WO2021002359A1 - Nucleic acid drug and use thereof - Google Patents

Abstract

The present inventors have identified antisense oligonucleotides (ASOs) which act on a p53 gene transcription product. By using these relatively short oligonucleotides, the amount of p53 mRNA can be reduced and the expression of the protein can be suppressed. These ASOs can be useful in treatment or prevention of acute kidney failure and alopecia.

Description

Nucleic acid medicine and its use Cross-reference of related applications

This application is an application that enjoys the priority benefit of Japanese Patent Application No. 2019-124901 (Filing date: July 4, 2019), which is incorporated herein by reference in its entirety.

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.

In the conventional drug discovery technology in drug development, many efforts to target proteins for drug discovery have been promoted, and drug discovery by drugs that act on proteins and peptides such as small molecule drugs and antibodies has been the main focus. In recent years, the causes of diseases such as genomic science and personalized medicine have been elucidated at the genetic level, so efforts are being made to use drug discovery targets as nucleic acids, and it is possible to respond to therapeutic targets that are difficult to target with small molecule drugs and antibody drugs. Therefore, nucleic acid drugs are expected as a new modality (drug discovery method).

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).

Acute renal failure (ARF) (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. 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. However, at present, 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.

U.S. Pat. No. 9,334,499 U.S. Pat. No. 7,910,566

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. 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:

[Aspect 1] 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. Contains, pharmaceutical composition.
[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.
[Aspect 14] The pharmaceutical composition according to aspect 13, wherein the cross-linked nucleic acid is LNA.
[Aspect 15] The pharmaceutical composition according to any of aspects 12 to 14, wherein the modified nucleoside bond is a phosphorothioate bond.
[Aspect 16] The pharmaceutical composition according to any of aspects 12 to 15, wherein all modified nucleoside bonds are phosphorothioate bonds.
[Aspect 17] The pharmaceutical composition according to any one of aspects 1 to 16, wherein all nucleoside bonds are phosphorothioate bonds.
[Aspect 18] The pharmaceutical composition according to any one of aspects 12 to 17, wherein the binding between modified nucleosides is chirally controlled.
[Aspect 19] The pharmaceutical composition according to any one of aspects 1 to 18, wherein one or both of the terminal hydroxyl groups of the oligonucleotide is modified.
[Aspect 20] The pharmaceutical composition according to any one of aspects 1 to 19, wherein a phosphate group is added to one or both of the terminal hydroxyl groups of the oligonucleotide.
[Aspect 21] The pharmaceutical composition according to any one of aspects 1 to 18, wherein one or both of the terminal hydroxyl groups of the oligonucleotide is not modified.
[Aspect 22] The pharmaceutical composition according to any one of aspects 1 to 18, wherein a phosphate group is not added to one or both of the terminal hydroxyl groups of the oligonucleotide.
[Aspect 23] The pharmaceutical composition according to any one of aspects 1 to 22, wherein the oligonucleotide is an oligonucleotide containing any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 125.
[Aspect 24] The pharmaceutical composition according to any one of aspects 1 to 22, wherein the oligonucleotide is an oligonucleotide consisting of any one sequence of SEQ ID NO: 1 to SEQ ID NO: 125.
[Aspect 25] 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 pharmaceutical composition according to any one of aspects 1 to 22, which is an oligonucleotide consisting of a sequence having 100% identity.
[Aspect 26] The pharmaceutical composition according to any one of aspects 1 to 25, which is a pharmaceutical composition for use in treating or preventing a disease or symptom in a subject.
[Aspect 27] The pharmaceutical composition according to aspect 26, wherein the disease or symptom is related to the expression of the p53 gene.
[Aspect 28] 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. The pharmaceutical composition.
[Aspect 29] The pharmaceutical composition according to any of aspects 1-28, which comprises a pharmaceutically acceptable excipient, buffer, and / or additive.
[Aspect 30] 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 in which all nucleoside linkages of Gapmer are phosphorothioate linkages.
[Aspect 31] 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. A pharmaceutical composition containing an oligonucleotide selected from the group consisting of -15-16 as an active ingredient.
[Aspect 32] An oligonucleotide selected from the group consisting of p53-ASO-15, p53-ASO-15-11, p53-ASO-15-12, and p53-ASO-15-16, or a salt thereof.

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. 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. The left side shows the result 1 day after transfection, and the right side shows the result 2 days after transfection. 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. 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). The present invention will be described in detail below.

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. Thus, 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. Such side effects are often attributed to p53-induced cell death of normal cells, and temporary suppression of p53 during the acute phase of anticancer therapy is a therapeutic strategy to avoid these serious toxicity. Has been proposed as. That is, it is understood by those skilled in the art that a disease or symptom related to induction of apoptosis can be treated or prevented by controlling the expression of the p53 protein by allowing a drug such as ASO to act on the transcript of the p53 gene. In the suppression of p53 gene expression, the amount of protein synthesized from the p53 gene through transcription and translation is 75% or less, 50% or less, 40% or less, 30% or less, 25, as compared with the case where no drug is allowed to act. It can be done by reducing to% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 3% or less. 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. In certain embodiments, 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. , Acute kidney injury, Chronic kidney disease (CKD), Side effects associated with anticancer drug therapy, Delayed transplantation function (DGF) in patients with renal transplantation, Spinal cord injury, Brain injury, Attack, Stroke, Neurodegenerative disease, Parkinson's disease, Alzheimer's disease Disease, tumor, burn, wound, hyperthermia, hypoxia, ischemia, organ transplant, bone marrow transplant (BMT), myocardial infarction / heart attack, cardiotoxicity, p53-positive cancer, or acute liver failure, but the target is Not limited to these.

The base sequence of the p53 gene and its mRNA is known and can be easily obtained from a database such as GenBank. For example, as the mRNA sequence of the human p53 gene, the sequence of NCBI accession number NM_000546.5 (FIG. 1; SEQ ID NO: 132) can be used.

Antisense oligonucleotide (ASO)
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. The mechanism by which the antisense effect is exerted includes any mechanism resulting from hybridization of the antisense oligonucleotide with the target nucleic acid, for example, the result or effect of the hybridization can be either degradation of the target or occupation of the target. It can be. In certain embodiments, 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. For example, 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. In particular, 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. be able to. Also, in certain embodiments, 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. In particular, 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". For example, 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. In certain embodiments, 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 (gap 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 (wing 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. In certain embodiments, 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, in certain embodiments, 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. Examples of methods using known chemical synthesis include a phosphoramidite method, a phosphorothioate method, a phosphotriester method, and the like. For example, ABI3900 high-throughput nucleic acid synthesizer (manufactured by Applied Biosystems) and 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. For example, during 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 μM.

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. Examples of oligonucleotides (also referred to as polynucleotides) include RNA, which is a polymer of ribonucleotides, DNA, which is a polymer of deoxyribonucleotides, polymers in which RNA and DNA are mixed, and 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. Further, the ASO used in the present invention may contain a morpholino nucleic acid and a peptide nucleic acid.

Modified nucleosides 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. For 2'-O-methoxyethyl (2'-MOE), nuclease resistance is further increased over 2'-OMe modification, and the binding affinity (ΔTm) 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), which links the 2'oxygen and 4'carbon of ribose, brings about a significant increase in binding affinity. In LNA, 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. However, 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.

Other 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. (For example, an amino group), or one substituted with an alkyl group having 1 to 6 carbon atoms or one protected with a protective group (for example, a methyl group or an acyl group), 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 (or modified bases) 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. For example, in an oligonucleotide having the sequences of SEQ ID NOs: 1-125, 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. In one embodiment of the invention, 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. , And 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. For example, 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} ) -α, β-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.

The phosphorothioate (PS) bond, one of the major modified nucleoside bonds, helps protect the oligonucleotide from degradation by nucleases. 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.

In some embodiments, 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. In the ASO of the present invention, 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.

GalNAc-linked oligonucleotides and PUFA-linked oligonucleotides are known as examples of terminal modification. By linking GalNAc to the terminal, the efficiency of delivery of the oligonucleotide to the liver can be enhanced. 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. For example, 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. 5671791, Hata et al. Biochemical and Biophysical Research Communivcations 1991, 179 , 528-534, Bayever et al. Leukemia & Lymphoma 1994, 12 , 223-231, Mahdi et al. Journal of Cell Science 1995, 108 , 1287-1293, Hirota et al. Jpn. J. Cancer Res. 1996, 87 , 735-742, Dai et al. Acta Pharmacologica Sinica 2006, 27 , 1453-1458, Robu et al. Pros Genetics 2007, 3 , e78, alachkar et al. Journal of Pharmaceutical and Biomedical Analysis, 2012, 71 , 228-232, Gorska et al. 2013, PLoS ONE 8 (11): e78863, Swiatkowska et al. PLOS ONE, 2015, 10 , e0141676, Swaitkowska et al. Acta Biochimica Polonica, 2016, 63 , 645-651. The sequence of 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 ™ for sequencing.

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.).

In addition, when sequencing, not only the knockdown efficiency of the target gene, but also toxicity, off-target effect, commonality among species, stability, intracellular uptake efficiency, and other factors are taken into consideration. Can be included.

Complementarity with Target Sequence 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. Here, 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. In certain embodiments, 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. The value of identity can be calculated according to the description of WO 2016/0277747 (the contents of which are incorporated herein by reference). Inhibition of expression or activity by ASO refers to reduction or inhibition of expression or activity, and expression or activity does not necessarily have to be completely eliminated.

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. As an active ingredient, 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. Regarding. Here, 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.

Techniques for the design, preparation and use of antisense oligonucleotides are well known to those of skill in the art, but can be referred to, for example, WO 2016/0277747 (the contents of which are incorporated herein by reference).

Delivery of Oligonucleotides DDS tools such as transfection reagents, liposomes, vectors, nanomicelles, and complementary nucleic acids can be used to deliver oligonucleotides to cells. In some aspects of the invention, the antisense oligonucleotide is essentially a single-stranded molecule. Here, 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. 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. .. In some embodiments of the present invention, 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. 2017/13112, 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. Furthermore, in some aspects of the invention, ligands such as tocopherol (Toc) and GalNAc may be linked to the nucleic acid region complementary to ASO. Moreover, in some embodiments, 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.

Examples of 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. are 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. In addition, 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.

Specific examples of the carrier include lactose, glycerin, liposomes, and nanomicelles. Depending on the properties of the nucleic acid used in the present invention and the carrier used, 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 μg / kg to 100 mg / kg per day for adults.

One aspect of the invention relates to 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. In this specification, 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. In some embodiments, a 14 mer ASO is preferably used.

In certain embodiments, 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.

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. For 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.

Use and Therapeutic Methods in the Manufacture of Medicines 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. In some embodiments, a 14 mer ASO is preferably used.

In addition, 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. Regarding the method. In some embodiments, a 14 mer ASO is preferably used.

In addition, 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.

Prevention and Treatment of Surgery-Related Acute Kidney Insufficiency Acute Kidney Insufficiency (ARF) 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.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited by these.

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. For example, G (L) represents LNA type guanosine. In addition, the acute accent flex "^" between bases (this symbol is also called a caret or hat) 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 (SEQ ID NO: 1)
5'-5 (L) G (L) t ^ t ^ g ^ t ^ t ^ t ^ t ^ c ^ a ^ g ^ G (L) A (L) -3'
p53-ASO-02 (SEQ ID NO: 2)
5'-T (L) G (L) a ^ a ^ c ^ c ^ a ^ t ^ t ^ g ^ t ^ t ^ 5 (L) A (L) -3'
p53-ASO-03 (SEQ ID NO: 3)
5'-G (L) T (L) g ^ a ^ a ^ c ^ c ^ a ^ t ^ t ^ g ^ t ^ T (L) 5 (L) -3'
p53-ASO-04 (SEQ ID NO: 4)
5'-5 (L) A (L) c ^ g ^ c ^ c ^ c ^ a ^ c ^ g ^ g ^ a ^ T (L) 5 (L) -3'
p53-ASO-05 (SEQ ID NO: 5)
5'-5 (L) T (L) c ^ a ^ c ^ g ^ c ^ c ^ c ^ a ^ c ^ g ^ G (L) A (L) -3'
p53-ASO-06 (SEQ ID NO: 6)
5'-5 (L) A (L) t ^ c ^ t ^ c ^ g ^ a ^ a ^ g ^ c ^ g ^ 5 (L) T (L) -3'
p53-ASO-07 (SEQ ID NO: 7)
5'-T (L) G (L) a ^ g ^ t ^ t ^ c ^ c ^ a ^ a ^ g ^ g ^ 5 (L) 5 (L) -3'
p53-ASO-08 (SEQ ID NO: 8)
5'-5 (L) 5 (L) t ^ t ^ g ^ a ^ g ^ t ^ t ^ c ^ c ^ a ^ A (L) G (L) -3'
p53-ASO-09 (SEQ ID NO: 9)
5'-G (L) G (L) a ^ g ^ c ^ c ^ c ^ a ^ g ^ c ^ a ^ g ^ 5 (L) T (L) -3'
p53-ASO-10 (SEQ ID NO: 10)
5'-5 (L) G (L) g ^ a ^ g ^ c ^ c ^ c ^ a ^ g ^ c ^ a ^ G (L) 5 (L) -3'
p53-ASO-11 (SEQ ID NO: 11)
5'-5 (L) T (L) c ^ c ^ c ^ a ^ g ^ c ^ c ^ c ^ g ^ a ^ A (L) 5 (L) -3'
p53-ASO-12 (SEQ ID NO: 12)
5'-5 (L) 5 (L) c ^ a ^ g ^ c ^ c ^ c ^ g ^ a ^ a ^ c ^ G (L) 5 (L) -3'
p53-ASO-13 (SEQ ID NO: 13)
5'-G (L) T (L) c ^ a ^ c ^ c ^ g ^ t ^ c ^ g ^ t ^ g ^ G (L) A (L) -3'
p53-ASO-14 (SEQ ID NO: 14)
5'-T (L) 5 (L) c ^ a ^ g ^ g ^ g ^ a ^ a ^ g ^ c ^ g ^ T (L) G (L) -3'
p53-ASO-15 (SEQ ID NO: 15)
5'-G (L) G (L) c ^ a ^ g ^ t ^ g ^ a ^ c ^ c ^ c ^ g ^ G (L) A (L) -3'
p53-ASO-16 (SEQ ID NO: 16)
5'-5 (L) 5 (L) c ^ a ^ c ^ a ^ g ^ c ^ t ^ g ^ c ^ a ^ 5 (L) A (L) -3'
p53-ASO-17 (SEQ ID NO: 17)
5'-G (L) T (L) c ^ a ^ t ^ g ^ t ^ g ^ c ^ t ^ g ^ t ^ G (L) A (L) -3'
p53-ASO-18 (SEQ ID NO: 18)
5'-A (L) G (L) t ^ g ^ t ^ t ^ t ^ c ^ t ^ g ^ t ^ c ^ A (L) T (L) -3'
p53-ASO-19 (SEQ ID NO: 19)
5'-G (L) G (L) c ^ a ^ c ^ c ^ a ^ c ^ c ^ a ^ c ^ a ^ 5 (L) T (L) -3'
p53-ASO-20 (SEQ ID NO: 20)
5'-5 (L) T (L) c ^ c ^ a ^ t ^ c ^ c ^ a ^ g ^ t ^ g ^ G (L) T (L) -3'
p53-ASO-21 (SEQ ID NO: 21)
5'-5 (L) T (L) t ^ c ^ a ^ g ^ g ^ t ^ g ^ g ^ c ^ t ^ G (L) G (L) -3'
p53-ASO-22 (SEQ ID NO: 22)
5'-T (L) T (L) t ^ a ^ t ^ g ^ g ^ c ^ g ^ g ^ g ^ a ^ G (L) G (L) -3'
p53-ASO-23 (SEQ ID NO: 23)
5'-G (L) A (L) g ^ t ^ t ^ t ^ t ^ t ^ t ^ a ^ t ^ g ^ G (L) 5 (L) -3'

Example 2: Evaluation of antisense against p53 D-MEM containing 10% FBS (Cat no.F7524, SIGMA, deactivated) and 1% antibody at 37 ° C. in a 5% CO ₂ atmosphere (Wako Pure Chemical Industries, Ltd.) Cat no.041-29775) HeLa cells were seeded in a petri dish with a diameter of 10 cm in medium, cultured near the confluence, and then released from the plate by trypsin treatment. In a 24-well plate, 2 × 10 ⁴ cell numbers / 500 μL / 1 well, total 2 wells (N = 2), were cultured overnight. On the next day, 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 μL per well to each of the above serum-free mediums, mix well by pipetting to a final 50 μL, 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. First, 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 μL of total RNA was prepared, heated at 65 ° C. for 5 minutes, and then 2 μL of 5xRT Master Mix included in the kit was added to each sample at 37 ° C. for 15 minutes. The reaction was carried out in 4 steps of 50 ° C., 5 minutes, 98 ° C., 5 minutes, and then 4 ° C. Next, 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 μL containing 400 nM of forward and reverse primers, 12.5 μL of TB Green Premix EX Tag II, and 2 μL 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.

SEQ ID NO: 126
p53 5'primer: 5'-CCTCTCCCCAGCCAAAGAAG-3'
SEQ ID NO: 127
p53 3'Primer: 5'-GCCTCATTCAGCTCTCGGAA-3'
SEQ ID NO: 128
GAPDH 5'primer: 5'-CCCACTCCTCCCACCTTTGAC-3'
SEQ ID NO: 129
GAPDH 3'Primer: 5'-ACCCTGTTGCTGTAGCCAAA-3'
SEQ ID NO: 130
UBC 5'Primer: 5'-ATTTGGGTCGCGGTTCTTG-3'
SEQ ID NO: 131
UBC 3'Primer: 5'-TGCCTTGACATTCTCGATGGT-3'

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"). The "/ GAPDH" value of p53 of each sample was divided by the "/ GAPDH" value of mock to obtain the value of "/ GAPDH (mock = 1)". Further, the value "/ UBC" obtained by dividing the "Rel.Qty" value of p53 of each sample and the mock by the "Rel.Qty" value of the corresponding UBC was obtained. The value of "/ UBC (mock = 1)" was obtained by dividing the calculated value of "/ UBC" of each sample by the value of "/ UBC" of mock. The average value of the calculated "/ GAPDH (mock = 1)" value and the "/ UBC (mock = 1)" value was obtained, and a bar graph was created (FIG. 2).

As a result, it was confirmed in about half of the samples that the expression level of p53 mRNA could be reduced by adding ASO-01 to 23 when a transfection reagent was used in HeLa cells (Fig. 2). Inhibitory activity of 50% or less was observed 1 day after culturing for each ASO of ASO-03-05,09,10,12,15,17,18,21,23.

As for ASO-15 and 23, as a result of homology search, it was found that there is no human gene sequence that hybridizes 100% other than the target p53 mRNA, and off-target effects may occur in these ASOs. Low sex.

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. As a result of transfecting the 15 mer series having a chain length extended to 15 mer with 20 nM, none showed a clearly stronger inhibitory effect than ASO-15, which is 14 mer. 38, 37, 15-40, 43 are also sequences close to ASO-15. Therefore, it may have a strong inhibitory effect. This time, 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. As a result of transfecting the 17mer series with the chain length extended to 17mer at 20 nM, none showed a clearly stronger inhibitory effect than ASO-15, which is 14 mer. 15-41 was about the same as ASO-15 for both Days 1 and 2. In addition, 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).

(SEQ ID NO: 95-111)

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.

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%.

The result after 2 days is shown in FIG. On Day 2, 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. At 48 hours after TF, 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 Δp53 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.

Although preferred embodiments of the present invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided solely for purposes of illustration, and those skilled in the art will appreciate it. It will be possible to make various modifications, modifications and substitutions without departing from the present invention. It should be understood that various alternative embodiments of the invention described herein can be used in practicing the invention. In addition, the content described in all publications, including the patents and patent application documents referred to in this specification, is incorporated by its citation in the same manner as the content specified in this specification. Should be interpreted.

We have identified antisense oligonucleotides (ASOs) 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.

Claims (32)

1. A pharmaceutical composition for inhibiting the expression of the p53 gene in cells, which comprises, as an active ingredient, an oligonucleotide containing a complementary region substantially complementary to at least a part of mRNA encoding the p53 gene. Composition.
2. The pharmaceutical composition according to claim 1, wherein the p53 gene is a human p53 gene.
3. The pharmaceutical composition according to claim 1 or 2, wherein the p53 gene has the sequence of SEQ ID NO: 132.
4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the oligonucleotide is essentially a single-strand molecule.
5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the oligonucleotide is an antisense oligonucleotide (ASO).
6. The pharmaceutical composition according to claim 5, wherein the antisense oligonucleotide (ASO) is a gapmer.
7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the oligonucleotide has a length of 12 to 18 bases.
8. The pharmaceutical composition according to claim 7, wherein the oligonucleotide has a length of 14 bases.
9. The pharmaceutical composition according to claim 8, wherein the oligonucleotide is a 2-10-2 gapmer.
10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the base in the oligonucleotide is 80% or more complementary to the p53 gene.
11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the base in the oligonucleotide is 100% complementary to the p53 gene.
12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the oligonucleotide contains a modified nucleoside and / or a bond between modified nucleosides.
13. The pharmaceutical composition according to claim 12, wherein the modified nucleoside is a bridged nucleic acid.
14. The pharmaceutical composition according to claim 13, wherein the cross-linked nucleic acid is LNA.
15. The pharmaceutical composition according to any one of claims 12 to 14, wherein the modified nucleoside bond is a phosphorothioate bond.
16. The pharmaceutical composition according to any one of claims 12 to 15, wherein all the modified nucleoside bonds are phosphorothioate bonds.
17. The pharmaceutical composition according to any one of claims 1 to 16, wherein all nucleoside bonds are phosphorothioate bonds.
18. The pharmaceutical composition according to any one of claims 12 to 17, wherein the binding between modified nucleosides is chirally controlled.
19. The pharmaceutical composition according to any one of claims 1 to 18, wherein one or both of the terminal hydroxyl groups of the oligonucleotide is modified.
20. The pharmaceutical composition according to any one of claims 1 to 19, wherein a phosphate group is added to one or both of the terminal hydroxyl groups of the oligonucleotide.
21. The pharmaceutical composition according to any one of claims 1 to 18, wherein one or both of the terminal hydroxyl groups of the oligonucleotide is not modified.
22. The pharmaceutical composition according to any one of claims 1 to 18, wherein a phosphate group is not added to one or both of the terminal hydroxyl groups of the oligonucleotide.
23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the oligonucleotide is an oligonucleotide containing any one sequence of SEQ ID NO: 1 to SEQ ID NO: 125.
24. The pharmaceutical composition according to any one of claims 1 to 22, wherein the oligonucleotide is an oligonucleotide consisting of any one sequence of SEQ ID NO: 1 to SEQ ID NO: 125.
25. The oligonucleotide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1 to 125. The pharmaceutical composition according to any one of claims 1 to 22, which is an oligonucleotide consisting of a sequence having sex.
26. The pharmaceutical composition according to any one of claims 1 to 25, which is a pharmaceutical composition for use in treating or preventing a disease or symptom in a subject.
27. The pharmaceutical composition according to claim 26, wherein the disease or symptom is related to the expression of the p53 gene.
28. The diseases or symptoms are 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 renal transplantation, spinal cord injury, brain injury, stroke, stroke, neurodegenerative disease, Parkinson's disease, Alzheimer's disease, tumor, burn, wound, high fever 26 or 27, which is selected from the group consisting of disease, hypoxia, ischemia, organ transplantation, bone marrow transplantation (BMT), myocardial infarction / heart attack, cardiotoxicity, p53-positive cancer, and acute liver failure. Pharmaceutical composition.
29. The pharmaceutical composition according to any one of claims 1 to 28, which contains a pharmaceutically acceptable excipient, buffer, and / or additive.
30. A pharmaceutical composition for use in the treatment or prevention of a disease or condition in a subject, which is essentially single-stranded, comprising a complementary region that is substantially complementary to at least a portion of the mRNA encoding the p53 gene. It contains an oligonucleotide as an active ingredient, the oligonucleotide is a gapmer having a length of 14 bases, and the wing regions on the 5'side and 3'side of the gapmer each consist of 2 bases of LNA, and all of the gapmers. A pharmaceutical composition in which the internucleoside bond of the nucleotide is a phosphorothioate bond.
31. 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-15-16. A pharmaceutical composition containing an oligonucleotide selected from the group consisting of the above as an active ingredient.
32. An oligonucleotide selected from the group consisting of p53-ASO-15, p53-ASO-15-11, p53-ASO-15-12, and p53-ASO-15-16, or a salt thereof.
    
    

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