CALM2 antisense oligonucleotide

Abstract

This invention provides a novel compound or a pharmacologically acceptable salt thereof that inhibits the expression of the CALM2 gene, which is known to be involved in congenital LQTS. [Solution] The present invention provides a compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides, wherein the modified oligonucleotide has a nucleic acid base sequence comprising at least 8 consecutive nucleic acid bases included in a specific sequence. The compound or a pharmacologically acceptable salt thereof makes it possible to treat diseases or conditions (particularly congenital long QT syndrome) in which inhibition of CALM2 gene expression by controlling CALM2 gene expression is effective.

Description

[Technical Field]

[0001] This application relates to an antisense oligonucleotide of CALM2. [Background technology]

[0002] Congenital long QT syndrome (congenital LQTS) is a fatal congenital arrhythmia disorder that prolongs the QT interval on an electrocardiogram, leading to syncope and sudden death due to ventricular arrhythmias (Non-Patent Literature 1). It has been reported that the cause is often a gene mutation in an ion channel or its regulatory protein expressed in the heart. While KCNQ1 and KCNH2, which are involved in potassium channels, and SCN5A, which is involved in sodium channels, were previously known as causative genes, calmodulin genes (CALM1-3) have recently been identified as the culprits.

[0003] Calmodulin is a ubiquitously expressed calcium-sensing protein, with three different genes (CALM1-3) encoding the same calmodulin protein with the same amino acid sequence. Calmodulin regulates various proteins, including multiple ion channels, and in particular controls the inactivation of L-type calcium channels (LTCCs) expressed in the heart (Non-Patent Literature 3).

[0004] Many patients with congenital LQTS caused by the calmodulin gene experience severe complications, including sudden death in childhood, and often do not survive to adulthood. Hetero-missense mutations in the CALM gene have been reported to be associated with the development of severe arrhythmias (Non-Patent Literature 2). It is thought that a missense mutation in one of the three different CALM genes exerts a dominant-negative effect, leading to severe symptoms (Non-Patent Literature 4).

[0005] Genome editing techniques targeting CALM2 are also being studied. Mutation allele-nonspecific knockdown using the CRISPRi system (Non-Patent Document 6) and mutation allele-specific knockout using the CRISPR / Cas9 system have been reported (Non-Patent Document 4). Mutation allele-specific knockout by the CRISPR / Cas9 system has been shown to be an effective gene therapy for patients with congenital LQTS caused by the dominant negative action of the CALM gene (Non-Patent Document 4).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document ]]2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0007] As a treatment method for congenital LQTS, for example, treatment using an implantable defibrillator for sudden death prevention is considered, but there are problems in that it is a countermeasure therapy, many of the patients are children and the physical and mental burden is large, and invasiveness and complications after implantation occur (Non-Patent Document 5). Moreover, neither the method using the CRISPRi system nor the CRISPR / Cas9 system has any clinical application examples, and there are said to be many issues in clinical applications. Therefore, there is a demand for the establishment of drug therapy for congenital LQTS that can overcome the problems of conventional surgical therapy or gene therapy. In particular, the need for new therapeutic drugs that can prevent lethal arrhythmia events and improve the prognosis is very high, and it is considered to have great clinical significance (Non-Patent Document 5). Currently, β-blockers are often administered to cases of LQT15 caused by CALM2 mutations, but it has been reported that the therapeutic effect is low (Non-Patent Document 5). Therefore, there is still a demand for the provision of drugs useful for the treatment, prevention, improvement, etc. of congenital LQTS. Thus, the present application aims to provide a novel compound that inhibits the expression of the CALM2 gene known to be involved in congenital LQTS or a pharmaceutically acceptable salt thereof.

Means for Solving the Problems

[0008] In view of the above problems, the present inventors intensively studied and found that a compound containing a modified oligonucleotide having a specific sequence or a pharmaceutically acceptable salt thereof has an excellent inhibitory effect on the expression of the CALM2 gene. That is, the present application provides the following gist.

[0009] [[ID=]21]1. A compound containing a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides or a pharmaceutically acceptable salt thereof, wherein the modified oligonucleotide has a nucleobase sequence containing at least 8 consecutive nucleobases included in any one of the nucleobase sequences of SEQ ID NOs: 3 to 73 (in one embodiment, 3 to 20), a compound or a pharmaceutically acceptable salt thereof. 2. The compound or the pharmaceutically acceptable salt thereof according to 1., wherein the modified oligonucleotide has a nucleobase sequence containing any one of the nucleobase sequences of SEQ ID NOs: 3 to 73 (in one embodiment, 3 to 20). 3. The modified oligonucleotide has a nucleic acid base sequence consisting of one of the nucleic acid base sequences of SEQ ID NOs: 3 to 73 (3 to 20 in one embodiment), the compound according to 1. or 2., or a pharmacoposly acceptable salt thereof. 4. Nucleic acid base position numbers of Sequence ID No. 1: 102-117, 160-175, 183-205, 212-227, 322-337, 365-405, 411-434, 464-494, 506-521, 606-635, 636-651, 692-707, 715-749, 754-801, 829-857, 862-934, 951-970, 995-1010, 1006-1021, 1036-1051, 1062-1077, 1081-11 A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 04, 1138-1166, 1188-1203, and 1239-1254, wherein the modified oligonucleotide has at least 80% complementarity to at least a portion of the acid base sequence of SEQ ID NO: 1. 5. A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by the positions 102-117, 160-175, 183-198, 212-227, 322-337, 365-380, 387-402, 479-494, 620-635, 715-730, 862-877, 896-934, 995-1010, 1062-1077, 1089-1104, 1140-1166 and 1239-1254 of SEQ ID NO: 1, wherein the modified oligonucleotide has at least 80% complementarity to at least a portion of the acid base sequence of SEQ ID NO: 1. 6. A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of the nucleic acid base sequence indicated by position numbers 896 to 934, preferably 907 to 922, of the nucleic acid base sequence of Sequence ID No. 1, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of Sequence ID No. 1. 7. Nucleic acid base position numbers of Sequence ID No. 2: 102-117, 160-175, 1624-1639, 1661-1676, 3355-3370, 5859-5881, 13959-13974, 14069-14084, 14228-14278, 14284-14307, 14764-14794, 14806-14821, 15824-15869, 15910-15925, 15933-15967, 15972-16019, 16047-16075, 16080-16152, 16169-16188, 16213-16239 A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 16254-16269, 16280-16295, 16299-16322, 16356-16384, 16406-16421 and 16457-16472, wherein the modified oligonucleotide has at least 80% complementarity to at least a portion of the acid base sequence of SEQ ID NO: 2. 8. Nucleic acid base position numbers of Sequence ID No. 2: 102-117, 160-175, 5859-5874, 13959-13974, 14069-14084, 14238-14253, 14260-14275, 14779-14794, 15838-15853, 15933-15948, 16080-16095, 16114-16152, 16213-16228, 16280-16295, 16307-16322, 16 A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 358 to 16384 and 16457 to 16472, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of SEQ ID NO: 2. 9. A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of the nucleic acid base sequence indicated by the positional numbers of the nucleic acid bases 16114 to 16152, preferably 16125 to 16140 of SEQ ID NO: 2, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of SEQ ID NO: 2. 10. The modified oligonucleotide is a compound according to any one of 1. to 9., or a pharmacoposly acceptable salt thereof, comprising a phosphorothioate bond. 11. The compound described in any one of 1. to 10. or a pharmacoposically acceptable salt thereof, wherein the modified oligonucleotide comprises at least one selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides. 12. The aforementioned 2'-4'-bridged nucleoside is LNA, ENA, cEt, BNA NC A compound described in 11, or a pharmacoposly acceptable salt thereof, which is at least one selected from the group consisting of AmNA, scpBNA, and GuNA. 13. The compound described in 12. or a pharmacoposically acceptable salt thereof, wherein the 2'-4'-bridged nucleoside is LNA. 14. The compound described in any one of 11. to 13. or a pharmacoposically acceptable salt thereof, wherein the 2'-modified nucleoside is at least one selected from the group consisting of 2'-O-MCE nucleoside, 2'-O-MOE nucleoside, 2'-O-NMA nucleoside, and 2'-O-Me nucleoside. 15. The compound described in 14. or a pharmacoposically acceptable salt thereof, wherein the 2'-modified nucleoside is at least one selected from the group consisting of 2'-O-MCE nucleoside and 2'-O-MOE nucleoside. 16. The modified oligonucleotide is a compound described in any one of 1 to 15, or a pharmacoposly acceptable salt thereof, comprising 5-methylcytosine. 17. The modified oligonucleotide comprises a gap segment, a 5' wing segment, and a 3' wing segment. The gap segment contains at least two deoxyribonucleosides, and the 5' and 3' ends of the gap segment are deoxyribonucleosides. The nucleoside at the 3' end of the 5' wing segment is a sugar-modified nucleoside and is linked to the 5' end of the gap segment. The nucleoside at the 5' end of the 3' wing segment is a sugar-modified nucleoside and is linked to the 3' end of the gap segment. A compound listed in any one of items 1-16, or a pharmacokinetically acceptable salt thereof. 18. The gap segment consists of 5 to 30 deoxyribonucleosides. The 5' wing segment and the 3' wing segment each independently consist of 1 to 10 sugar-modified nucleosides independently selected from the group consisting of LNA, 2'-O-MCE nucleosides, and 2'-O-MOE nucleosides, wherein each wing segment contains at least one phosphorothioate bond. The compound described in 17. or a pharmacoposically acceptable salt thereof, wherein the cytosine in the gap segment and each wing segment is 5-methylated. 19. The gap segment consists of 8 to 12 deoxyribonucleosides. The 5'-wing segment and the 3'-wing segment each consist of 2 to 5 sugar-modified nucleosides independently selected from the group consisting of LNA and 2'-O-MCE nucleosides. The gap segment contains at least one phosphorothioate bond. The compounds described in 18. or their pharmacokinetically acceptable salts. 20. The compound according to 19, wherein the 5' wing segment and the 3' wing segment are each independently selected from the group consisting of LL, LLL, VLL, LVL, LLV, LVV, VLV, VVL, VVLL, VLLL, LVLL, LLVL, LLLV, LVVV, VLVV, VVLV, and VVVL, where L represents LNA and V represents a 2'-O-MCE nucleoside. twenty one. The modified oligonucleotide has a continuous nucleoside length of 11 to 50, preferably 15 to 25, and is a compound according to any one of 1 to 20, or a pharmacoposly acceptable salt thereof. twenty two. The modified oligonucleotide is an antisense oligonucleotide, and the compound is one of the compounds described in 1. to 21. or a pharmacoposly acceptable salt thereof. twenty three. The compound containing the modified oligonucleotide is a compound described in any one of 1. to 22., or a pharmacoposically acceptable salt thereof, which includes the prodrug portion. twenty four. The compound containing the modified oligonucleotide is a compound described in any one of 1. to 23., or a pharmacoposly acceptable salt thereof, which contains a functional molecule. twenty five. The compound described in 24, or a pharmacoposly acceptable salt thereof, wherein the functional molecule is a group derived from a molecule having the function of delivering modified oligonucleotides to a target site. 26. The functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and their derivatives, and is a compound according to 24. or 25. or a pharmacoposly acceptable salt thereof. 27. The compound described in any one of 24. to 26. or a pharmacologically acceptable salt thereof, wherein the functional molecule is a lipid selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, and C5-30 unsaturated fatty acids. 28. The compound described in any one of 1 to 23, or a pharmacoposly acceptable salt thereof, wherein the compound comprises the modified oligonucleotide. 29. The salt described in any one of 1. to 28., wherein the pharmacologically acceptable salt is a sodium salt. 30. A pharmaceutical product containing any one of the compounds listed in items 1-29 or a pharmacologically acceptable salt thereof. 31. A pharmaceutical product for treating, preventing and / or improving a disease or condition in which the expression of the CALM2 gene is effectively inhibited, comprising any one of the compounds described in 1. to 29. or a pharmacologically acceptable salt thereof. 32. A CALM2 gene expression inhibitor comprising one of the compounds or pharmacologically acceptable salts described in any one of items 1 to 29. 33. A pharmaceutical product comprising any one of the compounds or pharmacologically acceptable salts described in 1. to 29. for the treatment, prevention, and / or improvement of congenital long QT syndrome. 34. A pharmaceutical product for treating, preventing, and / or improving calmodulinopathy, comprising any one of the compounds or pharmacologically acceptable salts described in 1. to 29. 35. A method for treating, preventing, and / or improving a disease or condition in which the expression inhibition of the CALM2 gene is effective, comprising the step of administering an effective amount of any one of the compounds or pharmacologically acceptable salts described in 1. to 29. to a subject in need thereof. 36. A method for inhibiting the expression of the CALM2 gene, comprising the step of administering an effective amount of any one of the compounds or pharmacologically acceptable salts described in 1. to 29. to a subject in need thereof. 37. A method for treating, preventing, and / or improving congenital long QT syndrome, comprising the step of administering an effective amount of any one of the compounds or pharmacologically acceptable salts described in 1. to 29. to a subject in need thereof. 38. A method for treating, preventing, and / or improving calmodulinopathy, comprising the step of administering an effective amount of any one of the compounds or pharmacologically acceptable salts described in 1. to 29. to a subject in need thereof. 39. A compound listed in any one of items 1 to 29, for use as a pharmaceutical. 40. A compound described in any one of 1 to 29, for use in the treatment, prevention, and / or improvement of a disease or condition in which inhibiting CALM2 gene expression is effective. 41. A compound described in any one of items 1 to 29, for use in inhibiting the expression of the CALM2 gene. 42. A compound described in any one of items 1 to 29, for use in the treatment, prevention, and / or improvement of congenital long QT syndrome. 43. A compound described in any one of 1 to 29, for use in the treatment, prevention, and / or improvement of calmodulinopathy. 44. The use of any one of the compounds or pharmacologically acceptable salts described in 1. to 29. in the manufacture of a pharmaceutical product for treating, preventing and / or improving a disease or condition in which the expression inhibition of the CALM2 gene is effective. 45. The use of any one of the compounds described in 1. to 29. or a pharmacologically acceptable salt in the manufacture of a CALM2 gene expression inhibitor. 46. Use of any one of the compounds or pharmacologically acceptable salts described in 1. to 29. in the manufacture of a medicament for the treatment, prevention, and / or improvement of congenital long QT syndrome. 47. Use of any one of the compounds or pharmacoposly acceptable salts described in 1. to 29. in the manufacture of a medicament for the treatment, prevention, and / or improvement of calmodulinopathy. [Effects of the Invention]

[0010] This invention provides a compound containing a novel modified oligonucleotide or a pharmacologically acceptable salt thereof. Because this compound or its pharmacologically acceptable salt exhibits excellent CALM2 gene expression inhibitory activity, it may be particularly useful in the treatment, improvement, and / or prevention of congenital long QT syndrome and calmodulinopathy. [Brief explanation of the drawing]

[0011] [Figure 1] This graph shows the effect of ASO according to this embodiment on the expression levels of CALM1, CALM2, and CALM3 in human liver cancer-derived cells. [Figure 2] These are the results of measuring action potentials in human iPS cell-differentiated cardiomyocytes derived from LQT15 patients, both in the presence and absence of ASO according to this embodiment. [Figure 3] This graph shows the effect of ASO according to this embodiment on the action potential duration in human iPS cell-differentiated cardiomyocytes derived from LQT15 patients. [Figure 4] This graph shows the effect of ASO according to this embodiment on the expression level of CALM2 in human iPS cell-differentiated cardiomyocytes derived from LQT15 patients. [Figure 5] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the heart of C57BL / 6J mice. [Figure 6] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the liver of C57BL / 6J mice. [Figure 7] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the kidneys of C57BL / 6J mice. [Figure 8] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the heart of C57BL / 6J mice. [Figure 9] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the heart of C57BL / 6J mice. [Figure 10] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the heart of C57BL / 6J mice. [Figure 11] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the liver of C57BL / 6J mice. [Figure 12] This graph shows the effect of administering ASO according to this embodiment on the expression level of CALM2 in the kidneys of C57BL / 6J mice. [Modes for carrying out the invention]

[0012] It is understood that both the above summary and the detailed description below are illustrative and descriptive only and do not limit the claimed invention in any way. In this specification, matters indicated in the singular include examples in the plural unless the context clearly indicates otherwise or is otherwise specifically stated. In this specification, antisense oligonucleotides may be referred to as "ASO".

[0013] Nucleic acids in nature are most fundamentally composed of adenosine (A), thymidine (T) (or uridine (U)), cytidine (C), and guanosine (G). These basic nucleic acids are often referred to as AT(U)GC, etc. Therefore, in this specification, when sequences are shown as "nucleic acid base sequences" or "sequence numbers" in relation to, for example, the sequence of the CALM2 gene, they are basically sequences composed of A, G, C, T, and U. On the other hand, the nucleic acids constituting ASO in this application include not only basic nucleic acids (AT(U)CG) but also those that have undergone structural modifications. Details of the modifications will be described later, but they include modifications to the sugar moiety, internucleoside bonds, and / or nucleic acid bases. Therefore, in this specification, if, for example, ASO in this application is described as a "compound" or a "compound to which a compound number (P number) has been assigned" and its nucleic acid base sequence is described by A, G, C, T, and U, then A, G, C, T, and U also include those which have undergone structural modifications.

[0014] Further details are provided below. Unless otherwise specified, the following terms have the following meanings:

[0015] "May be substituted" means either no substitution is made, or substitution is made.

[0016] "Nucleoside" is a term well known to those skilled in the art and is generally understood to be a molecule in which a sugar and a nucleic acid base are bonded, and which can be a constituent unit of a nucleic acid. In this specification, nucleoside is a broader concept and includes deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, which are described below. The nucleic acid base may be modified.

[0017] "Deoxyribonucleoside" refers to a molecule having a nucleic acid base at the 1' position of the carbon atom of 2'-deoxyribose. In this application, a deoxyribonucleoside may be a naturally occurring deoxyribonucleoside or a deoxyribonucleoside in which the nucleic acid base portion of a naturally occurring deoxyribonucleoside has been modified. Multiple types of modifications may be applied to a single deoxyribonucleoside. The modified deoxyribonucleosides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, and International Publication No. 2018 / 155450, etc.

[0018] "Ribonucleoside" refers to a molecule having a nucleic acid base at the 1' position of the carbon atom of ribose. In this application, a ribonucleoside may be a naturally occurring ribonucleoside or a ribonucleoside in which the nucleic acid base portion of a naturally occurring ribonucleoside has been modified. Multiple types of modifications may be applied to a single ribonucleoside. The modified ribonucleosides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, and International Publication No. 2018 / 155450, etc.

[0019] "Modified sugars" are (Z1) Molecules in which ribose or 2'-deoxyribose is partially substituted by one or more substituents. (Z2) Penta- or hexa-monosaccharides other than ribose and 2'-deoxyribose (e.g., hexitol, threose, etc.) (Z3) Molecules in which the entire ribose or 2'-deoxyribose, or the tetrahydrofuran ring thereof, is replaced with a 5- to 7-membered saturated or unsaturated ring (e.g., cyclohexane, cyclohexene, morpholine, etc.), or a substructure (e.g., a peptide structure) that can form a 5- to 7-membered ring by hydrogen bonding. or (Z4) Molecules in which ribose or 2'-deoxyribose is replaced with alkylene glycols having 2 to 6 carbon atoms (e.g., ethylene glycol, propylene glycol, etc.) It means... Modified sugars include "2'-modified sugars" and "2'-4'-bridged sugars," which are described below. Examples of modified sugars and sugar-modified nucleosides described later include sugars and sugar-modified nucleosides disclosed as suitably used in antisense methods in Japanese Patent Publication No. 10-304889, International Publication No. 2005 / 021570, Japanese Patent Publication No. 10-195098, Japanese Patent Publication No. 2002-521310, International Publication No. 2007 / 143315, International Publication No. 2008 / 043753, International Publication No. 2008 / 029619, International Publication No. 2008 / 049085, and International Publication No. 2017 / 142054 (hereinafter, these documents are referred to as "documents relating to antisense methods"), etc. Modified sugars and sugar-modified nucleosides are also disclosed in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, and International Publication No. 2018 / 155450, among others.

[0020] Examples of modified sugars partially substituted by a single substituent include ribose or 2'-deoxyribose in which any position of the sugar moiety is substituted with (i) or (ii) below. (I C 1-6 Alkyl group. (ii) Halogen atom, C 1-6 Alkoxy group, Halo C1-6 Alkoxy groups, mono- or di-C 1-6 C substituted with at least one selected from the group consisting of alkylamino groups, 5-10 membered heterocyclic groups, carboxyl groups, carbamoyl groups, or N-substituted carbamoyl groups. 1-6 Alkyl group. Here, examples of the N-substituted carbamoyl group include the N-methyl-carbamoyl group and the N-ethyl-carbamoyl group, where the methyl and ethyl groups of the N-methyl-carbamoyl group and the N-ethyl-carbamoyl group are 5-10 membered heterocyclic groups or mono- or di-C 1-6 It may be substituted with an alkylamino group. Specific examples of N-substituted carbamoyl groups include N-methylcarbamoyl group, N-ethylcarbamoyl group, N-dimethylaminoethyl-carbamoyl group, N-morpholinoethylcarbamoyl group, N-(2-pyridylethyl)carbamoyl group, and N-((benzimidazole-1-yl)ethyl)carbamoyl group.

[0021] "Sugar-modified nucleoside" refers to a molecule having the aforementioned "modified sugar" instead of the sugar portion of a deoxyribonucleoside or ribonucleoside. For example, it includes the "2'-modified nucleoside" and "2'-4'-bridged nucleoside" described later. If the modified sugar is (Z3) as defined above, sugar-modified nucleoside also includes molecules in which the modified sugar and a nucleic acid base are linked via a methylene chain or the like.

[0022] "2'-modified sugars" refer to non-crosslinked sugars in which the oxygen or carbon atom at the 2' position of ribose is modified, and include "2'-O-Me", "2'-O-MOE", "2'-O-MCE", "2'-O-NMA", "2'-DMAECE", "2'-MоrECE", "2'-PyECE", and "BimECE". "2'-modified nucleoside" refers to a molecule having a nucleic acid base at the 1' position of the 2'-modified sugar, and examples include "2'-O-Me nucleoside", "2'-O-MOE nucleoside", "2'-O-MCE nucleoside", "2'-O-NMA nucleoside", "2'-DMAECE nucleoside", "2'-MоrECE nucleoside", "2'-PyECE nucleoside", and "BimECE nucleoside".

[0023] "2'-O-Me" (also called 2'-O-methyl) refers to a sugar in which the hydroxyl group at the 2' position of ribose is replaced with a methoxy group. A "2'-O-Me nucleoside" (also called a 2'-O-methyl nucleoside) refers to a molecule that has a nucleic acid base at the 1' position of "2'-O-Me".

[0024] "2'-O-MOE" (also called 2'-O-methoxyethyl) refers to a sugar in which the hydroxyl group at the 2' position of ribose is replaced with a 2-methoxyethyloxy group. A "2'-O-MOE nucleoside" (also called a 2'-O-methoxyethyl nucleoside) refers to a molecule that has a nucleic acid base at the 1' position of "2'-O-MOE".

[0025] "2'-O-MCE" (also known as 2'-O-methylcarbamoylethyl) refers to a sugar in which the hydroxyl group at the 2' position of ribose is replaced with a methylcarbamoylethyloxy group. A "2'-O-MCE nucleoside" (also known as a 2'-O-methylcarbamoylethyl nucleoside) refers to a molecule that has a nucleic acid base at the 1' position of "2'-O-MCE".

[0026] "2'-O-NMA" refers to a sugar in which the hydroxyl group at the 2' position of ribose is replaced by a 2-[(methylamino)-2-oxoethyl]oxy group. "2'-O-NMA nucleoside" refers to a molecule that has a nucleic acid base at the 1' position of "2'-O-NMA".

[0027] "2'-O-AP" refers to a sugar in which the hydroxyl group at the 2' position of ribose is replaced by a 3-aminopropyloxy group. "2'-O-AP nucleoside" refers to a molecule that has a nucleic acid base at the 1' position of "2'-O-AP".

[0028] "2'-F" refers to a sugar in which the hydroxyl group at the 2' position of ribose is replaced by a fluorine atom. A "2'-F nucleoside" refers to a molecule that has a nucleic acid base at the 1' position of "2'-F".

[0029] "2'-DMAECE" is a modified sugar with the following structure: [ka]

[0030] "2'-MоrECE" is a modified sugar with the following structure: [ka]

[0031] "2'-PyECE" is a modified sugar with the following structure. [ka]

[0032] "BimECE" is a modified sugar with the following structure: [ka]

[0033] "2'-DMAECE nucleoside", "2'-MorECE nucleoside", "2'-PyECE nucleoside", and "BimECE nucleoside" refer to molecules that have a nucleic acid base at the 1' position of "2'-DMAECE", "2'-MorECE", "2'-PyECE", and "BimECE", respectively.

[0034] "2'-4' bridged sugar" means a sugar in which a bridging unit is substituted by substitution at two positions, the 2'-position and the 4'-position, in ribose. As the bridging unit, for example, C 2-6 an alkylene group (the alkylene group is unsubstituted or substituted by one or more substituents selected from the group consisting of a halogen atom, an oxo group, and a thioxo group, and one or two methylene groups of the alkylene group are either unsubstituted or independently -O-, -NR 1 -(R 1 represents a hydrogen atom, C 1-6 an alkyl group or a halo C 1-6 alkyl group) and -S- and is replaced by a group selected from the group consisting of).

[0035] "2'-4' bridged nucleoside" (2',4'-BNA) means a molecule having a nucleobase at the 1'-position of the 2'-4' bridged sugar. For example, α-L-methyleneoxy (4'-CH2-O-2') BNA or β-D-methyleneoxy (4'-CH2-O-2') BNA, also referred to as LNA (Locked Nucleic Acid (registered trademark)) described later, ethyleneoxy (4'-(CH2)2-O-2') BNA, also referred to as ENA, β-D-thio (4'-CH2-S-2') BNA, aminooxy (4'-CH2-O-N(R 11 ))-2') BNA (R 11 is H or CH3), 2',4'-BNA NC also referred to as oxyamino (4'-CH2-N(R< 12 )-O-2') BNA (R 12 is H or CH3), 2',4'-BNA COC , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH3)-O-2') BNA, also referred to as cEt, (4'-CH(CH2OCH3)-O-2') BNA, also referred to as cMOE-BNA, amide-type BNA (4'-C(=O)-N(R 13 )-2') BNA (R 13(4'-C(spiro-cyclopropyl)-O-2')BNA, also known as scpBNA, and GuNA, also known as (4'-CH2-N(R 14 )-2')BNA(R 14 C(=NH2) + )NHR 15 And R 15 Examples include H or CH3, and other BNAs known to those skilled in the art.

[0036] In the aforementioned "deoxyribonucleosides," "ribonucleosides," "2'-modified nucleosides," and "2'-4'-bridged nucleosides," the bond between the carbon atom at the 1' position and the nucleic acid base can be an α-glycosidic bond or a β-glycosidic bond, but it is usually a β-glycosidic bond. As the nucleotide nutrient (LNA), β-D-methyleneoxybNA is usually used.

[0037] "n-" stands for normal, "s-" for secondary, and "t-" for tertiary.

[0038] "Halogen atom" refers to a fluorine atom, chlorine atom, bromine atom, or iodine atom.

[0039] "C 1-6 "Alkyl group" refers to a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, and isohexyl group.

[0040] "Hello C 1-6 "Alkyl alkyl group" refers to the aforementioned "C 1-6 This refers to a group in which a hydrogen atom at any position of the alkyl group is substituted with one or more of the aforementioned halogen atoms.

[0041] "C 1-6An "alkylene group" refers to a divalent group obtained by removing one hydrogen atom from any position from a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. Examples include the methylene group, ethylene (ethanediyl) group, propane-1,3-diyl (trimethylene) group, propane-2,2-diyl group, 2,2-dimethyl-propane-1,3-diyl group, hexane-1,6-diyl (hexamethylene) group, and 3-methylbutane-1,2-diyl group. "C 2-6 The term "alkylene group" refers to the aforementioned "C 1-6 Among the "alkylene groups," this refers to a linear or branched divalent group having 2 to 6 carbon atoms, and examples include, excluding the methylene group, the aforementioned "C 1-6 It is similar to an alkylene group. "C 2-20 An "alkylene group" refers to a divalent group obtained by removing one hydrogen atom from any position from a linear or branched saturated hydrocarbon group with 2 to 20 carbon atoms. Similarly, "C 8-12 An "alkylene group" refers to a divalent group obtained by removing one hydrogen atom from any position on a straight-chain or branched saturated hydrocarbon group with 8 to 12 carbon atoms.

[0042] "C 2-20 An "alkenylene group" refers to a divalent group obtained by removing one hydrogen atom from any position from a straight-chain or branched unsaturated hydrocarbon group having 2 to 20 carbon atoms and containing at least one double bond.

[0043] "C 1-6 The term "alkoxy group" refers to the aforementioned "C 1-6 An alkyl group is a group that is bonded to an oxy group.

[0044] "Hello C 1-6 The term "alkoxy group" refers to the aforementioned "C 1-6 This refers to a group in which a hydrogen atom at any position of the "alkoxy group" is substituted with one or more of the aforementioned "halogen atoms".

[0045] "Mono- or di-C 1-6 An alkylamino group is a group in which one hydrogen atom of an amino group is replaced by one C 1-6A group replaced by an alkyl group, or two hydrogen atoms of an amino group that are the same or different C 1-6 This refers to a group that has been replaced with an alkyl group, such as methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, and N-ethyl-N-methylamino groups.

[0046] A "5-10 membered heterocyclic group" refers to a 5- to 10 membered monocyclic or fused polycyclic aromatic or aromatic heterocyclic group containing 1 to 4 heteroatoms selected from nitrogen, sulfur, and oxygen atoms in addition to carbon atoms as ring constituent atoms. Suitable examples of the aforementioned "5-10 member heterocyclic group" include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl, triazolyl, tetrazolyl, triazinyl, benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyradinyl, imidazopyridinyl, imidazopyridinyl, aziridinyl, and Examples include xylanil, azetidinil, oxetanil, thietanil, tetrahydrothienyl, tetrahydrofuranil, pyrrolinil, pyrrolidinil, oxopyrrolidinil, imidazolinil, oxoimidazolinil, imidazolidinil, oxazolinil, pyrazolinil, pyrazolinil, thiazolinil, thiazolidinil, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, piperidinil, piperazinil, tetrahydropyridinil, dihydropyridinil, tetrahydropyridazinil, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, and thiomorpholinil.

[0047] An "oxo group" refers to a group in which an oxygen atom is substituted via a double bond (=O). When an oxo group is substituted for a carbon atom, it forms a carbonyl group together with that carbon atom. A "thioxo group" refers to a group (=S) in which a sulfur atom is substituted via a double bond. When a thioxo group is substituted on a carbon atom, it forms a thiocarbonyl group together with that carbon atom.

[0048] "Nucleic acid bases" are purine bases or pyrimidine bases, and may be naturally occurring nucleic acid bases or modified naturally occurring nucleic acid bases. Examples of naturally occurring nucleic acid bases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The aforementioned "nucleic acid bases" include naturally occurring nucleic acid bases and the "modified nucleic acid bases" described below.

[0049] Examples of modifications to nucleic acid bases in "modified nucleic acid bases" include halogenation, methylation, ethylation, n-propylation, isopropylation, cyclopropylation, n-butylation, isobutylation, s-butylation, t-butylation, cyclobutylation, hydroxylation, amination, thioation, and demethylation. More specifically, examples include 5-methylation, 5-fluoration, 5-bromination, 5-iodation, and N4-methylation of cytosine; 2-thioation, 5-demethylation, 5-fluoration, 5-bromination, and 5-iodation of thymine; 2-thioation, 5-fluoration, 5-bromination, and 5-iodation of uracil; N6-methylation and 8-bromination of adenine; and N2-methylation and 8-bromination of guanine. Furthermore, examples of modifications to the nucleic acid base portion of nucleosides are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, and International Publication No. 2018 / 155450, among others.

[0050] The nucleic acid base in the nucleoside is preferably at least one selected from the group consisting of adenine, guanine, thymine, cytosine, uracil, and 5-methylcytosine.

[0051] "5-methylcytosine" refers to cytosine that has been 5-methylated, that is, cytosine that has a methyl group at the 5-position.

[0052] "Nucleic acid base sequence" refers to the sequence of nucleic acid bases from the 5' end to the 3' end of each nucleoside contained in an oligonucleotide.

[0053] "Continuous nucleic acid bases" refers to the sequence of a portion of nucleic acid bases that are continuous in the aforementioned "nucleic acid base sequence," from the 5' end to the 3' end.

[0054] "Nucleoside-inter-nucleoside bond" refers to a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. "Nucleoside-inter-nucleoside bonds" include phosphodiester bonds and "modified nucleoside-inter-nucleoside bonds" as described below.

[0055] "Modified nucleoside bonds" refer to modified phosphodiester bonds, such as phosphorothioate bonds, methylphosphonate bonds (including chiral-methylphosphonate bonds), methylthiophosphonate bonds, phosphorodithioate bonds, phosphoramidate bonds, phosphorodiamidate bonds, phosphoramidothioate bonds, and boranophosphate bonds. Examples of phosphodiester bond modifications are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, etc., and can be used for modified phosphodiester bonds.

[0056] "Modified nucleoside" refers to a nucleoside that has a modified sugar moiety and / or a modified nucleic acid base.

[0057] "Oligononucleotide" means a molecule having a structure in which two or more identical or different "nucleosides" are independently linked to each other by the aforementioned "nucleoside bonds" (for example, phosphodiester bonds or modified phosphodiester bonds). "Modified oligonucleotide" means an oligonucleotide that contains at least one selected from the group consisting of modified nucleoside bonds, modified sugars, and modified nucleic acid bases. A "compound containing a modified oligonucleotide" means a compound that contains a modified oligonucleotide in its chemical structure, and may also be the modified oligonucleotide itself. Examples of compounds containing a modified oligonucleotide include compounds in which a functional molecule, as described later, is directly or indirectly bound to the modified oligonucleotide, compounds containing a prodrug moiety, as described later, or the modified oligonucleotide itself.

[0058] "DNA" means a polynucleotide or oligonucleotide in which two or more identical or different "deoxyribonucleosides" are linked together by "nucleoside bonds". "RNA" means a polynucleotide or oligonucleotide in which two or more identical or different "ribonucleosides" are linked together by "nucleoside bonds".

[0059] "Antisense effect" refers to the control of the function of a target RNA by hybridizing a target RNA, selected in response to a target gene, with an oligonucleotide having a sequence complementary to its sub-sequence. For example, if the target RNA is mRNA, hybridization can inhibit the translation of the target RNA, alter splicing functions such as exon skipping, or degrade the target RNA due to recognition of the hybridized portion. In one embodiment, the target RNA is "CALM2 mRNA" and / or "CALM2 pre-mRNA".

[0060] An "antisense oligonucleotide" (ASO) is an oligonucleotide that produces the aforementioned antisense effect. Examples include, but are not limited to, DNA, gapmers, and mixmers; RNA or oligonucleotides designed to normally produce an antisense effect may also be used.

[0061] "Hybridization" refers to the act of forming a double helix with oligonucleotides or parts thereof that contain complementary sequences, and the phenomenon of oligonucleotides or parts thereof containing complementary sequences forming a double helix.

[0062] "Complementary" means that two nucleic acid bases can form a Watson-Crick base pair (native base pair) or a non-Watson-Crick base pair (Hoogsteen base pair, etc.) via hydrogen bonding. Two oligonucleotides or parts thereof can "hybridize" if their sequences are complementary. Two oligonucleotides or parts thereof do not need to be perfectly complementary to hybridize, but the complementaryity required for two oligonucleotides or parts thereof to hybridize is preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher (e.g., 95%, 96%, 97%, 98%, or 99% or higher). Sequence complementarity is determined by using a computer program that automatically identifies subsequences of oligonucleotides. OligoAnalyzer is one such software, for example, provided by Integrated DNA Technologies. This program is also available on their website.

[0063] "Gapmer" refers to an oligonucleotide that includes the "gap segment," "5' wing segment," and "3' wing segment," which will be described later.

[0064] The "gap segment" is a region containing "at least four consecutive nucleosides recognized by RNaseH," and is not particularly limited as long as it contains four or more consecutive nucleosides that are recognized by RNaseH, but the consecutive nucleosides are preferably selected independently from deoxyribonucleosides and sugar-modified nucleosides. Preferably, the gap segment contains at least two deoxyribonucleosides, and the nucleosides at the 5' and 3' ends of the gap segment are deoxyribonucleosides.

[0065] The "5' wing segment" is a region attached to the 5' side of the gap segment that contains "at least one nucleoside" but does not contain "at least four consecutive nucleosides recognized by RNaseH". Here, the sugar moiety of the nucleoside at the 3' end of the 5' wing segment is different from the sugar moiety of the nucleoside at the 5' end of the gap segment. The boundary between the 5' wing segment and the gap segment is identified by the difference in sugar moieties. (For example, the nucleoside at the 5' end of the gap segment is a deoxyribonucleoside, and the nucleoside at the 3' end of the 5' wing segment is a sugar-modified nucleoside.) The nucleoside at the 3' end of the 5' wing segment is generally a sugar-modified nucleoside. The 5' wing segment is not particularly limited as long as it satisfies the above definition, but the at least one nucleoside is preferably independently selected from deoxyribonucleosides and sugar-modified nucleosides, and includes at least one sugar-modified nucleoside. Preferably, the nucleoside at the 3' end of the 5' wing segment is a sugar-modified nucleoside and is linked to the 5' end of the gap segment. The "3' wing segment" is a region attached to the 3' side of the gap segment that contains "at least one nucleoside" but does not contain "at least four consecutive nucleosides recognized by RNaseH". Here, the sugar moiety of the 5'-terminal nucleoside of the 3' wing segment is different from the sugar moiety of the 3'-terminal nucleoside of the gap segment. The difference in sugar moieties confirms the boundary between the 3' wing segment and the gap segment. (For example, the 3'-terminal nucleoside of the gap segment is a deoxyribonucleoside, and the 5'-terminal nucleoside of the 3' wing segment is a sugar-modified nucleoside.) The 5'-terminal nucleoside of the 3' wing segment is generally a sugar-modified nucleoside. The 3' wing segment is not particularly limited as long as it satisfies the above definition, but the at least one nucleoside is preferably independently selected from deoxyribonucleosides and sugar-modified nucleosides, and includes at least one sugar-modified nucleoside. Preferably, the 5'-terminal nucleoside of the 3' wing segment is a sugar-modified nucleoside and is linked to the 3' end of the gap segment.

[0066] RNaseH is generally known as a ribonuclease that recognizes double helix formed by the hybridization of DNA and RNA in living organisms, cleaves the RNA, and produces single-stranded DNA. RNaseH can recognize not only double helix formed by the hybridization of DNA and RNA, but also double helix in which at least one of the nucleic acid base portion, phosphodiester bond portion, or sugar portion of at least one of the DNA or RNA is modified. For example, it can recognize double helix formed by the hybridization of phosphorothioate-modified DNA and RNA. Therefore, DNA can be recognized by RNaseH when hybridized with RNA. Similarly, RNA can be cleaved by RNaseH when hybridized with DNA. The same applies when at least one of the nucleic acid base portion, phosphodiester bond portion, and sugar portion of DNA or RNA is modified. For example, a typical example is an oligonucleotide in which the phosphodiester bond portion of DNA is modified with a phosphorothioate. Examples of DNA and / or RNA modifications that can be recognized by RNaseH are described, for example, in Nucleic Acids Research, 2014, 42, pp 5378-5389; Bioorganic & Medicinal Chemistry Letters, 2008, 18, pp 2296-2300; Molecular BioSystems, 2009, 5, pp 838-843; Nucleic Acid Therapeutics, 2015, 25, pp 266-274; and The Journal of Biological Chemistry, 2004, 279, pp 36317-36326. The RNaseH used in this application is preferably a mammalian RNaseH, more preferably a human RNaseH, and particularly preferably human RNaseH1.

[0067] "At least four consecutive nucleosides recognized by RNaseH" includes four or more consecutive nucleosides and is not particularly limited as long as it is recognized by RNaseH, but examples include "at least four consecutive deoxyribonucleosides". The number of nucleosides constituting "at least four consecutive nucleosides recognized by RNaseH" is, for example, 5 to 30, preferably 5 to 15, more preferably 8 to 12, and particularly preferably 10. A person skilled in the art can determine whether a given sequence of at least four consecutive nucleosides is "at least four consecutive nucleosides recognized by RNaseH" by the structure of the sugar moiety of the sequence of nucleosides.

[0068] Calmodulin is a ubiquitously expressed calcium-sensing protein that regulates various proteins, including multiple ion channels. Calmodulin is involved in a wide range of processes, including inflammation, metabolism, apoptosis, muscle contraction, intracellular transport, short-term memory, long-term memory, nerve growth, and immune responses. In particular, it promotes the inactivation of L-type calcium channels (LTCCs) expressed in the heart.

[0069] Three distinct genes are known to encode calmodulin: CALM1, CALM2, and CALM3. These three genes encode calmodulin with the same amino acid sequence.

[0070] "CALM2 mRNA" refers to mRNA that encodes calmodulin and has been transcribed and spliced ​​from the CALM2 gene, while "CALM2 pre-mRNA" refers to pre-mRNA that encodes calmodulin and has been transcribed from the CALM2 gene. "CALM2 nucleic acid" includes, for example, "CALM2 mRNA" and "CALM2 pre-mRNA".

[0071] "CALM2 mRNA" is represented, for example, by SEQ ID NO: 1, and "CALM2 pre-mRNA" is represented, for example, by SEQ ID NO: 2. Both "CALM2 mRNA" and "CALM2 pre-mRNA" have nucleosides linked to each other by phosphodiester bonds, and thymine is usually replaced by uracil. "CALM2 mRNA" and "CALM2 pre-mRNA" do not have any other modified sugars, modified nucleic acid bases, or modified nucleoside bonds.

[0072] "CALM1 mRNA" refers to mRNA that encodes calmodulin and has been transcribed and spliced ​​from the CALM1 gene, while "CALM1 pre-mRNA" refers to pre-mRNA that encodes calmodulin and has been transcribed from the CALM1 gene. "CALM1 nucleic acid" includes, for example, "CALM1 mRNA" and "CALM1 pre-mRNA".

[0073] "CALM3 mRNA" refers to mRNA that encodes calmodulin and has been transcribed and spliced ​​from the CALM3 gene, while "CALM3 pre-mRNA" refers to pre-mRNA that encodes calmodulin and has been transcribed from the CALM3 gene. "CALM3 nucleic acid" includes, for example, "CALM3 mRNA" and "CALM3 pre-mRNA".

[0074] "Gene expression" refers to the conversion of genetic coding information into structures or functions within a cell. Examples of such structures, though not limited to them, include the products of transcription and translation (mRNA, pre-mRNA, proteins, etc.).

[0075] Long-QT syndrome (LQTS) is an arrhythmia disorder characterized by a prolonged QT interval on an electrocardiogram (ECG) compared to that of a healthy person. LQTS can cause syncope or sudden death due to ventricular arrhythmias. The QT interval is the time from the beginning of the QRS wave to the end of the T wave on an ECG, and represents the contraction and relaxation time of the ventricles. The normal value of the QT interval varies with heart rate, so a corrected QT interval (QTc = QT / √RR), which is corrected for heart rate using Bazett's formula, is usually used. The normal value of the corrected QT interval is between 360 and 440 milliseconds. QT prolongation is when the corrected QT interval is longer than the normal value. Congenital long QT syndrome (congenital LQTS) is a congenital arrhythmia disorder among the aforementioned LQTS conditions, primarily caused by dysfunction of ion channels. While most cases of congenital LQTS are hereditary (familial), CALM2 mutations are often "de novo mutations," meaning that neither parent has a genetic mutation. Congenital LQTS is classified into six types, LQT1 through LQT16, based on the differences in the mutated gene. Diagnosis of congenital LQTS is made by a physician according to guidelines, but it is generally understood as a disorder characterized by a longer corrected QT interval than in healthy individuals and a mutation in a gene associated with congenital LQTS.

[0076] "LQT15" is a congenital long QT syndrome caused by a mutation in CALM2. Known CALM2 mutations include a mutation in which the 98th amino acid from the N-terminus of CALM2 changes from asparagine (N) to serine (S), and a mutation in which the 130th amino acid changes from aspartic acid (D) to glycine (G). "LQT14" is a congenital long QT syndrome caused by a mutation in CALM1, and "LQT16" is a congenital long QT syndrome caused by a mutation in CALM3. Calmodulinopathy is a disorder primarily characterized by arrhythmias, caused by mutations in the gene encoding calmodulin. In addition to long QT syndrome (LQTS), other reported symptoms include catecholamine-induced polymorphic ventricular tachycardia (CPVT) and idiopathic ventricular fibrillation (IVF).

[0077] Next, preferred embodiments of modified oligonucleotides or compounds containing modified oligonucleotides will be described. In the following explanation, the term "modified oligonucleotide" will be used, but it goes without saying that these explanations can also be applied by appropriately replacing "modified oligonucleotide" with "compound containing a modified oligonucleotide" or, in a preferred embodiment, "antisense oligonucleotide" (ASO).

[0078] The modified oligonucleotides in this application preferably target CALM2 mRNA and / or pre-mRNA. The modified oligonucleotides do not need to hybridize with the entirety of CALM2 mRNA and / or pre-mRNA; usually, it is sufficient for them to hybridize with at least a portion, and not with the other portion. For example, CALM2 gene expression is regulated by hybridization of a portion of CALM2 mRNA and / or pre-mRNA with an oligonucleotide having a sequence complementary to a partial sequence of CALM2 mRNA and / or pre-mRNA (such as a gapmer or an oligonucleotide designed to produce an antisense effect).

[0079] The complementarity between the nucleic acid base sequence of the modified oligonucleotide in this application and the partial sequence of CALM2 mRNA and / or pre-mRNA is preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, or 99% or more). For the modified oligonucleotide in this application and at least a portion of the partial sequence of CALM2 mRNA and / or pre-mRNA to hybridize, their sequences do not need to be perfectly complementary, but it is even more preferable that they be perfectly complementary.

[0080] The modified oligonucleotide in this application has a gap segment, a 5' wing segment, and a 3' wing segment. The gap segment consists of at least five nucleosides independently selected from the group consisting of deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, with its 3' and 5' ends independently being deoxyribonucleosides. Preferably, the gap segment contains "at least four consecutive deoxyribonucleosides".

[0081] The number of nucleosides contained in the gap segment is preferably 5 to 30, more preferably 5 to 15, even more preferably 8 to 12, even more preferably 9 to 11, and particularly preferably 10. In one embodiment, the nucleosides contained in the gap segment are deoxyribonucleosides.

[0082] The nucleoside-to-nucleoside bonds contained in the gap segment are preferably selected independently from phosphodiester bonds and phosphorothioate bonds. The nucleoside bonds contained in the gap segment preferably contain at least one phosphorothioate bond, more preferably 50% or more are phosphorothioate bonds, more preferably 75% or more are phosphorothioate bonds, even more preferably 80% or more are phosphorothioate bonds, even more preferably 90% or more are phosphorothioate bonds, and particularly preferably all are phosphorothioate bonds.

[0083] The 5' wing segment consists of at least one nucleoside independently selected from the group consisting of deoxyribonucleosides, ribonucleosides, and glycosylated nucleosides, wherein its 3' end bound to the gap segment is a glycosylated nucleoside and does not contain "at least four consecutive nucleosides recognized by RNaseH". The 3' wing segment consists of at least one nucleoside independently selected from the group consisting of deoxyribonucleosides, ribonucleosides, and glycosylated nucleosides, wherein its 5' end bound to the gap segment is a glycosylated nucleoside and does not contain "at least four consecutive nucleosides recognized by RNaseH". The 5' wing segment and the 3' wing segment preferably do not contain "at least four consecutive deoxyribonucleosides".

[0084] In the following, properties common to both the 5' wing segment and the 3' wing segment will be referred to simply as wing segments. The number of nucleosides contained in each wing segment is 1 to 15, preferably 1 to 10, more preferably 1 to 7, even more preferably 2 to 6, even more preferably 3 to 5, and particularly preferably 3.

[0085] The sugar-modified nucleoside contained in the wing segment is preferably a nucleoside whose affinity for the partial sequence of CALM2 mRNA and / or CALM2 pre-mRNA has been increased by substitution or the like, or a nucleoside whose resistance to nucleases has been increased. More preferably, it is independently selected from 2'-modified nucleosides and 2',4'-BNA. The 2'-modified nucleoside included in the wing segment is preferably independently selected from the group consisting of 2'-O-Me nucleoside, 2'-O-MOE nucleoside, 2'-O-AP nucleoside, 2'-F nucleoside, 2'-O-NMA nucleoside, 2'-O-MCE nucleoside, 2'-DMAECE nucleoside, 2'-MоrECE nucleoside, 2'-PyECE nucleoside, and BimECE nucleoside. At least one, more preferably at least one independently selected from the group consisting of 2'-O-Me nucleoside, 2'-O-MOE nucleoside, 2'-O-NMA nucleoside and 2'-O-MCE nucleoside, even more preferably at least one independently selected from 2'-O-MOE nucleoside and 2'-O-MCE nucleoside, and particularly preferably 2'-O-MCE nucleoside. The 2',4'-BNA contained in the wing segment is preferably LNA, ENA, cEt, BNA NC It is at least one independently selected from the group consisting of AmNA, scpBNA, and GuNA, and more preferably LNA. The sugar-modified nucleoside contained in the wing segment is more preferably at least one independently selected from 2'-O-MOE nucleoside, LNA, and 2'-O-MCE nucleoside, more preferably 2'-O-MOE nucleoside, 2'-O-MCE nucleoside, or LNA and 2'-O-MCE nucleoside, and particularly preferably LNA and 2'-O-MCE nucleoside.

[0086] Each wing segment preferably consists of 1 to 10 nucleosides independently selected from the group consisting of sugar-modified nucleosides and deoxyribonucleosides, and includes at least one sugar-modified nucleoside. More preferably, it consists of 2 to 6 nucleosides independently selected from the group consisting of sugar-modified nucleosides and deoxyribonucleosides, and includes at least two sugar-modified nucleosides. Even more preferably, it consists of 2 to 6 nucleosides independently selected from the group consisting of 2'-modified nucleosides and 2',4'-BNA, and even more preferably, it consists of 3 to 5 nucleosides independently selected from the group consisting of 2'-modified nucleosides and 2',4'-BNA. Each wing segment consists more specifically of 1 to 10, preferably 2 to 6, nucleosides independently selected from LNA, 2'-O-MCE nucleosides, and 2'-O-MOE nucleosides, and more preferably of 2 to 5, more preferably 3, nucleosides independently selected from LNA and 2'-O-MCE nucleosides. Even more preferably of 2 LNA and 1 or 2 2'-O-MCE nucleosides, and particularly preferably of 2 LNA and 1 2'-O-MCE nucleoside. In another embodiment, particularly preferably of 2 to 5, most preferably of 3 LNA. In yet another embodiment, particularly preferably of 3 to 6, most preferably of 5 2'-O-MCE nucleosides. In another preferred embodiment, each wing segment is an oligonucleotide comprising 2 to 5 nucleosides independently selected from the group consisting of 2',4'-BNA and deoxyribonucleosides, and containing at least 2 2',4'-BNA, for which reference can be found in International Publication No. 2016 / 127002, etc. The preferred LNA contained in each wing segment is β-D-methyleneoxy BNA.

[0087] In some embodiments, the 5'-wing segment and the 3'-wing segment each independently contain 3, 4, 5, or 6 glycosylated nucleosides, and the gap segment consists of 8, 9, 10, 11, 12, 13, or 14 deoxyribonucleosides. The number of nucleosides in such a gapmer can be expressed as (5'-wing segment - gap segment - 3'-wing segment) and includes 5-10-5, 5-11-4, 4-11-5, 4-12-4, 3-14-3, 6-8-6, 3-12-3, 3-10-3, 4-10-4, 3-10-4, 4-10-3, 3-9-3, 4-9-4, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, and 4-8-4.

[0088] In some embodiments, the modified oligonucleotide has at least 11, 12, 13, 14, or 15 consecutive nucleosides, the gap segment contains at least 5, 6, 7, 8, or 9 consecutive nucleosides, and the 5' wing segment and the 3' wing segment are any of (i) to (iv) below. (i) The 5' wing segment and the 3' wing segment each independently contain 3, 4, 5, or 6 2'-O-MOE nucleosides. (ii) The 5' wing segment and the 3' wing segment each independently contain 3, 4, 5, or 6 2'-O-MCE nucleosides. (iii) The 5' wing segment and the 3' wing segment each independently contain 2, 3, 4, 5, or 6 LNAs. (iv) Each 5' wing segment and 3' wing segment independently comprises 3 or 4 nucleosides selected from 2'-O-MCE nucleosides and LNAs, with at least one 2'-O-MCE nucleoside and at least one LNA.

[0089] The 5'-wing segment and the 3'-wing segment of the modified oligonucleotide of the present invention are preferably selected from the group consisting of LL, LLL, VLL, LVL, LLV, LVV, VLV, VVL, VVLL, VLVL, LVLV, LLVV, VLLL, LVLL, LLVL, LLLV, LVVV, VLVV, VVLV, and VVVL. More preferably, the 5' wing segment is selected from the group consisting of LL, LLL, VLL, VLV, VVL, VVLL, VLVL, VLLL, VLVV, VVLV, and VVVL, and the 3' wing segment is selected from the group consisting of LL, LLL, LLV, VLV, LVV, LLVV, LVLV, XLLV, VVLV, VLVV, and LVVV. More preferably, the 5' wing segment is selected from the group consisting of LL, LLL, VLL, VLV, VVL, and VLVL, and the 3' wing segment is selected from the group consisting of LL, LLL, LLV, VLV, LVV, and LVLV. In other preferred embodiments, the 5' wing segment is selected from the group consisting of LLL, VLL, VVL, LVL, and VLVL, and the 3' wing segment is selected from the group consisting of LLL, LLV, LVV, LVL, and LVLV. Particularly preferred, the 5' wing segment is LLL and the 3' wing segment is LLL. In other embodiments, particularly preferably, the 5' wing segment is VLL and the 3' wing segment is LLV. Here, L and V in the 5'-wing segment and 3'-wing segment are sugar-modified nucleosides having different modified sugars, with the left side representing the 5' side and the right side representing the 3' side. Preferably, L represents a 2'-4' crosslinked nucleoside and V represents a 2'-modified nucleoside. Particularly preferably, L represents LNA and V represents an MCE nucleoside.

[0090] The nucleoside bonds contained in the wing segment are preferably selected independently of phosphodiester bonds and phosphorothioate bonds. The nucleoside-to-nucleoside bonds in the 5' wing segment preferably contain at least one phosphorothioate bond, preferably 50% or more are phosphorothioate bonds, more preferably 60% or more are phosphorothioate bonds, even more preferably 75% or more are phosphorothioate bonds, even more preferably 80% or more are phosphorothioate bonds, even more preferably 90% or more are phosphorothioate bonds, and particularly preferably all are phosphorothioate bonds. The 3' wing segment is similar to the 5' wing segment. In another aspect, it is preferable that all nucleoside bonds contained in the wing segment are phosphodiester bonds, from the viewpoint of reducing toxicity.

[0091] In the modified oligonucleotide of the present application, the 3' end of the 5' wing segment and the 5' end of the gap segment are preferably linked by a phosphodiester bond or a modified phosphodiester bond. More preferably, the 3' end of the 5' wing segment and the 5' end of the gap segment are linked by a modified phosphodiester bond. Even more preferably, the 3' end of the 5' wing segment and the 5' end of the gap segment are linked by a phosphorothioate bond.

[0092] The modified oligonucleotides in this application are modified oligonucleotides having a continuous nucleoside length of 8 to 80, preferably 11 to 50, more preferably 15 to 25, even more preferably 16 to 20, and particularly preferably 16.

[0093] The modified oligonucleotides in this application preferably have a nucleic acid base sequence containing at least 8, more preferably 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleic acid bases, which are included in any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 73. The modified oligonucleotide in this application more preferably has a nucleic acid base sequence containing one of the nucleic acid base sequences of SEQ ID NOs: 3 to 73. The modified oligonucleotide in this application more preferably has a nucleic acid sequence consisting of one of the nucleic acid sequences of SEQ ID NOs: 3 to 73.

[0094] In one embodiment, the modified oligonucleotide in the present application preferably has a nucleic acid base sequence containing at least 8 consecutive nucleic acid bases, more preferably 8, 9, 10, 11, 12, 13, 14, or 15, which are included in any one of the nucleic acid base sequences of SEQ ID NOs: 3-30, 32-36, 38-46, or 48-71. In one embodiment, the modified oligonucleotide in the present application more preferably has a nucleic acid sequence containing one of the nucleic acid sequences of SEQ ID NOs: 3-30, 32-36, 38-46, and 48-71. In one embodiment, the modified oligonucleotide in the present application more preferably has a nucleic acid base sequence consisting of one of the nucleic acid base sequences of SEQ ID NOs: 3-30, 32-36, 38-46, and 48-71.

[0095] In another embodiment, the modified oligonucleotide in the present application preferably has a nucleic acid sequence containing at least eight, more preferably eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen consecutive nucleic acid bases, which are included in any one of the nucleic acid sequences of SEQ ID NOs: 3 to 20. In another embodiment, the modified oligonucleotide in the present application more preferably has a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 3 to 20. In another embodiment, the modified oligonucleotide in the present application more preferably has a nucleic acid sequence consisting of one of the nucleic acid sequences of SEQ ID NOs: 3 to 20.

[0096] In yet another embodiment, the nucleic acid base sequence of the modified oligonucleotide in the present application preferably includes at least 8 consecutive nucleic acid bases, more preferably 8, 9, 10, 11, 12, 13, 14, or 15, which are included in the nucleic acid base sequence of SEQ ID NO: 4. In yet another embodiment, the nucleic acid base sequence of the modified oligonucleotide in the present application preferably includes at least the nucleic acid base sequence of SEQ ID NO: 4. In yet another embodiment, the modified oligonucleotide in the present application more preferably has a nucleic acid sequence consisting of the nucleic acid sequence of SEQ ID NO: 4.

[0097] The modified oligonucleotide in this application is preferably CALM2 represented by Sequence ID No. 1. It is complementary to at least a portion of the nucleic acid sequences selected from the group consisting of nucleic acid sequences indicated by the positional numbers of the mRNA nucleic acid bases 102-117, 160-175, 183-205, 212-227, 322-337, 365-405, 411-434, 464-494, 506-521, 606-635, 636-651, 692-707, 715-749, 754-801, 829-857, 862-934, 951-970, 995-1010, 1006-1021, 1036-1051, 1062-1077, 1081-1104, 1138-1166, 1188-1203 and 1239-1254.

[0098] In one embodiment, the modified oligonucleotide in the present application is preferably complementary to at least a portion of a nucleic acid sequence selected from the group consisting of nucleic acid sequences represented by the positional numbers of nucleic acid bases 102-117, 160-175, 183-198, 212-227, 322-337, 365-380, 387-402, 479-494, 620-635, 715-730, 862-877, 896-934, 995-1010, 1062-1077, 1089-1104, 1140-1166 and 1239-1254 of the CALM2 mRNA represented by SEQ ID NO: 1.

[0099] More preferably, the modified oligonucleotide in this application is complementary to at least a portion of the nucleic acid base sequence indicated by the nucleic acid base positions 896-934 of the CALM2 mRNA represented by SEQ ID NO: 1.

[0100] More preferably, the modified oligonucleotide in this application is complementary to at least a portion of the nucleic acid base sequence indicated by the nucleic acid base positions 907-922 of the CALM2 mRNA represented by SEQ ID NO: 1.

[0101] The complementarity between the modified oligonucleotide in this application and the corresponding portion of the CALM2 mRNA represented by SEQ ID NO: 1 (for example, each of the target regions described above) is at least 80%, preferably 85%, more preferably 90%, and even more preferably 95%, 96%, 97%, 98%, or 99%.

[0102] The modified oligonucleotides in this application are preferably those with nucleic acid base position numbers 102-117, 160-175, 1624-1639, 1661-1676, 3355-3370, 5859-5881, 13959-13974, 14069-14084, 14228-14278, 14284-14307, 14764-14794, 14806-14821, 15824-15869, 15910-15925, and 15933-1596 of the CALM2 pre-mRNA represented by SEQ ID NO: 2. 7. It is complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 15972~16019, 16047~16075, 16080~16152, 16169~16188, 16213~16239, 16254~16269, 16280~16295, 16299~16322, 16356~16384, 16406~16421 and 16457~16472.

[0103] In one embodiment, the modified oligonucleotide in the present application is preferably complementary to at least a portion of a nucleic acid sequence selected from the group consisting of nucleic acid sequences represented by the nucleic acid base position numbers 102-117, 160-175, 5859-5874, 13959-13974, 14069-14084, 14238-14253, 14260-14275, 14779-14794, 15838-15853, 15933-15948, 16080-16095, 16114-16152, 16213-16228, 16280-16295, 16307-16322, 16358-16384 and 16457-16472 of the CALM2 pre-mRNA represented by SEQ ID NO: 2.

[0104] The modified oligonucleotide in this application is more preferably complementary to at least a portion of a nucleic acid sequence selected from the group consisting of nucleic acid sequences indicated by the nucleic acid base positions 16114 to 16152 of the CALM2 pre-mRNA represented by Sequence ID No. 2.

[0105] The modified oligonucleotide in this application is more preferably complementary to at least a portion of the nucleic acid base sequence indicated by the nucleic acid base positions 16125 to 16140.

[0106] The complementarity between the modified oligonucleotide in this application and the corresponding portion of the CALM2 pre-mRNA represented by Sequence ID No. 2 (e.g., each of the target regions described above) is at least 80%, preferably 85%, more preferably 90%, and even more preferably 95%, 96%, 97%, 98%, or 99%.

[0107] A modified oligonucleotide having a nucleic acid base sequence containing at least eight consecutive nucleic acid bases from any one of the nucleic acid base sequences of SEQ ID NOs. 3 to 73 (SEQ ID NOs. 3 to 20 in one embodiment), and having a length of 8 to 80 consecutive nucleosides, preferably has 2 to 5 nucleosides selected from the group consisting of LNA and 2'-O-MCE nucleosides in each wing segment, more preferably has 1 or 2 LNA and 1 or 2 2'-O-MCE nucleosides, even more preferably has 2 LNA and 1 2'-O-MCE nucleoside, and particularly preferably has 3 LNA.

[0108] In this application, a functional molecule may be directly or indirectly bound to the modified oligonucleotide. The binding between the functional molecule and the modified oligonucleotide may be direct or indirect through another substance, but it is preferable that the oligonucleotide and the functional molecule are bound by a covalent bond, an ionic bond, or a hydrogen bond. From the viewpoint of high binding stability, it is more preferable that the binding is direct by a covalent bond or that the binding is via a linker (binding group) by a covalent bond.

[0109] When the functional molecule is covalently bonded to a modified oligonucleotide, it is preferable that the functional molecule is directly or indirectly bonded to the 3' or 5' end of the modified oligonucleotide molecule. The bond between the linker or functional molecule and the terminal nucleoside of the modified oligonucleotide molecule is selected depending on the functional molecule. Preferably, the linker or functional molecule and the terminal nucleoside of the modified oligonucleotide molecule are linked by a phosphodiester bond or a modified phosphodiester bond, and more preferably by a phosphodiester bond. The linker or functional molecule may be directly linked to the oxygen atom at the 3' position of the 3'-terminal nucleoside of the modified oligonucleotide molecule or to the oxygen atom at the 5' position of the 5'-terminal nucleoside.

[0110] The structure of the "functional molecule" (conjugate) is not particularly restricted, and its binding imparts a desired function to the modified oligonucleotide. Desired functions include labeling, purification, and delivery to a target site. Examples of molecules that impart labeling function include fluorescent proteins and luciferase compounds. Examples of molecules that impart purification function include biotin, avidin, His-tagged peptides, GST-tagged peptides, and FLAG-tagged peptides.

[0111] Furthermore, from the viewpoint of delivering the modified oligonucleotide of this application to a target site (e.g., the heart) with high specificity and efficiency, and very effectively controlling the expression of the CALM2 gene by the modified oligonucleotide, it is preferable that a molecule having the function of delivering the modified oligonucleotide to the target site is bound to the modified oligonucleotide as a functional molecule. For example, the molecule having the delivery function can be found in European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp 78-92, Expert Opinion on Drug Delivery, 2014, 11, pp 791-822, Nucleic Acids Research, 2019, 47, 12, pp 6045-6058, International Publication No. 2017 / 053995, etc.

[0112] Examples of functional molecules include sugars, lipids, peptides, proteins, and their derivatives.

[0113] For example, lipids are functional molecules that can efficiently deliver the modified oligonucleotides of the present invention to the heart and other organs. Examples of lipids include cholesterol; vitamins such as vitamin E (tocopherols, tocotrienols), vitamin A, vitamin D, and vitamin K; steroids such as glucocorticoids, mineralocorticoids, estrogen, androgen, and progesterone; fatty acids such as C5-30 saturated fatty acids and C5-30 unsaturated fatty acids; intermediate metabolites such as acylcarnitine and acyl-CoA; glycolipids; glycerides; and their derivatives.

[0114] The lipids used are preferably fatty acids such as C5-30 saturated fatty acids and C5-30 unsaturated fatty acids. Here, C5-30 saturated fatty acids are straight-chain or branched saturated fatty acids with 5 to 30 carbon atoms, and specific examples include palmitic acid and stearic acid. C5-30 unsaturated fatty acids are straight-chain or branched unsaturated fatty acids having at least one carbon-carbon double bond and with 5 to 30 carbon atoms, and specific examples include oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. The lipids used are more preferably C15-25 saturated fatty acids or C15-25 unsaturated fatty acids, and even more preferably C15-25 saturated fatty acids. C15-25 saturated fatty acids are linear or branched saturated fatty acids having 15 to 25 carbon atoms, and C15-25 unsaturated fatty acids are linear or branched unsaturated fatty acids having at least one carbon-carbon double bond and having 15 to 25 carbon atoms. The lipids used are more preferably linear fatty acids among C15-25 saturated fatty acids or C15-25 unsaturated fatty acids, and even more preferably linear C15-25 saturated fatty acids, and particularly preferably palmitic acid. Other lipids used are preferably cholesterol or vitamin E, more preferably tocopherols, even more preferably tocopherol, and particularly preferably α-tocopherol, from the viewpoint of improving accumulation in the heart.

[0115] As a functional molecule capable of delivering the modified oligonucleotide of the present invention with high specificity to the liver, a sugar derivative that interacts with the asialocrycoprotein receptor may be used. "Asialoglycoprotein receptors" are present on the surface of liver cells and recognize galactose residues of asialoglycoproteins, taking the molecules into the cell and breaking them down. "Sugar derivatives that interact with asialoglycoprotein receptors" are preferably compounds that have a structure similar to galactose residues and are taken into the cell through interaction with asialoglycoprotein receptors. Examples include GalNAc (N-acetylgalactosamine) derivatives, galactose derivatives, and lactose derivatives.

[0116] Functional molecules that can deliver the modified oligonucleotides of this invention to organs with high specificity and efficiency by interacting with various proteins on the cell surface of each organ include receptor ligands, antibodies, and peptides or proteins derived from their fragments.

[0117] The functional molecule and the modified oligonucleotide may be linked via a linker, and the linker is not particularly limited as long as it can stably link the functional molecule and the oligonucleotide, as long as it can perform the function that the functional molecule has as a modified oligonucleotide molecule. Examples of the linker include oligonucleotides with 1 to 20 nucleosides or groups derived from nucleosides, polypeptides with 1 to 20 amino acids or groups derived from amino acids, alkylenes with 1 to 20 carbon atoms and alkenylenes with 2 to 20 carbon atoms, polyalkylene glycol groups, etc. The oligonucleotides with 1 to 20 nucleosides or groups derived from nucleosides are divalent groups obtained by removing hydrogen atoms, etc., from the 3' and 5' ends of oligonucleotides with 1 to 20 nucleosides or nucleosides (nucleosides if the number of nucleosides is 1). For oligonucleotides having 1 to 20 nucleosides or groups derived from nucleosides, see, for example, International Publication No. 2017 / 053995. International Publication No. 2017 / 053995 describes, for example, a 3-base linker having a TCA motif, a 1 to 5-base linker without a TCA motif, etc. For polypeptides having 1 to 20 amino acids or groups derived from amino acids, the groups are divalent groups obtained by removing two groups selected from hydroxyl, hydrogen, and amino, etc., from polypeptides having 1 to 20 amino acids or amino acids (or amino acids if the number of amino acids is 1). The alkylene and alkenylene may be linear or branched. For example, the polyalkylene glycol group is given by formula: H-(OW-)n-OH (In the formula, W is independently an alkylene having 1 to 10 carbon atoms, where the alkylene may be independently substituted with hydroxyl and / or amino, and n is an integer from 1 to 20.) This group is obtained by removing a hydroxyl group and two hydrogen atoms selected from the hydroxyl group from a polyalkylene glycol represented by [formula]. W is preferably independently an alkylene having 2 to 4 carbon atoms, where the alkylene may be independently substituted with hydroxyl and / or amino. W is more preferably independently an alkylene having 2 or 3 carbon atoms, where the alkylene may be independently substituted with hydroxyl. W is more preferably independently an ethane-1,2-diyl or propane-1,2-diyl, where the ethane-1,2-diyl and propane-1,2-diyl may be substituted with a hydroxyl group. n is preferably an integer from 1 to 10, more preferably an integer from 1 to 6, even more preferably an integer from 2 to 5, and particularly preferably 4.

[0118] The linker between the functional molecule and the modified oligonucleotide is preferably an alkylene having 1 to 20 carbon atoms or an alkenylene having 2 to 20 carbon atoms, more preferably an alkylene having 2 to 10 carbon atoms, even more preferably an alkylene having 6 carbon atoms, and particularly preferably a 1,6-hexane-diyl group. Other preferred linkers are polyalkylene glycol groups.

[0119] Compounds containing modified oligonucleotides in this application may also contain a prodrug moiety. A prodrug is a derivative of a pharmaceutical compound that has a group that can be chemically or metabolically broken down, and is derived into a pharmacologically active pharmaceutical compound by solvolysis or in vivo breakdown under physiological conditions. Methods for selecting and manufacturing appropriate prodrug derivatives are described, for example, in Design of Prodrugs (Elsevier, Amsterdam, 1985). For example, as a prodrug moiety, there are acyloxy derivatives produced by reacting the hydroxyl group of a modified oligonucleotide (or a compound containing a modified oligonucleotide) with an appropriate acyl halide, an appropriate acid anhydride, or an appropriate alkyloxycarbonyl halide compound, such as -OC(=O)C2H5, -OC(=O)(t-Bu), and -OC(=O)C 15 H 31Examples include -OC(=O)-(m-CO2Na-Ph), -OC(=O)CH2CH2CO2Na, -OC(=O)CH(NH2)CH3, -OC(=O)CH2N(CH3)2, or -O-CH2OC(=O)CH3.

[0120] In this application, the prodrugs of compounds containing modified oligonucleotides also include complexes formed by the hybridization of two or more oligonucleotides. Examples of such preferred structures (prodrugs) include double-stranded oligonucleotides containing ribonucleosides (e.g., RNA), peptide nucleic acids (PNA), or deoxyribonucleosides (e.g., DNA) that are complementary to the modified oligonucleotide (or compound containing a modified oligonucleotide) (e.g., International Publication Nos. 2013 / 089283, 2017 / 068791, 2017 / 068790, or 2018 / 003739), and single-stranded oligonucleotides in which RNA complementary to the modified oligonucleotide is linked by a linker (e.g., International Publication Nos. 2017 / 131124, 2018 / 143475). The linker may be an oligonucleotide linker or a linker containing a non-nucleoside structure. Single-stranded oligonucleotides, in which a modified oligonucleotide is directly linked to complementary RNA, are also examples (International Publication No. 2019 / 022196).

[0121] Compounds containing modified oligonucleotides in this application also include compounds in which two identical or different modified oligonucleotides are linked together. For structures linking two modified oligonucleotides, see, for example, International Publication No. 2017 / 131124 and International Publication No. 2018 / 143475.

[0122] Compounds containing modified oligonucleotides in this application exist not only through tautomerism and geometric isomerism, but also as mixtures thereof or mixtures of their respective isomers. Furthermore, if a chiral center is present, or if a chiral center is formed as a result of isomerization, the invention also includes the existence of each optical isomer and mixtures in any ratio. In the case of compounds having two or more chiral centers, diastereomers based on each optical isomer also exist. The present invention also includes compounds containing all of these types in any ratio. Furthermore, optically active compounds can be obtained by methods well known for this purpose.

[0123] For example, if a compound containing a modified oligonucleotide in this application contains a modified phosphodiester bond (e.g., a phosphorothioate bond) and the phosphorus atom is a chiral atom, then both forms of oligonucleotides with controlled phosphorus atom stereochemistry and forms of oligonucleotides with uncontrolled phosphorus atom stereochemistry are included within the scope of the present invention.

[0124] Compounds containing modified oligonucleotides in this application, their prodrugs, or pharmaceutically acceptable salts thereof may exist in any crystalline form or as any hydrate depending on the manufacturing conditions, and these crystalline forms, hydrates, and mixtures thereof are also included within the scope of the present invention. They may also exist as solvates containing organic solvents such as acetone, ethanol, 1-propanol, and 2-propanol, and all of these forms are also included within the scope of the present invention.

[0125] Compounds containing modified oligonucleotides in this application may be converted to pharmacologically acceptable salts as needed, or liberated from the resulting salts. Examples of pharmacologically acceptable salts of the compounds include salts with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (calcium, etc.), magnesium, ammonium, organic bases (triethylamine, trimethylamine, etc.), amino acids (glycine, lysine, glutamic acid, etc.), inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.), or organic acids (acetic acid, citric acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.). In particular, the substructure represented by -P(=O)(OH)- is -P(=O)(OH) - It may be converted to an anionic substructure represented by -P(=O)(SH)- and form salts with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (calcium, etc.), magnesium, or ammonium. In addition, the substructure represented by -P(=O)(SH)- that forms a phosphorothioate bond may be converted to -P(=O)(S - It may be converted to an anionic substructure represented by )- and similarly form salts with alkali metals, alkaline earth metals, or ammonium, etc. The same applies to other modified phosphodiester bonds. Pharmacologically acceptable salts are, in particular, sodium salts.

[0126] Compounds containing modified oligonucleotides as described in this application can be prepared by those skilled in the art by appropriately selecting known methods. For example, those skilled in the art can design the nucleoside sequence of a modified oligonucleotide based on information of the nucleoside sequence of a target RNA and synthesize it using a commercially available automated nucleic acid synthesizer (e.g., Applied Biosystems, Beckman, GeneDesign). They can also be synthesized by an enzymatic reaction. Examples of such enzymes include, but are not limited to, polymerases, ligases, and restriction enzymes. That is, the method for producing compounds containing modified oligonucleotides as described in this application may include a step of extending the nucleoside chain at the 3' or 5' end.

[0127] Many methods for linking functional molecules with oligonucleotides are well known in this field, and can be found, for example, in European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp 78-92, Expert Opinion on Drug Delivery, 2014, 11, pp 791-822. For example, after linking a functional molecule with a linker by a known method, it can be converted to an amidite using an amiditation reagent, or to an H-phosphonate using an H-phosphonate reagent, and then linked to an oligonucleotide.

[0128] The obtained oligonucleotide can be purified by reverse-phase column chromatography or the like to prepare the compound containing the modified oligonucleotide described in this application.

[0129] Compounds containing modified oligonucleotides in this application, or pharmacologically acceptable salts thereof, may be included as active ingredients in pharmaceuticals. Compounds containing modified oligonucleotides or their pharmacologically acceptable salts can effectively inhibit the expression of the CALM2 gene and can therefore be used in pharmaceuticals to treat, prevent, and / or improve diseases or conditions in which the inhibition of CALM2 gene expression is effective. The diseases that can be treated, prevented, and / or improved by pharmaceuticals containing compounds containing modified oligonucleotides or their pharmacologically acceptable salts are not particularly limited as long as the inhibition of CALM2 gene expression is effective, but examples include congenital long QT syndrome (especially LQT15), calmodulinopathy, and other diseases involving calmodulin, such as inflammatory diseases, metabolic diseases, apoptosis, muscle contraction, diseases related to intracellular transport of calcium ions, diseases related to short-term and / or long-term memory, diseases related to nerve growth, and immune diseases. In particular, compounds containing modified oligonucleotides or their pharmacologically acceptable salts are effective in treating, preventing, and / or improving congenital long QT syndrome and calmodulinopathy.

[0130] The present invention can also provide compositions containing the modified oligonucleotide as an active ingredient, for example, used to inhibit the expression of the CALM2 gene by an antisense effect. In particular, the compounds containing the modified oligonucleotide in the present invention are useful as components of pharmaceutical compositions for treating, preventing, and / or improving diseases in which inhibition of CALM2 gene expression is effective, such as congenital long QT syndrome (especially LQT15) and calmodulinopathy.

[0131] Pharmaceutical compositions containing compounds comprising modified oligonucleotides or pharmaceutically acceptable salts thereof as described in this application can be formulated by known pharmaceutical methods. For example, the pharmaceutical compositions can be used enterally (oral, etc.) or non-enterally (parenterally) as injections, capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublingual preparations, chewable preparations, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, topical preparations, ointments, hard ointments, poultices, transdermal preparations, lotions, inhalants, aerosols, suppositories, etc.

[0132] These formulations can be appropriately combined with pharmacologically acceptable carriers, specifically sterile water or physiological saline, vegetable oil, solvents, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavoring agents, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonic agents, analgesics, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, viscosity modifiers, flavoring and odor modifiers, solubilizers, or other additives.

[0133] There are no particular restrictions on the route of administration of a pharmaceutical composition containing a compound containing a modified oligonucleotide or a pharmacoposly acceptable salt thereof as described in this application, and can be enteral (e.g., oral) or non-enteral. More preferably, it can be administered orally, intravenously, intra-arterially, intraperitoneally, subcutaneously, intradermally, intra-airway, rectally, intramuscularly, intrathecally, intraventricularly, nasally, and intravitreously, and administered by infusion. More preferably, it can be administered intravenously and subcutaneously, and particularly preferably intravenously.

[0134] Pharmaceutical compositions (formulations) for subcutaneous or intravenous administration containing a compound containing a modified oligonucleotide as described in this application, or a pharmacokinetically acceptable salt thereof, can be manufactured by conventional methods. For example, a preparation containing a compound containing a modified oligonucleotide as described in this application, or a pharmacokinetically acceptable salt thereof, with additives such as buffers and isotonic agents, is completely dissolved in sterile water for injection and then filtered and sterilized. If this preparation is filled into a sterile syringe, a pre-filled syringe preparation can be manufactured; if it is filled into a sterile vial, an injection preparation can be manufactured; and if it is filled into a sterile vial and then freeze-dried, a preparation for immediate preparation can be manufactured. Instead of filter sterilization, final sterilization such as autoclaving or gamma irradiation may be performed.

[0135] Various mammalian diseases or conditions can be treated, prevented, and / or improved by compounds containing the modified oligonucleotides described herein or their pharmaceutically acceptable salts. For example, but not limited to, diseases or conditions of mammals including humans, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats, rabbits, chimpanzees, or other rodent species such as bovines, ovines, equines, canines, felines, and mice. The mammal is particularly preferably a human.

[0136] When administering a compound containing a modified oligonucleotide (ASO) as described in this application, or a pharmacologically acceptable salt thereof, to a mammal including a human, the dosage is preferably an effective dose. An effective dose means a sufficient amount of the compound to produce the desired pharmacological effect in an individual requiring the drug, and varies depending on the age, weight, symptoms, health status of the individual whose disease or condition is to be treated, prevented and / or improved, the type of compound or pharmacologically acceptable salt thereof used, and the amount it is incorporated into the pharmaceutical composition, and is selected as appropriate. The dosage is preferably 0.0001 mg / kg / day to 100 mg / kg / day in terms of the modified oligonucleotide (particularly ASO) as described in this application.

[0137] Preferred embodiments of the uses of modified oligonucleotides or their pharmacologically acceptable salts in this application include the following: A method for controlling the expression of the CALM2 gene, comprising the step of contacting a cell with a compound containing a modified oligonucleotide as described in this application or a pharmacoposly acceptable salt thereof. A method for controlling the expression of the CALM2 gene in a subject, comprising the step of administering a pharmaceutical composition containing an effective amount of a compound containing the modified oligonucleotide of the present invention or a pharmacoposly acceptable salt thereof to a subject requiring such treatment. The use of compounds containing the modified oligonucleotides of this application or pharmacologically acceptable salts thereof for controlling the expression of the CALM2 gene. Use of a compound containing the modified oligonucleotide in this application or a pharmacoposly acceptable salt thereof for manufacturing a pharmaceutical product for controlling the expression of the CALM2 gene. Compounds containing the modified oligonucleotides described herein, or pharmacologically acceptable salts thereof, for controlling the expression of the CALM2 gene. [Examples]

[0138] The present invention will be described in more detail below based on examples, but the following examples do not limit the scope of the present invention in any way, and the scope of the present invention should be determined based on the appended claims.

[0139] In the table (1 to 12) in the examples, "Compound No." refers to the compound number, and "Chemical Structure" refers to the chemical structure of each compound. In the sequence notation in the examples (Tables 1, 5, 6, and 10), unless otherwise specified, "(L)" represents LNA (β-D-methyleneoxy BNA), "(V)" represents a 2'-O-MCE nucleoside, lowercase letters represent a deoxyribonucleoside, "^" represents a phosphorothioate bond, "5(x)" indicates that the nucleic acid base of the deoxyribonucleoside is 5-methylcytosine, and "5" in "5(V)" and "5(L)" indicates that the nucleic acid base of the nucleoside is 5-methylcytosine.

[0140] [Manufacturing Example 1] The antisense oligonucleotides listed in Table 1 (compounds represented by chemical structures corresponding to compound numbers) were prepared using the nS-8II automated nucleic acid synthesizer (Gene Design Co., Ltd.).

[0141] [Table 1]

[0142] Table 2 shows the target location in the human CALM2 mRNA or pre-mRNA sequence for each antisense oligonucleotide shown in Table 1, as well as the corresponding sequence number and nucleic acid sequence for each antisense oligonucleotide. In the sequence notation in Table 2, "SEQ1 START" means "SEQ ID NO: 1 Start Site" and indicates the position number of the 5' nucleoside targeted by the antisense oligonucleotide in the human CALM2 mRNA sequence. "SEQ1 END" means "SEQ ID NO: 1 Stop Site" and indicates the position number of the 3' nucleoside targeted by the antisense oligonucleotide in the human CALM2 mRNA sequence. "SEQ2 START" refers to the "SEQ ID NO: 2 Start Site" and indicates the position number of the 5' nucleoside targeted by the antisense oligonucleotide in the human CALM2 pre-mRNA sequence. "SEQ2 END" refers to the "SEQ ID NO: 2 Stop Site" and indicates the position number of the 3' nucleoside targeted by the antisense oligonucleotide in the human CALM2 pre-mRNA sequence. Each antisense oligonucleotide targets either human CALM2 mRNA designated herein as Sequence ID No. 1, or / or human CALM2 pre-mRNA designated herein as Sequence ID No. 2. "SEQ No." indicates the sequence number, and "BASE SEQUENCE" indicates the nucleic acid base sequence of the antisense oligonucleotide.

[0143] [Table 2]

[0144] [Evaluation Example 1] Antisense suppression of human CALM2 in HepG2 cells HepG2 cells were seeded at a density of 10,000 cells / well in a 96-well plate. Approximately 24 hours later, antisense oligonucleotides (manufactured in Manufacturing Example 1) were added using Lipofectamine® 3000 (Thermo Fisher Scientific) to a final concentration of 50 nM (transfection). After 24 hours, RNA was isolated from the cells using the RNeasy Mini Kit (QIAGEN), and then reverse transcribed using PrimeScript® RT Master Mix (Perfect Real Time) (Takara Bio) to produce cDNA. The CALM2 gene expression level was measured using the produced cDNA by quantitative real-time PCR with TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the amount of mRNA of the housekeeping gene Peptidylprolyl isomerase A [PPIA] was also quantified, and the amount of CALM2 mRNA relative to the amount of PPIA mRNA was evaluated as the CALM2 expression level. The results are shown in Table 3 as percentage expression of CALM2 compared to untreated control cells.

[0145] [Table 3]

[0146] [Evaluation Example 2] Dose-dependent antisense suppression of human CALM1 / CALM2 / CALM3 in HepG2 cells HepG2 cells were seeded at a density of 10,000 cells / well in a 96-well plate. Approximately 24 hours later, P19710002 was added using Lipofectamine® 3000 (Thermo Fisher Scientific) to achieve final concentrations of 0.1 nM, 1 nM, 10 nM, and 100 nM (transfection). After 24 hours, RNA was isolated from the cells using the RNeasy Mini Kit (QIAGEN), and then reverse transcribed using PrimeScript® RT Master Mix (Perfect Real Time) (Takara Bio) to produce cDNA. Using the produced cDNA, the expression levels of the CALM1, CALM2, and CALM3 genes were measured by quantitative real-time PCR using TaqMan® Gene Expression Assays (Thermo Fisher Scientific). Real-time PCR was used to simultaneously quantify the mRNA levels of the housekeeping gene PPIA. The mRNA levels of CALM1, CALM2, and CALM3 relative to the PPIA mRNA levels were evaluated as the expression levels of CALM1, CALM2, and CALM3. The results are shown in Figure 1 as the percentage expression of CALM2 relative to untreated control cells (control).

[0147] As is clear from Figure 1, P19710002 suppressed CALM2 expression in a dose-dependent manner. On the other hand, it was shown that it did not suppress the expression of its family genes, CALM1 and CALM3.

[0148] [Evaluation Example 3] Antisense suppression of mouse CALM2 in 3T3-L1 cells 3T3-L1 cells were seeded at a density of 10,000 cells / well in a 96-well plate. After approximately 24 hours, P19710002 was added to the cells to achieve final concentrations of 0.1 nM, 1 nM, 10 nM, and 100 nM (Free-Uptake). Five days later, RNA was isolated from the cells using the RNeasy Mini Kit (QIAGEN), and then reverse-transcribed to produce cDNA using PrimeScript® RT Master Mix (Perfect Real Time) (Takara Bio). Using the produced cDNA, the mouse CALM2 gene expression level was measured by quantitative real-time PCR using TaqMan® Gene Expression Assays (Thermo Fisher Scientific). Real-time PCR also quantified the mRNA amount of the housekeeping gene PPIA, and the CALM2 mRNA amount relative to the PPIA mRNA amount was evaluated as the CALM2 expression level. The results are shown in Table 4 as the percentage expression of CALM2 compared to untreated control cells. In Table 4, "concentration" refers to the concentration of P19710002.

[0149] [Table 4]

[0150] [Reference Example 1] Establishment of iPS cells derived from LQT15 patients Blood was collected from LQT15 patients in whom the CALM2 gene mutation (c.293A >G, p. N98S) had been identified, and LQT15 patient-derived iPS cells were generated in the same manner as in Non-Patent Document 4.

[0151] [Reference Example 2] Generation of differentiated cardiomyocytes from LQT15 patient-derived iPS cells iPS cells established in Reference Example 1 were differentiated into cardiomyocytes using a two-dimensional differentiation induction method called the GiWi method (Lian et al, Nat Protoc. 2013 Jan;8 (1): 162-75). iPS cells cultured on plates coated with Matrigel Growth Factor Reduced® diluted in IMDM (Iscove's Modified Dulbecco's Media) medium were cultured for 1 day in a medium (insulin-free) containing RPMI 1640 supplemented with B-27®, to which CHIR99021[6-{2-[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-pyrimidine-2-ylamino]-ethylamino}-nicotinonitrile] was added. From day 3 after the start of differentiation, IWP-2[[N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide] was added and cultured for 2 days. From day 7 after the start of differentiation, the cells were cultured in a medium containing RPMI 1640 supplemented with B-27(registered trademark) and insulin, and beating cardiomyocytes appeared from day 12 onwards.

[0152] [Evaluation Example 4] Functional evaluation of LQT15 patient-derived human iPS cell-differentiated cardiomyocytes 50,000 cardiomyocytes differentiated in Reference Example 2 were suspended in 4 μL of culture medium and seeded onto a 96-well plate coated with Matrigel Growth Factor Reduced® diluted in 4 μL of IMDM (Iscove's Modified Dulbecco's Media) to create sheet-like iPS cell-derived cardiomyocytes. After approximately 24 hours, P19710002 was added to achieve a final concentration of 1 μM (Free-uptake). After approximately 120 hours, Fluovolt (Thermo Fisher Scientific), a membrane potential-sensitive dye, was loaded at 37°C for 30 minutes, and the action potential was measured using an imaging system (microscope: Nikon, high-speed optical measurement system: BrainVision) at 37°C and 0.5 Hz pacing. The results are shown in Figures 2 and 3. The results for the case without P19710002 were also measured simultaneously and are shown in Figures 2 and 3 as ASO(-). In Figures 2 and 3, "ms" indicates milliseconds.

[0153] As is clear from Figures 2 and 3, P19710002, an antisense oligonucleotide of CALM2, has a 90% action potential duration (APD 90 It was suggested that the term be shortened.

[0154] [Evaluation Example 5] Antisense suppression of human CALM2 in human iPS cell-differentiated cardiomyocytes derived from LQT15 patients. Cardiomyocytes differentiated in Reference Example 2 were seeded into a 96-well plate at a density of 200,000 cells per well. After approximately 24 hours, P19710002 was added to achieve a final concentration of 1 μM (Free-uptake). After approximately 120 hours, RNA was extracted from the cells using NucleoSpin RNA Plus XS (Takara Bio), and cDNA was synthesized by reverse transcription using the Transcriptor First Strand cDNA Synthesis Kit (Roche). The expression level of the mouse CALM2 gene was measured using the synthesized cDNA by quantitative real-time PCR using the TaqMan method. In real-time PCR, the mRNA amount of the housekeeping gene GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was also quantified simultaneously, and the CALM2 mRNA amount relative to the GAPDH mRNA amount was evaluated as the CALM2 expression level. The results are shown in Figure 4. Note that ASO(-) indicates the case where P19710002 was not added.

[0155] [Manufacturing Example 2] The antisense oligonucleotides listed in Table 5 (compounds represented by the chemical structure corresponding to the compound number) were prepared using the nucleic acid automated synthesizer nS-8II (Gene Design Co., Ltd.). In the sequence notation in Table 5, "C16-" indicates that the portion obtained by removing a hydrogen atom from the hydroxyl group at the 5' end is shown in formula (I) below. [ka] (In the formula, * indicates the binding position with the oligonucleotide.) This means that it is bound to the group represented by . Note that the nucleic acid sequence of P19710036 is Sequence ID No. 4.

[0156] [Table 5]

[0157] [Evaluation Example 6] CALM2 Antisense Suppression in Mice C57BL / 6J mice (male, 6 weeks old, Charles River Pharmaceuticals Japan) were intravenously administered P19710002 and P19710036 dissolved in physiological saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) at doses of 1.9 μmol / kg or 9.5 μmol / kg per mouse, equivalent to the amount of antisense oligonucleotide. P19710036 was also administered intravenously at 0.4 μmol / kg. As a control, only physiological saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) was administered. Five days after administration, heart, liver, and kidney tissues were collected under isoflurane anesthesia. RNA was isolated from each organ using the RNeasy Mini Kit (Qiagen), and cDNA was obtained using PrimeScript® RT Master Mix (Perfect Real Time) (Takara Bio). Using the prepared cDNA, the mouse CALM2 gene expression level was measured by quantitative real-time PCR using TaqMan® Gene Expression Assays (Thermo Fisher Scientific). Real-time PCR was used to simultaneously quantify the mRNA level of the housekeeping gene PPIA, and the ratio of CALM2 mRNA to PPIA mRNA was used to evaluate the CALM2 expression level. The results are shown as percentage expression of CALM2 relative to the untreated control group (control) in Figures 5 (heart), 6 (liver), and 7 (kidney).

[0158] As is clear from Figures 5-7, P19710002 and P19710036 suppressed CALM2 expression in the heart, liver, and kidneys.

[0159] [Manufacturing Example 3] The antisense oligonucleotides listed in Table 6 (compounds represented by chemical structures corresponding to compound numbers) were prepared using the nS-8II automated nucleic acid synthesizer (manufactured by Gene Design).

[0160] [Table 6]

[0161] Table 7 shows the target location in the human CALM2 mRNA or pre-mRNA sequence for each antisense oligonucleotide shown in Table 6, as well as the corresponding sequence number and nucleic acid sequence for each antisense oligonucleotide. Note that the notation in Table 7 is the same as in Table 2.

[0162] [Table 7]

[0163] [Evaluation Example 7] Antisense suppression of human CALM2 in HepG2 cells Using the same evaluation method as in Evaluation Example 1, the final concentration of the antisense oligonucleotide (manufactured in Manufacturing Example 3) was set to 30 nM, and the amount of CALM2 mRNA relative to the amount of PPIA mRNA was evaluated as the CALM2 expression level. The results are shown in Table 8 as the percentage expression of CALM2 compared to untreated control cells.

[0164] [Table 8]

[0165] [Evaluation Example 8] Antisense suppression of human CALM2 in HepG2 cells Using the same evaluation method as in Evaluation Example 1, antisense oligonucleotides (manufactured in Manufacturing Examples 1 and 3) were tested at various concentrations. The final concentrations were set to 0.3 nM, 1 nM, and 30 nM, and the amount of CALM2 mRNA relative to the amount of PPIA mRNA was evaluated as the CALM2 expression level. The results were calculated as the concentration at which the RNA transcript level in untreated control cells reached 50% (IC50 value), and are shown in Table 9.

[0166] [Table 9]

[0167] [Evaluation Example 9] CALM2 antisense suppression in mice The same evaluation method as in Evaluation Example 6 was used. P19710002, P19710025, P19710031, and P19710034 were intravenously administered so that the dosage per mouse个体 was 9.5 μmol / kg in terms of the amount of antisense oligonucleotide. As a control, only physiological saline (Otsuka Shoyo Chuzai, Otsuka Pharmaceutical Factory) was administered. Five days after the administration, the amount of CALM2 mRNA relative to the amount of PPIA mRNA in the heart tissue was evaluated as the expression level of CALM2. The results are shown in Fig. 8 as the percentage expression of CALM2 relative to the untreated control group (control).

[0168] As is clear from Fig. 8, P19710002, P19710025, P19710031, and P19710034 suppressed the expression of CALM2 in the heart.

[0169] [Production Example 4] The antisense oligonucleotides (compounds represented by the chemical structures corresponding to the compound numbers) described in Table 10 were prepared using a nucleic acid automatic synthesizer nS-8II (manufactured by Gene Design). [[ID=1十一]]

[0170] [Table 10]

[0171] [[ID=二十二]]The positions targeted by each antisense oligonucleotide shown in Table 10 in the sequence of human CALM2 mRNA or pre-mRNA, the sequence numbers and nucleotide sequences corresponding to each antisense oligonucleotide are shown in Table 11. A "-" (hyphen) indicates that the antisense oligonucleotide does not target the sequence of its mRNA or pre-mRNA with 100% complementarity. Otherwise, the notations in Table 11 are the same as in Table 2.

[0172] [[]END]] [Table 11] [[]END]]

[0173] [Evaluation Example 10] Antisense suppression of human CALM2 in HepG2 cells Using the same evaluation method as in Evaluation Example 1, the final concentration of the antisense oligonucleotide (manufactured in Manufacturing Example 4) was set to 10 nM, and the amount of CALM2 mRNA relative to the amount of PPIA mRNA was evaluated as the CALM2 expression level. The results are shown in Table 12 as the percentage expression of CALM2 compared to untreated control cells.

[0174] [Table 12]

[0175] [Manufacturing Example 5] The antisense oligonucleotides listed in Table 13 (compounds represented by the chemical structure corresponding to the compound number) were prepared using the nucleic acid automated synthesizer nS-8II (Gene Design Co., Ltd.). In the sequence notation in Table 13, "Toc-TEG-" represents the portion obtained by removing a hydrogen atom from the hydroxyl group at the 5' end, as shown in formula (II) below. [ka] (In the formula, * indicates the binding position with the oligonucleotide, the tocopherol portion is the DL isomer, and the carbon atom to which the hydroxymethyl group is bound is a mixture of the (R) and (S) isomers.) This means that it is bound to the group represented by (in the formula). Note that the nucleic acid sequence of P20710091 is Sequence ID No. 4.

[0176] [Table 13]

[0177] [Evaluation Example 11] CALM2 antisense suppression in mice The same evaluation method as in Evaluation Example 6 was used. P19710002 and P20710091 were administered intravenously to mice at a dose equivalent to 1.9 μmol / kg in terms of antisense oligonucleotide amount. As a control, only physiological saline (Otsuka Saline Injection, Otsuka Pharmaceutical Co., Ltd.) was administered. The amount of CALM2 mRNA relative to the amount of PPIA mRNA in cardiac tissue at 5, 10, and 20 days after administration was evaluated as the CALM2 expression level. The results are shown in Figure 9 as the percentage expression of CALM2 compared to the untreated control group (control).

[0178] As is clear from Figure 9, P19710002 and P20710091 suppressed CALM2 expression in the heart.

[0179] [Evaluation Example 12] CALM2 antisense suppression in mice The same evaluation method as in Evaluation Example 6 was used. P19710068, P19710069, and P19710071 were administered intravenously to mice at a dose of 1.9 μmol / kg or 9.5 μmol / kg in terms of antisense oligonucleotide amount. As a control, only physiological saline (Otsuka Saline Injection, Otsuka Pharmaceutical Co., Ltd.) was administered. Five days after administration, the amount of CALM2 mRNA relative to the amount of PPIA mRNA in cardiac, hepatic, and renal tissue was evaluated as the CALM2 expression level. The results are shown as percentage expression of CALM2 compared to the untreated control group (control) in Figures 10 (heart), 11 (liver), and 12 (kidney).

[0180] As is clear from Figures 10-12, P19710068, P19710069, and P19710071 suppressed CALM2 expression in the heart, liver, and kidneys.

[0181] All publications or any part thereof cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0182] Compounds containing modified oligonucleotides in this application, or pharmacologically acceptable salts thereof, can inhibit the expression of the CALM2 gene and may therefore be useful in the treatment, prevention, and / or improvement of diseases and conditions in which inhibition of CALM2 gene expression is effective, particularly congenital long QT syndrome and calmodulinopathy.

[0183] This application is based on Japanese Patent Applications No. 2020-036715 and No. 2020-138019, which are incorporated herein by reference.

Claims

1. A compound comprising a modified oligonucleotide having a continuous nucleoside length of 8 to 80, or a pharmacologically acceptable salt thereof, wherein the modified oligonucleotide has a nucleic acid base sequence comprising at least eight continuous nucleic acid bases included in any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 73, or a pharmacologically acceptable salt thereof.

2. The compound according to claim 1 or a pharmacoposly acceptable salt thereof, wherein the modified oligonucleotide has a nucleic acid base sequence comprising any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 73.

3. The compound according to claim 1 or 2, or a pharmacokinetically acceptable salt thereof, wherein the modified oligonucleotide has a nucleic acid base sequence consisting of any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 73.

4. Nucleic acid base position numbers of Sequence ID No. 1: 102-117, 160-175, 183-205, 212-227, 322-337, 365-405, 411-434, 464-494, 506-521, 606-635, 636-651, 692-707, 715-749, 754-801, 829-857, 862-934, 951-970, 995-1010, 1006-1021, 1036-1051, 1062-1077, 1081-11 A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 04, 1138-1166, 1188-1203, and 1239-1254, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of SEQ ID NO:

1.

5. A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by the positions 102-117, 160-175, 183-198, 212-227, 322-337, 365-380, 387-402, 479-494, 620-635, 715-730, 862-877, 896-934, 995-1010, 1062-1077, 1089-1104, 1140-1166 and 1239-1254 of SEQ ID NO: 1, wherein the modified oligonucleotide has at least 80% complementarity to at least a portion of the acid base sequence of SEQ ID NO:

1.

6. A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of the nucleic acid base sequence represented by position numbers 896 to 934, preferably 907 to 922, of the nucleic acid base sequence of Sequence ID No. 1, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of Sequence ID No.

1.

7. Nucleic acid base position numbers of Sequence ID No. 2: 102-117, 160-175, 1624-1639, 1661-1676, 3355-3370, 5859-5881, 13959-13974, 14069-14084, 14228-14278, 14284-14307, 14764-14794, 14806-14821, 15824-15869, 15910-15925, 15933-15967, 15972-16019, 16047-16075, 16080-16152, 16169-16188, 16213-16239 A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 16254-16269, 16280-16295, 16299-16322, 16356-16384, 16406-16421 and 16457-16472, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of SEQ ID NO:

2.

8. Nucleic acid base position numbers of Sequence ID No. 2: 102-117, 160-175, 5859-5874, 13959-13974, 14069-14084, 14238-14253, 14260-14275, 14779-14794, 15838-15853, 15933-15948, 16080-16095, 16114-16152, 16213-16228, 16280-16295, 16307-16322, 16 A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 358 to 16384 and 16457 to 16472, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of SEQ ID NO:

2.

9. A compound or a pharmacologically acceptable salt thereof comprising a modified oligonucleotide having a length of 8 to 80 consecutive nucleosides that is complementary to at least a portion of the nucleic acid base sequence represented by the nucleic acid base positions 16114 to 16152, preferably 16125 to 16140, of SEQ ID NO: 2, wherein the modified oligonucleotide has at least 80% complementarity with at least a portion of the acid base sequence of SEQ ID NO:

2.

10. The modified oligonucleotide comprises a phosphorothioate bond, the compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof.

11. The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, wherein the modified oligonucleotide comprises at least one selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides.

12. The aforementioned 2'-4'-bridged nucleoside is LNA, ENA, cEt, BNA ＮＣ The compound according to claim 11 or a pharmaceutically acceptable salt thereof, which is at least one selected from the group consisting of AmNA, scpBNA, and GuNA.

13. The compound according to claim 12 or a pharmaceutically acceptable salt thereof, wherein the 2'-4'-bridged nucleoside is LNA.

14. The compound according to any one of claims 11 to 13 or a pharmaceutically acceptable salt thereof, wherein the 2'-modified nucleoside is at least one selected from the group consisting of 2'-O-MCE nucleoside, 2'-O-MOE nucleoside, 2'-O-NMA nucleoside, and 2'-O-Me nucleoside.

15. The compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein the 2'-modified nucleoside is at least one selected from the group consisting of 2'-O-MCE nucleoside and 2'-O-MOE nucleoside.

16. The compound according to any one of claims 1 to 15 or a pharmacoposly acceptable salt thereof, wherein the modified oligonucleotide comprises 5-methylcytosine.

17. The modified oligonucleotide includes a gap segment, a 5' wing segment, and a 3' wing segment. The gap segment contains at least two deoxyribonucleosides, and the 5' and 3' ends of the gap segment are deoxyribonucleosides. The nucleoside at the 3' end of the 5' wing segment is a sugar-modified nucleoside and is linked to the 5' end of the gap segment. The nucleoside at the 5' end of the 3' wing segment is a sugar-modified nucleoside and is linked to the 3' end of the gap segment. A compound according to any one of claims 1 to 16 or a pharmacokinetically acceptable salt thereof.

18. The gap segment consists of 5 to 30 deoxyribonucleosides. The 5' wing segment and the 3' wing segment each independently consist of 1 to 10 sugar-modified nucleosides independently selected from the group consisting of LNA, 2'-O-MCE nucleosides, and 2'-O-MOE nucleosides, wherein each wing segment contains at least one phosphorothioate bond. The compound according to claim 17 or a pharmacoposly acceptable salt thereof, wherein the cytosine in the gap segment and each wing segment is 5-methylated.

19. The gap segment consists of 8 to 12 deoxyribonucleosides. The 5'-wing segment and the 3'-wing segment each consist of 2 to 5 sugar-modified nucleosides independently selected from the group consisting of LNA and 2'-O-MCE nucleosides. The gap segment contains at least one phosphorothioate bond. The compound according to claim 18 or a pharmaceutically acceptable salt thereof.

20. The compound according to claim 19, wherein the 5' wing segment and the 3' wing segment are each independently selected from the group consisting of LL, LLL, VLL, LVL, LLV, LVV, VLV, VVL, VVLL, VLVVL, LVLV, LLVLL, LVLL, LLVL, LLLV, LVVV, VLVV, VVLV, and VVVL, where L represents LNA and V represents a 2'-O-MCE nucleoside.

21. The compound according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, wherein the modified oligonucleotide has a continuous nucleoside length of 11 to 50, preferably 15 to 25.

22. The compound according to any one of claims 1 to 21 or a pharmacoposly acceptable salt thereof, wherein the modified oligonucleotide is an antisense oligonucleotide.

23. The compound comprising the modified oligonucleotide comprises a prodrug portion, as described in any one of claims 1 to 22, or a pharmacokinetically acceptable salt thereof.

24. The compound comprising the modified oligonucleotide comprises a functional molecule, as described in any one of claims 1 to 23, or a pharmacokinetically acceptable salt thereof.

25. The compound according to claim 24 or a pharmacoposly acceptable salt thereof, wherein the functional molecule is a group derived from a molecule having the function of delivering modified oligonucleotides to a target site.

26. The compound according to claim 24 or 25 or a pharmaceutically acceptable salt thereof, wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and derivatives thereof.

27. The compound according to any one of claims 24 to 26 or a pharmacoposly acceptable salt thereof, wherein the functional molecule is a lipid selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, and C5-30 unsaturated fatty acids.

28. The compound according to any one of claims 1 to 23, or a pharmacokinetically acceptable salt thereof, wherein the compound comprises the modified oligonucleotide.

29. The salt according to any one of claims 1 to 28, wherein the pharmacologically acceptable salt is a sodium salt.

30. A pharmaceutical product comprising a compound or a pharmacokinetically acceptable salt described in any one of claims 1 to 29.

31. A pharmaceutical agent for treating, preventing and / or improving a disease or condition in which the expression of the CALM2 gene is effectively inhibited, comprising a compound or a pharmacologically acceptable salt described in any one of claims 1 to 29.

32. An inhibitor of CALM2 gene expression comprising a compound or a pharmacologically acceptable salt according to any one of claims 1 to 29.

33. A pharmaceutical agent for treating, preventing, and / or improving congenital QT prolongation syndrome, comprising a compound or a pharmacologically acceptable salt described in any one of claims 1 to 29.

34. A pharmaceutical agent for treating, preventing, and / or improving calmodulinopathy, comprising a compound or a pharmacoposly acceptable salt according to any one of claims 1 to 29.

35. A method for treating, preventing and / or improving a disease or condition in which an inhibitory effect on the expression of the CALM2 gene is effective, comprising the step of administering an effective amount of a compound or a pharmacologically acceptable salt described in any one of claims 1 to 29 to a subject in need thereof.

36. A method for inhibiting the expression of the CALM2 gene, comprising the step of administering an effective amount of a compound or a pharmacologically acceptable salt described in any one of claims 1 to 29 to a subject in need thereof.

37. A method for treating, preventing, and / or improving congenital long QT syndrome, comprising the step of administering an effective amount of a compound or a pharmacologically acceptable salt described in any one of claims 1 to 29 to a subject in need thereof.

38. A method for treating, preventing, and / or improving calmodulinopathy, comprising the step of administering an effective amount of a compound or a pharmacologically acceptable salt described in any one of claims 1 to 29 to a subject in need thereof.

39. A compound according to any one of claims 1 to 29, for use as a pharmaceutical.

40. A compound according to any one of claims 1 to 29, for use in the treatment, prevention and / or improvement of a disease or condition in which the expression inhibition of the CALM2 gene is effective.

41. A compound according to any one of claims 1 to 29, for use in inhibiting the expression of the CALM2 gene.

42. A compound according to any one of claims 1 to 29, for use in the treatment, prevention, and / or improvement of congenital long QT syndrome.

43. A compound according to any one of claims 1 to 29, for use in the treatment, prevention, and / or improvement of calmodulinopathy.

44. Use of a compound or pharmacoposly acceptable salt according to any one of claims 1 to 29 in the manufacture of a pharmaceutical product for treating, preventing and / or improving a disease or condition in which the expression inhibition of the CALM2 gene is effective.

45. Use of a compound or a pharmacologically acceptable salt according to any one of claims 1 to 29 in the production of a CALM2 gene expression inhibitor.

46. Use of a compound or pharmacologically acceptable salt according to any one of claims 1 to 29 in the manufacture of a medicament for the treatment, prevention and / or improvement of congenital long QT syndrome.

47. Use of a compound or pharmacoposly acceptable salt according to any one of claims 1 to 29 in the manufacture of a medicament for the treatment, prevention and / or improvement of calmodulinopathy.

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