Rel.Qty” value is a value obtained by correcting the PCR analysis results of the p53 gene and the UBC gene with the GAPDH analysis result of the housekeeping gene set as a control ("/ GAPDH").
  • Example 3 Evaluation of ASO with the target site shifted back and forth (with transfection reagent) Based on the design of p53-ASO-15, the antisense sequence (ASO-24, ASO-15-18 to ASO-15-28) in which the position of ASO hybridizing to the target mRNA is shifted to the 5'side or the 3'side. SEQ ID NOs: 24-35) were designed. The chain length is 14 mer, which is the same as p53-ASO-15. In the sequence shown below, the left side is the 3'end and the right side is the 5'end, and the lowercase letters represent DNA, the uppercase letters represent LNA, and the uppercase letter C represents LNA-type 5-methylcytidine (hereinafter). Similarly).
  • the assay was performed in the same manner as in Example 2, and ASO was transfected into HeLa cells at a final concentration of 20 nM to evaluate the expression inhibitory effect. The results are shown in FIG. Those whose design position was shifted to the 5'side or 3'side from the center (15-18 to 15-28) had a weaker suppression effect than ASO-15.
  • Example 4 Based on the design of antisense shortened p53-ASO-15, which has been found to have inhibitory activity on p53, the position of ASO that hybridizes to the target mRNA was designed to be shifted to the 5'side or 3'side. (ASO-25 to ASO-36; SEQ ID NOs: 36 to 47). The chain length was 13 mer, which was 1 nt shorter than p53-ASO-15.
  • the assay was performed in the same manner as in Example 2, and ASO was transfected into HeLa cells at a final concentration of 20 nM to evaluate the expression inhibitory effect.
  • the results are shown in FIG. On Day 1, none showed a stronger inhibitory effect than ASO-15, which is 14 mer. 25 and 26 show an inhibitory effect of about 40%, which is the strongest in the 13mer series. These two ASOs are ASOs with the green part in the center and have an arrangement close to ASO-15, so they may show some effect. On Day 2, 25 and 33 showed a slightly weaker effect than ASO-15.
  • Example 5 Based on the design of antisense elongated p53-ASO-15, which has been found to have inhibitory activity on p53, the position of ASO was shifted to the 5'side or 3'side (ASO-37 to ASO). -49, ASO-15-40; SEQ ID NOs: 48-61). The chain length was 15 mer, which was 1 nt longer than p53-ASO-15.
  • the assay was performed in the same manner as in Example 2, and ASO was transfected into HeLa cells at a final concentration of 20 nM to evaluate the expression inhibitory effect.
  • the results are shown in FIG.
  • QPI1002 was also transfected with 20 nM, but showed the same or slightly weaker inhibitory effect as ASO-15.
  • Example 6 Based on the design of further lengthened p53-ASO-15 of antisense, which was found to have inhibitory activity on p53, the position of ASO was shifted to the 5'side or 3'side (ASO-50 ⁇ ). ASO-63, ASO-15-10; SEQ ID NOs: 62-76). The chain length is 16 mer, which is 2 nt longer than p53-ASO-15.
  • the assay was performed in the same manner as in Example 2, and ASO was transfected into HeLa cells at a final concentration of 20 nM to evaluate the expression inhibitory effect.
  • the results are shown in FIG. As a result of transfecting the 16mer series with the chain length extended to 16mer at 20 nM, none showed a clearly stronger inhibitory effect than ASO-15, which is 14 mer.
  • Example 7 Based on the design of p53-ASO-15, which is a further lengthened antisense that has been found to have inhibitory activity on p53, the position of ASO was shifted to the 5'side or 3'side (ASO-64-). ASO-78, ASO-15-41; SEQ ID NOs: 77-92). The chain length is 17 mer, which is 3 nt longer than p53-ASO-15 (hereinafter referred to as the 17 mer series).
  • the assay was performed in the same manner as in Example 2, and the 17mer series was transfected with ASO into HeLa cells at a final concentration of 20 nM to evaluate the expression inhibitory effect.
  • the results are shown in FIG.
  • 15-41 was about the same as ASO-15 for both Days 1 and 2.
  • ASO-70 showed a slightly weaker effect than ASO-15 only on Day 1. After all, the effect of ASO centering on the green part is high.
  • Example 8 Based on the design of further lengthened p53-ASO-15 of antisense, which was found to have inhibitory activity on p53, the position of ASO was shifted to the 5'side or 3'side (ASO-79-). ASO-94, ASO- (95), ASO-15-11; SEQ ID NOs: 93-109). The chain length is 18 mer, which is 4 nt longer than p53-ASO-15 (hereinafter 18 mer series).
  • the assay was performed in the same manner as in Example 2, and the 18mer series was transfected with ASO into HeLa cells at a final concentration of 20 nM to evaluate the expression inhibitory effect.
  • the results are shown in FIG. As a result of transfecting the 18mer series with the chain length extended to 18mer at 20 nM, none showed a clearly stronger inhibitory effect than ASO-15, which is 14 mer.
  • Example 9 Effect of modification of wing region Based on the design of p53-ASO-15, the number of LNAs located at both ends was changed and the effect on the expression suppression effect was investigated (ASO-15-29 to ASO-15). -33; SEQ ID NOs: 110-114). In addition, ASO in which LNAs at both ends were changed to 2'-O-methoxyethyl (MOE) was also evaluated for its inhibitory effect at the same time.
  • MOE 2'-O-methoxyethyl
  • the assay was performed in the same manner as in Example 2. The results are shown in FIG. 15-29 to 15-32 with the LNA (capital letters) shifted or increased are about the same as or slightly weaker than the ASO-15. 15-33 with 2'-MOE modification (capital italics) showed almost no inhibitory effect.
  • Example 10 Effect of other modifications The chain length was extended by 2 nt to the 5'side, 3'side, or both ends centering on p53-ASO-15. In addition, 15-16 in which all the bases were PS-bonded and 15-17 in which the gap region was narrowed were also evaluated at the same time (ASO-15-07 to ASO-15-17; SEQ ID NOs: 115 to 125).
  • the assay was performed in the same manner as in Example 2, and HeLa cells were transfected with a final concentration of 100 nM to evaluate the expression inhibitory effect.
  • the results are shown in FIG.
  • the base is extended in the black direction of the upper bar.
  • the same effect as ASO-15 was exhibited only when it was extended to the 3'side (the core sequence of ASO-15 was present at the 5'end).
  • 15-16 which had PS bonds between all bases including those between LNAs, showed a stronger inhibitory effect than the original.
  • 15-17 which increased the number of LNAs, had a slightly weaker inhibitory effect than ASO-15. It was p53-ASO-15-16 with PS bonds between the bases that showed a clearly stronger inhibitory effect than p53-ASO-15.
  • the structure of p53-ASO-15-16 (SEQ ID NO: 124) is 5'-G (L) ⁇ G (L) ⁇ c ⁇ a ⁇ g ⁇ t ⁇ g ⁇ a ⁇ c ⁇ c ⁇ c ⁇ g ⁇ G It can be written as (L) ⁇ A (L) -3'.
  • Example 11 P53-ASO-15-16-32 with cholesterol added to the 3'end of cholesterol- added p53-ASO-15-16 was transfected into HeLa cells at a final concentration of 20 nM, and the same assay as in Example 2 was performed. went. The results are shown in FIG. P53ASO-15-16-32 with cholesterol added to the 3'end markedly reduced p53 mRNA levels as compared to Mock or the negative control NTS1. Compared with 15-16, the inhibitory effect was slightly weakened on the first day after transfection, but had the same effect on the second day. From this result, it was shown that the inhibitory effect was maintained even when cholesterol was added as a ligand.
  • Example 12 p53-induced reporter gene assay
  • the amount of p53 protein was measured by a luciferase assay using a reporter plasmid with a p53 response element.
  • the expression-suppressing effect of p53-ASO-15 and Quark's siRNA QP1002 was evaluated by a luciferase assay. The result after one day is shown in FIG. ASO showed an effect from 1.2 nM and suppressed its expression in a concentration-dependent manner up to 100 nM.
  • the expression of QPI1002 (siRNA) was suppressed in a concentration-dependent manner, and the effect leveled off at concentrations above 3.7 nM with an effect of about 80%.
  • ASO-15 showed an inhibitory effect of about 50% at 3.7 nM.
  • QPI1002 showed an inhibitory effect of about 70% at 3.7 nM, and the inhibitory effect was almost leveled off. At concentrations lower than 1.2 nM, there was little difference between the two.
  • the IC 50 was 4.0 nM for ASO and 2.6 nM for QPI1002.
  • Example 13 Evaluation of concentration dependence by qPCR p53ASO-15 or QPI-1002 was TF to HCT116 cells at a concentration of 0.046 to 100 nM, and the expression inhibitory effect was measured by performing the same assay as in Example 2. The results are shown in FIG. At 24 hours after TF, p53-ASO-15 showed almost no inhibitory effect at 0.046 to 11 nM, and showed an inhibitory effect of 24% at 33 nM and 49% at 100 nM. The inhibitory effect of QPI-1002 under the same conditions showed almost no inhibitory effect at 0.046 to 11 nM, and showed an inhibitory effect of 23% at 33 nM and 59% at 100 nM.
  • p53-ASO-15 showed almost no inhibitory effect at 0.046 to 3.7 nM, and showed an inhibitory effect of 10% at 11 nM, 22% at 33 nM, and 52% at 100 nM.
  • the inhibitory effect of QPI-1002 under the same conditions showed almost no inhibitory effect at 0.046 to 3.7 nM, 15% at 10 nM, 31% at 33 nM, and 79% at 100 nM.
  • the sample described as ⁇ p53 uses RNA extracted from HCT116 cells in which the p53 gene is knocked out.
  • Example 14 Evaluation by Western blotting HCT116 cells were transfected (TF) with ASO or QPI-1002 at a concentration of 1 to 100 nM, and the cells were then recovered 48 hours later. The collected cells were divided into two groups, one of which prepared a whole cell lysate and the other of which prepared total RNA (Fig. 15). Western blotting was performed using the prepared whole cell lysate to measure p53 protein levels. In addition, real-time PCR was performed using the prepared total RNA to measure the p53 mRNA level. The results are shown in FIG. The p53 protein level was correlated with the mRNA level and was so attenuated that the p53 band was undetectable at 100 nM when ASO-15, 15-16 was used. On the other hand, QPI-1002 was at a level that could be detected even at a concentration of 100 nM, although the band intensity was weakened.
  • ASOs antisense oligonucleotides that act on transcripts of the p53 gene. By using these relatively short oligonucleotides, the amount of p53 mRNA can be reduced and protein expression can be suppressed. These ASOs may be useful in the treatment or prevention of acute renal failure, alopecia and the like.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Neurosurgery (AREA)
  • Biochemistry (AREA)
  • Neurology (AREA)
  • Cardiology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Dermatology (AREA)
  • Hospice & Palliative Care (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Transplantation (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Urology & Nephrology (AREA)
  • Psychology (AREA)

Abstract

Les présents inventeurs ont identifié des oligonucléotides antisens (ASO) qui agissent sur un produit de transcription du gène p53. À l'aide de ces oligonucléotides relativement courts, la quantité d'ARNm de p53 peut être réduite et l'expression de la protéine peut être inhibée. Ces ASO peuvent être utiles dans le traitement ou la prévention de l'insuffisance rénale aiguë et de l'alopécie.
PCT/JP2020/025676 2019-07-04 2020-06-30 Médicament d'acide nucléique et son utilisation Ceased WO2021002359A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2021500250A JP6934695B2 (ja) 2019-07-04 2020-06-30 核酸医薬とその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019124901 2019-07-04
JP2019-124901 2019-07-04

Publications (1)

Publication Number Publication Date
WO2021002359A1 true WO2021002359A1 (fr) 2021-01-07

Family

ID=74101332

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/025676 Ceased WO2021002359A1 (fr) 2019-07-04 2020-06-30 Médicament d'acide nucléique et son utilisation

Country Status (2)

Country Link
JP (1) JP6934695B2 (fr)
WO (1) WO2021002359A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021157730A1 (fr) * 2020-02-06 2021-08-12 株式会社Ｖｅｒｉｔａｓ Ｉｎ Ｓｉｌｉｃｏ Médicament à base d'acide nucléique et utilisation associée

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CACERES G. ET AL.: "TP53 suppression promotes erythropoiesis in del(5q) MDS, suggesting a targeted therapeutic strategy in lenalidomide-resistant patients.", PNAS, vol. 110, no. 40, 2013, pages 16127 - 16132, XP055783288 *
CERALINE J. ET AL.: "INACTIVATION OF p53 IN NORMAL HUMAN CELLS INCREASES G2/ M ARREST AND SENSITIVITY TO DNA-DAMAGING AGENTS.", INT. J. CANCER, vol. 75, 1998, pages 432 - 438, XP055783295 *
CORTES J. ET AL.: "Phase 2 Randomized Study of p53 Antisense Oligonucleotide (Cenersen) Plus Idarubicin With or Without Cytarabine in Refractory and Relapsed Acute Myeloid Leukemia.", CANCER, vol. 118, 2012, pages 418 - 427, XP055783290 *
DMITRIEVA N. ET AL.: "p53 Protects renal inner medullary cells from hypertonic stress by restricting DNA replication.", AM J PHYSIOL RENAL PHYSIOL, vol. 281, 2001, pages F522 - F530, XP055783296 *
OKIBA SATOSHI ET AL: "Design of antisense oligonucleotide", FOLIA PHARMACOLOGICA JAPONICA, vol. 148, 2016, pages 100 - 104 *
PETTY RD. ET AL.: "Expression of the p53 tumour suppressor gene product is a determinant of chemosensitivity.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 199, no. 1, 1994, pages 264 - 270, XP001016198 *
TAKEBA Y. ET AL.: "Irinotecan Activates p53 With Its Active Metabolite, Resulting in Human Hepatocellular Carcinoma Apoptosis.", J PHARMACOL SCI, vol. 104, 2007, pages 232 - 242, XP055783292 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021157730A1 (fr) * 2020-02-06 2021-08-12 株式会社Ｖｅｒｉｔａｓ Ｉｎ Ｓｉｌｉｃｏ Médicament à base d'acide nucléique et utilisation associée

Also Published As

Publication number Publication date
JPWO2021002359A1 (ja) 2021-09-13
JP6934695B2 (ja) 2021-09-15

Similar Documents

Publication Publication Date Title
JP7174384B2 (ja) キメラ２重鎖核酸
JP7554488B2 (ja) 血液脳関門通過型ヘテロ２本鎖核酸
WO2020196662A1 (fr) Complexe d'acide nucléique double brin et son utilisation
JP7129702B2 (ja) オーバーハングを有する二本鎖核酸複合体
JP6440170B2 (ja) キメラ一本鎖アンチセンスポリヌクレオチドおよび二本鎖アンチセンス剤
WO2023022229A1 (fr) Acide héteronucléique modifié contenant de l'acide nucléique morpholino
EP4206213A1 (fr) Composés et procédés de modulation de smn2
KR20220069103A (ko) 최소 플루오린 함량을 갖는 작은 간섭 rna의 화학적 변형
KR20210142699A (ko) Dux4의 발현을 조절하기 위한 화합물, 방법 및 의약조성물
JPWO2019181946A1 (ja) 毒性が軽減した核酸
JP2024514880A (ja) Ｐｎｐｌａ３発現を調節するための組成物及び方法
JP2025156522A (ja) 修飾ヘテロ核酸
JP7737667B2 (ja) 核酸複合体
WO2021157730A1 (fr) Médicament à base d'acide nucléique et utilisation associée
JP6934695B2 (ja) 核酸医薬とその使用
JPWO2019044974A1 (ja) スモールガイドアンチセンス核酸とその使用
WO2021187392A1 (fr) Acide hétéro-nucléique contenant un acide nucléique morpholino
CN121057820A (zh) 使用环状前药核酸递送rna治疗药物
KR20250125967A (ko) 진보된 rna 표적화 (arnatar)
EP4522748A1 (fr) Compositions et procédés d'inhibition de l'expression de snca
TW202313977A (zh) 作為新穎基因靜默技術的短雙股ｄｎａ及其應用
EP4446414A1 (fr) Complexe d'oligonucléotide antisens
WO2023127848A1 (fr) Médicament à base d'acide nucléique ciblant des troubles liés à l'ebv
WO2023042876A1 (fr) Acide hétéronucléique comprenant un nucléoside 2'-modifié
WO2022250050A1 (fr) Acide hétéro-nucléique à teneur en ｓｃｐｂｎａ ou ａｍｎａ

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2021500250

Country of ref document: JP

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20835586

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20835586

Country of ref document: EP

Kind code of ref document: A1