Prophylactic or therapeutic agent for benign adult-type familial myoclonic epilepsy

Abstract

The preventive or therapeutic agent for benign adult familial myoclonic epilepsy (BAFME) according to the present invention contains an antisense oligonucleotide that comprises at least one of the base sequences of SEQ ID NOS: 1 to 4. In the antisense oligonucleotide, an oligonucleotide comprising at least one of the base sequences of SEQ ID NOS: 1 to 4 may contain at least one RNase H inactive nucleotide analog. The RNase H inactive nucleotide analog may be β-D-oxy-L-LNA, β-D-ENA, R cEt and 2'-OMe-nucleotide analogs and / or MOE-nucleotide analog.

Description

[Technical Field]

[0001] The present invention relates to a preventive or therapeutic agent for benign adult familial myoclonus epilepsy, and more specifically, to a preventive or therapeutic agent for benign adult familial myoclonus epilepsy comprising an antisense oligonucleotide. [Background technology]

[0002] Benign adult familial myoclonic epilepsy (BAFME) is a type of progressive myoclonic epilepsy, characterized primarily by myoclonus (involuntary movements of a part of the body) and epileptic seizures (generalized seizures, seizures of loss of consciousness).

[0003] The initial symptoms of progressive myoclonic epilepsy are generalized seizures or myoclonus in the whole or part of the body. However, because consciousness is preserved during myoclonus alone, it can sometimes be overlooked. As the condition progresses, symptoms such as unsteadiness while walking, memory loss, dementia-like symptoms, and psychiatric symptoms may appear. Eventually, irreversible abnormalities occur in specific areas of the brain.

[0004] BAFME (Beneficial Aortic Fibre Syndrome) is traditionally considered "benign" and "non-progressive" because it develops in adulthood, initially presents with mild symptoms, and progresses slowly. However, recent research has revealed that it is progressive, with symptoms worsening particularly with age, and a decline in activities of daily living (ADL). Epidemiologically, it is more common in Japan and China, with 60 families reported in Japan. The incidence rate is 1 in 35,000.

[0005] BAFME is also known as Familial Cortical Myoclonic Tremor Associated with Epilepsy (FCMTE) or Familial Adult Myoclonic Epilepsy (FAME). BAFME is classified into seven types, from type 1 to type 7, based on age of onset, disease progression, and linkage analysis. Hereinafter, these will be referred to as BAFME1, BAFME2, ..., BAFME7.

[0006] Ishiura et al. (Non-Patent Literature 1) identified, using data from Japanese patients, that the cause of BAFME1, BAFME6, and BAFME7 is the elongation of a 5-base repeat sequence in the non-coding region within the introns of the genes SAMD12, TNRC6A, and PAPGEF2, respectively, located on different autosomes. Cen et al. (Non-Patent Literature 2) confirmed this using data from Chinese patients.

[0007] Table 1 below summarizes the findings for BAFME1, BAFME6, and BAFME7, including the chromosomal location of the causative gene, the name of the causative gene, the number of patients (number of families in parentheses), the repetitive sequence listed in the human genome reference sequence Hg19, and the composition of the extended repetitive sequence in the Japanese patient genome. The subscript numbers in Table 1 for the reference sequence and extended repetitive sequence indicate the number of repeats of the repetitive sequence in parentheses. The subscript "exp" in Table 1 for the extended repetitive sequence indicates that the 5-base repeat sequence in parentheses has been repeated many times and extended. The number of repeats varies from patient to patient and from gene locus to gene. For example, in BAFME1, repeat counts ranging from 440 to 3680 were observed. It is also known that patients with a higher number of repeats tend to develop epilepsy and myoclonus at a younger age.

[0008] [Table 1]

[0009] In BAFME1, RNA aggregates are detected in cortical neurons and Purkinje cells by fluorescence in situ hybridization (hereinafter referred to as "FISH") using a probe for the repeat sequence TTTCA (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Ishiura, H. et al., Nature Genetics, 50: 581-590. (2018) [Non-Patent Document 2] Cen, Z. et al., Brain, 141: 2280-2288 (2018) [Overview of the project] [Problems that the invention aims to solve]

[0011] There is still no established causal therapy for BAFME, and treatment is currently symptomatic, involving the continuous use of medications to alleviate or eliminate epileptic seizures and myoclonus.

[0012] The object of the present invention is to provide a preventive or therapeutic agent for BAFME as a causal therapy for BAFME. [Means for solving the problem]

[0013] To achieve the above objective, the inventors conducted research and discovered that a disease model system using iPS cells derived from patients with BAFME1 disease is suitable for screening ASOs with high RNA aggregate removal activity, and completed the present invention using this assay system.

[0014] The present invention provides a preventive or therapeutic agent for BAFME. The preventive or therapeutic agent of the present invention comprises an ASO containing at least one nucleotide sequence of SEQ ID NOs: 1 to 4.

[0015] In the prophylactic or therapeutic agent of the present invention, the oligonucleotide consisting of at least one nucleotide sequence of SEQ ID NO: 1 to 4 may contain at least one RNase H-inactive nucleotide analog.

[0016] In the prophylactic or therapeutic agent of the present invention, the RNase H-inactive nucleotide analog may be at least one nucleotide analog selected from nucleotide analogs having a modified 2'-position of ribose and nucleotide analogs having a crosslinking modification between the 2'-position and the 4'-position of ribose.

[0017] In the prophylactic or therapeutic agent of the present invention, the nucleotide analog having a modified 2'-position of ribose may be at least one nucleotide analog selected from the group consisting of 2'-OMe-nucleotide analogs and MOE-nucleotide analogs.

[0018] In the prophylactic or therapeutic agent of the present invention, the nucleotide analog having a crosslinking modification between the 2'-position and the 4'-position of ribose may be at least one nucleotide analog selected from the group consisting of β-D-oxy-L-LNA, β-D-ENA, and R-type cEt.

[0019] In the prophylactic or therapeutic agent of the present invention, at least two adjacent nucleotides of the oligonucleotide consisting of any one of the nucleotide sequences of SEQ ID NO: 1 to 4 among the ASOs may be linked by a modified internucleoside linkage.

[0020] In the prophylactic or therapeutic agent of the present invention, all adjacent nucleotides of the oligonucleotide consisting of any one of the nucleotide sequences of SEQ ID NO: 1 to 4 among the ASOs may be linked by a modified internucleoside linkage.

[0021] In the prophylactic or therapeutic agent of the present invention, the modified internucleoside linkage may be at least one linkage selected from the group consisting of phosphorothioate linkage, phosphorodithioate linkage, and boranophosphate linkage.

[0022] In the preventive or therapeutic agent of the present invention, the oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4 among the ASOs may be in the RNase H active form.

[0023] In the preventive or therapeutic agent of the present invention, the RNase H-activated oligonucleotide may be an oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4, in which the 1st to 5th nucleotides from the 5' end and the 16th to 20th nucleotides from the 5' end are at least two RNase H-inactive nucleotide analogs.

[0024] In the preventive or therapeutic agent of the present invention, the RNase H-activated oligonucleotide may be an oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4, in which the 1st to 5th nucleotides from the 5' end and the 16th to 20th nucleotides from the 5' end are all of the same type of RNase H-inactive nucleotide analog.

[0025] In the preventive or therapeutic agent of the present invention, the RNase H-activated oligonucleotide is an oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4, wherein the 1st to 5th nucleotides from the 5' end and the 16th to 20th nucleotides from the 5' end are RNase H-inactive nucleotide analogs, and the 6th to 15th nucleotides from the 5' end may be an RNase H-activated nucleotide.

[0026] In the preventive or therapeutic agent of the present invention, at least one of the RNase H-inactive nucleotide analogs of the ASO containing the RNase H-active oligonucleotide may be β-D-ENA.

[0027] In the preventive or therapeutic agent of the present invention, all of the RNase H inactive nucleotide analogs of the ASO containing the RNase H active oligonucleotide are β-D-ENA, and all of the modified nucleoside bonds may be phosphorothioate bonds.

[0028] In the preventive or therapeutic agent of the present invention, all of the RNase H inactive nucleotide analogs of the ASO containing the RNase H active oligonucleotide may be β-D-ENA, all of the RNase H active nucleotides may be deoxyribonucleotides, and all of the nucleoside bonds of the RNase H active oligonucleotide may be phosphorothioate bonds.

[0029] In the preventive or therapeutic agent of the present invention, the ASO, which includes an oligonucleotide consisting of the base sequence of SEQ ID NO: 1, 2, or 3, may be in an RNase H-inactive form.

[0030] In the preventive or therapeutic agent of the present invention, all nucleotides containing thymine in the oligonucleotide consisting of the base sequence of SEQ ID NO: 1 or 2 are nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked, and the nucleotides containing the 5th, 10th, 15th and 20th adenines from the 5' end of the oligonucleotide consisting of the base sequence of SEQ ID NO: 1 or 2 are nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked, and all other nucleotides may be nucleotide analogs in which the 2' position of ribose is modified.

[0031] In the preventive or therapeutic agent of the present invention, at least one nucleotide analog in which the 2' and 4' positions of the ribose of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 or 2 are cross-linked is β-D-ENA, and at least one nucleotide analog in which the 2' position of the ribose is modified is a 2'-OMe-nucleotide analog.

[0032] In the preventive or therapeutic agent of the present invention, all nucleotide analogs in which the 2' and 4' positions of the ribose of the oligonucleotide consisting of the base sequence of SEQ ID NO: 1 or 2 are cross-linked are β-D-ENA, and all nucleotide analogs in which the 2' position of the ribose is modified are 2'-OMe-nucleotide analogs.

[0033] In the preventive or therapeutic agent of the present invention, all of the oligonucleotides comprising the base sequence of SEQ ID NO: 3, and the nucleotides containing thymine, are nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked, and all other nucleotides may be nucleotide analogs in which the 2' position of ribose is modified.

[0034] In the preventive or therapeutic agent of the present invention, at least one nucleotide analog in which the 2' and 4' positions of ribose are cross-linked is β-D-ENA, and at least one nucleotide analog in which the 2' position of ribose is modified is a 2'-OMe-nucleotide analog.

[0035] In the preventive or therapeutic agent of the present invention, all nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked are β-D-ENA, and the nucleotide analog in which the 2' position of ribose is modified may be a 2'-OMe-nucleotide analog.

[0036] In the preventive or therapeutic agent of the present invention, all nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked are β-D-ENA, the nucleotide analog in which the 2' position of ribose is modified is a 2'-OMe-nucleotide analog, and all nucleoside bonds of the RNase H-inactive oligonucleotide may be phosphorothioate bonds.

[0037] The present invention provides an assay system for a BAFME1 type disease preventive or therapeutic agent, which includes undifferentiated iPS cells established from a human suffering from BAFME1 type disease. [Brief explanation of the drawing]

[0038] [Figure 1] A schematic diagram showing the relative positional relationship of the target site of the ASO in the SAMD12 gene pre-mamma and the classification of the ASO into RNase H active or inactive forms. (See Table 3 for details of the ASO.) [Figure 2A] This graph shows the results of an experiment in which the ASO of the present invention and a negative control ASO (Control Gapmer) were introduced into undifferentiated iPS cells (HPS3905) derived from BAFME patients by lipofection. The height of the bar graph represents the percentage of iPS cell nuclei 48 hours after lipofection. [Figure 2B] This graph shows the results of an experiment in which the ASO of the present invention and a negative control ASO (Control Gapmer) were introduced into undifferentiated iPS cells (HPS3905) derived from BAFME patients by lipofection. The height of the bar graph represents the percentage of iPS cell nuclei in which RNA aggregates were detected by a Cy3 probe 48 hours after lipofection. [Figure 2C] This graph shows the results of an experiment in which the ASO of the present invention and a negative control ASO (Control Gapmer) were introduced into undifferentiated iPS cells (HPS3905) derived from BAFME patients by lipofection. The height of the bar graph represents the percentage of iPS cell nuclei in which RNA aggregates were detected by the Cy5 probe 48 hours after lipofection. [Figure 3A] A schematic diagram showing the general procedure for creating the cerebral organoid according to the present invention. [Figure 3B] Multiplex immunohistochemical fluorescence microscopy images of cerebral organoid sections cultured for 70 days using HPS3905 iPS cells derived from a BAFME patient. The scale bar represents 50 μm. [Figure 3C]Fluorescence microscopy images of cerebral organoid sections cultured for 70 days using HPS3905 iPS cells derived from a BAFME patient, after FISH using a Cy3 probe and DAPI counterstaining. The scale bar represents 10 μm. [Figure 3D] This graph shows the percentage of cell nuclei in which RNA aggregates were detected using a Cy3 probe, among the cell nuclei of cerebral organoid sections cultured for 70 days using iPS cell line HPS3905 (BAFME1) derived from a BAFME patient and iPS cell line 201B7 (HC) derived from a healthy individual. [Figure 3E] Graphs showing the effect of the ASO of this invention (ASO-4M, same as 3009-2M) or the negative control ASO (Control ASO, same as Control Mixmer) on RNA aggregate formation in cerebral organoids created from iPS cells derived from BAFME patients. The graphs show the percentage of cell nuclei in which RNA aggregates were detected with a Cy3 probe among the cell nuclei of cerebral organoids cultured for 5 months from BAFME patient-derived iPS cell line HPS3905, after 1 week of lipofection administration of ASO-4M or Control ASO and fixation. [Modes for carrying out the invention]

[0039] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME comprising an ASO having at least one nucleotide sequence of SEQ ID NOs: 1 to 4.

[0040] The nucleotide sequences of oligonucleotides described herein are listed as Sequence IDs 1 to 7 in the sequence listing attached to this specification. The correspondence between the Sequence IDs and the nucleotide sequences is shown in Table 2.

[0041] [Table 2]

[0042] In Table 2, Sequence IDs 1 to 7 are the nucleotide sequences of ASO administered to iPS cells in the examples described herein. In the sequence listings attached to this specification, Sequence IDs 1 to 7 are listed as DNA. However, the sugar portion of the nucleoside constituting the oligonucleotide contained in the BAFME prophylactic or therapeutic agent of the present invention is not necessarily limited to deoxyribose, but may also be the sugar portion of a modified nucleotide as described below.

[0043] Of the ASOs administered to iPS cells in the examples herein, 3009-1M and 3009-4G are ASOs complementary to the RNA corresponding to the TTTTA repeat sequence in the fourth intron of the SAMD12 gene. 3009-2M and 3009-5G are ASOs complementary to the RNA corresponding to the TTTCA repeat sequence in the fourth intron of the SAMD12 gene. 3009-3M and 3009-6G are ASOs complementary to both the RNA corresponding to the TTTTA repeat sequence and the RNA corresponding to the TTTCA repeat sequence in the fourth intron of the SAMD12 gene. SAMD12-1M and SAMD12-1G are ASOs complementary to the RNA corresponding to the non-repeatable sequence 5' (upstream) from the TTTTA repeat sequence start site in the fourth intron of the SAMD12 gene. SAMD12-2M and SAMD12-2G, and SAMD12-3M and SAMD12-3G are ASOs complementary to RNAs corresponding to different non-repeatable sequences on the 3' side (downstream) of the TTTTA repeat termination site in the fourth intron of the SAMD12 gene. ASOs whose names contain the letter "M" are RNase H-inactive ASOs, while ASOs whose names contain the letter "G" are RNase H-active ASOs. Table 3 below shows the names, types, structures, and target sites in the fourth intron of the SAMD12 gene of the ASOs used in the examples described herein.

[0044] [Table 3]

[0045] In all ASOs in Table 3, adjacent nucleotides are linked by phosphorothioate bonds, and the bases "A", "G", "C", and "T" represent nucleotides containing adenine, guanine, cytosine, and thymine, respectively. Underlined nucleotides represent β-D-ENA nucleic acid (hereinafter referred to as "ENA"). In RNase H-activated ASOs, ununderlined nucleotides are deoxyribonucleotides. In RNase H-inactive ASOs, ununderlined nucleotides are 2'-OMe-nucleotide analogs. Nucleotide analogs that can be used in the ASOs of the present invention are described in detail below.

[0046] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an ASO, wherein the oligonucleotide comprising at least one base sequence of SEQ ID NOs: 1 to 4 contains at least one RNase H-inactive nucleotide analog.

[0047] A particular nucleotide analog is said to be RNase H inactive if, when a single-stranded nucleic acid consisting of the nucleotide analog, and a hetero-double-stranded nucleic acid formed by the pairing of the single-stranded nucleic acid with a complementary single-stranded RNA, are reacted with RNase H, the phosphodiester bond of the RNA is not hydrolyzed. Conversely, a particular nucleotide analog is said to be RNase H active if, when a single-stranded nucleic acid consisting of the nucleotide analog, and a hetero-double-stranded nucleic acid formed by the pairing of the single-stranded nucleic acid with a complementary single-stranded RNA, are reacted with RNase H, the phosphodiester bond of the RNA is hydrolyzed.

[0048] In this specification, "a particular oligonucleotide containing at least one RNase H-inactive nucleotide analog" means that at least one nucleotide of the oligonucleotide is a nucleotide analog described below. Furthermore, in this specification, "a particular nucleotide being an RNase H-inactive nucleotide" means that the nucleotide has the same base portion, and the sugar portion is the sugar portion of an RNase H-inactive nucleotide analog described below.

[0049] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an ASO, wherein the RNase H inactive nucleotide analog is at least one nucleotide analog selected from the group consisting of a nucleotide analog in which the 2' position of ribose is modified, and a nucleotide analog in which the 2' and 4' positions of ribose are cross-linked.

[0050] The nucleotide analogs in which the 2' position of ribose is modified in the ASO of the above embodiment include, but are not limited to, 2'-F nucleotide analogs, 2'-OMe nucleotide analogs, MOE nucleotide analogs, 2'-(3-hydroxy)propyl nucleotide analogs, and 2'-AP nucleotide analogs.

[0051] The nucleotide analogs in which the 2' position of ribose is modified are, for example, those shown in the following chemical formula (1) [wherein the formula, the substituent X attached to the 2' position of ribose is selected from a fluoro group, a methoxy group, an O-methoxyethyl group, a 2'-(3-hydroxy)propyl group and a 2'-(3-amino)propyl group, and Z and Z * B is independently selected from internucleotide links, R, terminal groups, or protecting groups, B is selected from natural or unnatural nucleotide base parts (nucleic acid bases), and R is hydrogen, a hydroxyl group, C 1-4 Alkyl and C 1-4 It is expressed as [selected from alkoxy groups].

[0052] [ka]

[0053] In chemical formula (1), the nucleotide analog with substituent X attached to the 2' position of ribose being a fluoro group is the 2'-F nucleotide analog; the nucleotide analog with substituent X attached to the 2' position of ribose being a methoxy group is the 2'-OMe nucleotide analog; the nucleotide analog with substituent X attached to the 2' position of ribose being an O-methoxyethyl group is the MOE nucleotide analog; the nucleotide analog with substituent X attached to the 2' position of ribose being a 2'-(3-hydroxy)propyl group is the 2'-(3-hydroxy)propyl nucleotide analog; and the nucleotide analog with substituent X attached to the 2' position of ribose being a 2'-(3-amino)propyl group is the 2'-AP nucleotide analog.

[0054] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME comprising an ASO, wherein the nucleotide analog obtained by modifying the 2' position of ribose is at least one nucleotide analog selected from the group consisting of 2'-OMe-nucleotide analogs and MOE-nucleotide analogs.

[0055] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME comprising an ASO in which the nucleotide analog obtained by modifying the 2' position of ribose is a 2'-OMe-nucleotide analog.

[0056] The nucleotide analogs in which the ribose between the 2' and 4' positions of the oligonucleotide in the above embodiment is cross-linked include, but are not limited to, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE.

[0057] Nucleotide analogs in which the 2'-position and the 4'-position of the ribose are crosslinked and modified are called locked nucleic acids or LNAs because the conformation between the 2'-position and the 4'-position is fixed (locked). Alternatively, they are also called BNAs or bicyclic nucleic acids because they are bridged. Nucleotide analogs in which the 2'-position and the 4'-position of the ribose are crosslinked and modified include oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE. Among these, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, and ENA have stereoisomers of β-D-configuration and α-L-configuration, and cEt and cMOE have R-type and S-type stereoisomers with the carbon atom bridging between the 2'-position and the 4'-position of the ribose as an asymmetric center.

[0058] Nucleotide analogs in which the 2'-position and the 4'-position of the ribose are crosslinked and modified, for example, in the following chemical formulas (2) and (3), the divalent atomic group "-Y-" bonded to the 2'-position of the ribose is selected from "-O-", "-S-", "-CH2-S-", "-N(H)-", "-N(R a )-", "-CH2-N(H)-", and "-CH2-N(R a )-", Z and Z * are independently selected from among internucleotide linkages, R b , terminal groups, or protecting groups, B is selected from natural or unnatural nucleotide base moieties (nucleobases), R a is independently selected from hydrogen and C 1-4 alkyl, and R b has a structure selected from hydrogen, a hydroxyl group, a C 1-4 alkyl group, and a C 1-4 alkoxy group.

[0059] Oxy-LNA, thio-LNA, and amino-LNA with a β-D- configuration are represented by general formula (2), and oxy-LNA, thio-LNA, and amino-LNA with an α-L- configuration are represented by general formula (3). In chemical formulas (2) and (3), the nucleotide analog in which the divalent atomic group "-Y-" attached to the 2' position of ribose is "-O-" is oxy-LNA, the nucleotide analog in which the divalent atomic group "-Y-" attached to the 2' position of ribose is "-S-" or "-CH2-S-" is thio-LNA, and the divalent atomic group "-Y-" attached to the 2' position of ribose is "-N(H)-" or "-N(R) a )-", "-CH2-N(H)-" and "-CH2-N(R a )-" (R here a is C 1-4 The nucleotide analog selected from (selected from alkyl groups) is amino-LNA.

[0060] [ka]

[0061] [ka]

[0062] 5'-methyl-LNA with a β-D configuration is represented by the following chemical formula (4), and 5'-methyl-LNA with an α-L configuration is represented by the following chemical formula (5).

[0063] [ka]

[0064] [ka]

[0065] ENA with a β-D configuration is represented by the following chemical formula (6), and ENA with an α-L configuration is represented by the following chemical formula (7).

[0066] [ka]

[0067] [ka]

[0068] R-type cEt and cMOE are represented by the following general formula (8), and S-type cEt and cMOE are represented by the following general formula (9). In chemical formulas (8) and (9), W is selected from a methyl group and a methoxymethyl group, and Z and Z * R is a nucleotide linkage, where R is hydrogen, hydroxyl group, C 1-4 Alkyl and C 1-4 B is independently selected from among alkoxy groups, terminal groups, or protecting groups, and has a structure selected from natural or unnatural nucleotide base moieties (nucleic acid bases).

[0069] [ka]

[0070] [ka]

[0071] In chemical formulas (8) and (9), the nucleotide analog with W being a methyl group is cEt, and the nucleotide analog with W being a methoxymethyl group is cMOE.

[0072] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an ASO, wherein the nucleotide analog obtained by cross-linking the 2' and 4' positions of ribose is at least one nucleotide analog selected from the group consisting of β-D-oxy-L-LNA, β-D-ENA, and R-type cEt.

[0073] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing ASO, wherein the nucleotide analog obtained by cross-linking the 2' and 4' positions of ribose is β-D-ENA.

[0074] Among nucleotide analogs, ENA is a thermodynamically stable nucleotide analog with high resistance to nuclease degradation, and ASOs incorporating ENA have high affinity for complementary RNA chains (Morita, K. et al., Bioorganic & Medicinal Chemistry, 11: 2211-2226. (2003)).

[0075] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an ASO, wherein at least two adjacent nucleotides of an oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4 are linked by modified nucleoside bonds.

[0076] The nucleoside monomers of the oligonucleotides contained in the preventive or therapeutic agent of the present invention are linked via internucleoside bonds. Optionally, each monomer is linked to a 3'-adjacent monomer via an internucleoside bond. The terminal 5' monomer of the oligonucleotide may or may not contain a 5' terminal group or a conjugation group, but does not contain an internucleoside bond.

[0077] The term "nucleoside linkage" refers to a group that can covalently link two nucleosides to form a dinucleotide. Natural nucleotides are linked by phosphodiester bonds. In contrast, modified nucleoside links include, but are not limited to, phosphorothioate, phosphorodithioate, and boranophosphate links. Generally, oligonucleotides containing modified nucleoside links have higher membrane permeability and greater resistance to intracellular and extracellular nucleases than oligonucleotides linked only by phosphodiester bonds. On the other hand, heteroduplex nucleic acids formed by intracellular hybridization of oligonucleotides containing modified nucleoside links and RNA are degraded by RNase H, similar to heteroduplex nucleic acids formed by intracellular hybridization of oligonucleotides linked only by phosphodiester bonds and RNA. Note that stereoisomers exist for modified nucleoside links. For example, phosphorothioate links have stereoisomers with respect to the sulfur atom. The oligonucleotides contained in the preventive or therapeutic agent of the present invention may be synthesized such that specific stereoisomer-modified nucleoside bonds are used in specific nucleotide linkages of the oligonucleotide, or they may be synthesized such that some or all of the nucleotide linkages are racemic.

[0078] In this specification, when a particular nucleotide is described as an RNase H-active nucleotide, an RNase H-inactive nucleotide, or a nucleotide with deoxyribose as its sugar moiety, the linkage between that nucleotide and adjacent nucleotides may be modified nucleoside linkages. For example, in the sequence listings attached to this specification, sequences SEQ ID NOs. 1 to 7 are listed as DNA. However, at least two adjacent nucleotides of the oligonucleotide contained in the BAFME prophylactic or therapeutic agent of this embodiment may be linked by modified nucleoside linkages.

[0079] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an ASO, wherein all adjacent nucleotides of an oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4 are linked by modified nucleoside bonds.

[0080] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an ASO, wherein the modified nucleoside bond is at least one group selected from the group consisting of phosphorothioate bonds, phosphorodithioate bonds, and boranophosphate bonds.

[0081] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME comprising an ASO, wherein the modified nucleoside bond is a phosphorothioate bond.

[0082] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an oligonucleotide comprising any of the base sequences of SEQ ID NOs: 1 to 4, which is an RNase H-active ASO.

[0083] RNase H-active ASOs are those in which, when an ASO forms a heteroduplex with a target RNA, the RNase H activity cleaves the nucleosides of the target RNA, thereby degrading the target RNA. In contrast, RNase H-inactive ASOs are those in which, when an ASO forms a heteroduplex with a target RNA, the RNase H activity does not cleave the nucleosides of the target RNA, thus preventing the degradation of the target RNA. RNase H-active ASOs have a structure consisting of several consecutive RNase H-active nucleotides. The structure of RNase H-active ASOs includes not only structures consisting solely of RNase H-active nucleotides, but also structures in which RNase H-inactive nucleotides are linked to the 5' and / or 3' ends of several consecutive RNase H-active nucleotides. Among the structures of RNase H-active ASOs, the structure in which RNase H-inactive nucleotides are linked to the 5' and 3' ends of several consecutive RNase H-active nucleotides is specifically called a gapmer structure.

[0084] In gapmer structures, the sequence of several RNase H-inactive nucleotides at the 5' and 3' ends of the oligonucleotide complementary to the target RNA of the ASO is called the "wing region." The RNase H-active nucleotides in the central region sandwiched between the wing regions are called the "gap region." The oligonucleotides in the wing region have high binding affinity to the complementary RNA, thus forming a stable double helix with the target RNA. Since the oligonucleotides in the gap region are a sequence of RNase H-active nucleotides, the heteroduplex formed by the pairing of the oligonucleotides in the gap region with the target RNA is degraded by RNase H. Therefore, it is preferable to adopt a gapmer structure for ASOs of the type where the degradation of the double helix with RNA is what causes the drug effect. The length of the wing region can be easily determined by those skilled in the art according to the total number of nucleotides in the ASO, and can be 3, 4, 5, 6, 7, or 8 or more nucleotides. The length of the gap region can also be easily determined by those skilled in the art according to the total number of nucleotides in the ASO. According to Monia, BP et al. (J. Biological. Chem., 268: 14514 (1993)), the degradation activity by RNase H against heteroduplex nucleic acids consisting of a gapmer ASO in which at least five consecutive RNase H-active nucleotides are sandwiched between wing regions of RNase H-inactive nucleotides and RNA complementary to the ASO exceeded 75%. However, the degradation activity by RNase H against heteroduplex nucleic acids consisting of a gapmer ASO in which four or fewer consecutive RNase H-active nucleotides are sandwiched between wing regions of RNase H-inactive nucleotides and RNA complementary to the ASO was less than 20%. Therefore, the length of the gap region can be set to at least five nucleotides.

[0085] In the present invention, whether a particular type of nucleotide analog is RNase H active or RNase H inactive can be determined, for example, by the following procedure. An oligonucleotide with a gapmer structure is prepared in which a gap region consisting of 10 consecutive nucleotide analogs of the particular type is sandwiched between wing regions consisting of 5 known RNase H inactive nucleotides, such as 2'-OMe-nucleotide analogs. A heteroduplex is prepared by pairing 300 picomoles of the gapmer oligonucleotide with 1500 picomoles of RNA that pairs with the oligonucleotide. The heteroduplex and E. coli RNase H 3.0 units are added to a reaction solution for RNase H, for example, a reaction solution consisting of 50 mM Tris-HCl (pH 8.0), 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol, to a total volume of 150 μL, and the mixture is reacted at 37°C for 60 minutes. The reaction product is analyzed by agarose gel electrophoresis.

[0086] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing an RNase H-activated ASO, wherein the oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4 has 1 to 5 nucleotides from the 5' end and 16 to 20 nucleotides from the 5' end being RNase H-inactive nucleotide analogs.

[0087] Since the base sequences of Sequence IDs 1 to 4 all have 20 nucleotides in total, in this embodiment, the number of nucleotides in the 5' and 3' wing regions is 5 each. When the ASO contained in the BAFME preventive or therapeutic agent of the present invention consists of any of the base sequences of Sequence IDs 1 to 4, the number of nucleotides in the ASO is the same as the number of nucleotides in any of the aforementioned Sequence IDs 1 to 4. However, when the number of nucleotides in the ASO contained in the BAFME preventive or therapeutic agent of the present invention is greater than the number of nucleotides in any of the aforementioned Sequence IDs 1 to 4, it should be noted that in this embodiment, the "5' end" in "the 1st to 5th nucleotides from the 5' end" and "the 16th to 20th nucleotides from the 5' end" refers not to the 5' end of the ASO, but to the nucleotide in the ASO base sequence that corresponds to the 5' end of any of the aforementioned Sequence IDs 1 to 4.

[0088] In this embodiment, the "RNase H inactive nucleotide analog" is, as described in other embodiments of the present invention, at least one nucleotide analog selected from the group consisting of nucleotide analogs modified at the 2' position of ribose and nucleotide analogs modified by crosslinking between the 2' and 4' positions of ribose. Nucleotide analogs modified at the 2' position of ribose include, but are not limited to, 2'-F nucleotide analogs, 2'-OMe nucleotide analogs, MOE nucleotide analogs, 2'-(3-hydroxy)propyl nucleotide analogs, and 2'-AP nucleotide analogs. Nucleotide analogs modified by crosslinking between the 2' and 4' positions of ribose include, but are not limited to, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE.

[0089] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME comprising the RNase H-active ASO, wherein the RNase H-inactive nucleotide analog is β-D-ENA.

[0090] β-D-ENA exhibits high affinity for complementary nucleic acids and is highly resistant to nucleases, making it preferable for use in the wing region of gapmer-type structures.

[0091] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME comprising an oligonucleotide consisting of the base sequence of SEQ ID NO: 1, 2, or 3, which is in the RNase H inactive form.

[0092] RNase H-inactive ASOs refer to single-stranded oligonucleotides consisting solely of RNase H-inactive nucleotides, or single-stranded oligonucleotides containing a mixture of RNase H-active and RNase H-inactive nucleotides, wherein the heteroduplex nucleic acid formed when the single-stranded oligonucleotide pairs with complementary RNA is not degraded by RNase H. According to Monia, BP et al. (J. Biological. Chem., 268: 14514 (1993)), oligonucleotides containing a mixture of RNase H-active and RNase H-inactive nucleotides may contain five or fewer RNase H-active nucleotides, for example, only five, four, three, or two consecutive nucleotides. The oligonucleotide consisting of the nucleotide sequence of SEQ ID NO 1, 2, or 3 among the ASOs contained in the BAFME prophylactic or therapeutic agent of this embodiment is one of the following: it contains 5 or 4 or fewer RNase H-active nucleotides, for example, only 5, 4, 3, or 2 consecutive RNase H-active nucleotides; all RNase H-active nucleotides are sandwiched between one or more RNase H-inactive nucleotides; or it contains no RNase H-active nucleotides at all and consists only of RNase H-inactive nucleotides.

[0093] One embodiment of the present invention is All nucleotides containing thymine in the oligonucleotide consisting of the base sequence of SEQ ID NO: 1 or 2 are nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked. Among oligonucleotides consisting of the base sequence of SEQ ID NO: 1 or 2, the nucleotides containing adenine at the 5th, 10th, 15th, and 20th positions from the 5' end are nucleotide analogs in which the ribose is cross-linked between the 2' and 4' positions. All other nucleotides are nucleotide analogs in which the 2' position of ribose is modified. This is a prophylactic or therapeutic agent for BAFME containing RNase H-inactive ASO.

[0094] In this embodiment as well, when the ASO contained in the BAFME preventive or therapeutic agent of the present invention consists of the base sequence of SEQ ID NO: 1 or 2, the number of nucleotides in the ASO is the same as the number of bases in the base sequence of SEQ ID NO: 1 or 2. However, when the number of nucleotides in the ASO contained in the BAFME preventive or therapeutic agent of the present invention is greater than the number of bases in the base sequence of SEQ ID NO: 1 or 2, it should be noted that in this embodiment, the "5' end" of "the 5th, 10th, 15th, and 20th adenines from the 5' end" refers not to the 5' end of the ASO, but to the nucleotide in the ASO base sequence that corresponds to the 5' end of the base sequence of SEQ ID NO: 1 or 2.

[0095] In this embodiment, the "RNase H inactive nucleotide analog" is, as described in other embodiments of the present invention, at least one nucleotide analog selected from the group consisting of nucleotide analogs modified at the 2' position of ribose and nucleotide analogs modified by crosslinking between the 2' and 4' positions of ribose. Nucleotide analogs modified at the 2' position of ribose include, but are not limited to, 2'-F nucleotide analogs, 2'-OMe nucleotide analogs, MOE nucleotide analogs, 2'-(3-hydroxy)propyl nucleotide analogs, and 2'-AP nucleotide analogs. Nucleotide analogs modified by crosslinking between the 2' and 4' positions of ribose include, but are not limited to, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE.

[0096] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME comprising an RNase H-inactive ASO of an oligonucleotide having the base sequence of SEQ ID NO 1 or 2, wherein all nucleotide analogs with cross-linking modification between the 2' and 4' positions of ribose are β-D-ENA, and the nucleotide analog with modification at the 2' position of ribose is a 2'-OMe-nucleotide analog.

[0097] β-D-ENA exhibits high affinity for complementary nucleic acids and is highly resistant to nucleases, making it preferable for use in RNase H-inactive ASOs. 2'-OMe-nucleotide analogs are more chemically stable than DNA and RNA and are resistant to degradation by DNA and RNA-specific nucleases, making them preferable for use in RNase H-inactive ASOs.

[0098] One embodiment of the present invention is All cytosine and thymine nucleotides in the oligonucleotide consisting of the base sequence of Sequence ID No. 3 are nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked. All other nucleotides are nucleotide analogs in which the 2' position of ribose is modified. This is a prophylactic or therapeutic agent for BAFME containing RNase H-inactive ASO.

[0099] In this embodiment, the "RNase H inactive nucleotide analog" is, as described in other embodiments of the present invention, at least one nucleotide analog selected from the group consisting of nucleotide analogs modified at the 2' position of ribose and nucleotide analogs modified by crosslinking between the 2' and 4' positions of ribose. Nucleotide analogs modified at the 2' position of ribose include, but are not limited to, 2'-F nucleotide analogs, 2'-OMe nucleotide analogs, MOE nucleotide analogs, 2'-(3-hydroxy)propyl nucleotide analogs, and 2'-AP nucleotide analogs. Nucleotide analogs modified by crosslinking between the 2' and 4' positions of ribose include, but are not limited to, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE.

[0100] One embodiment of the present invention is a prophylactic or therapeutic agent for BAFME containing the RNase H inactive ASO, wherein all nucleotide analogs with cross-linking modification between the 2' and 4' positions of ribose are β-D-ENA, and the nucleotide analog with modification at the 2' position of ribose is a 2'-OMe-nucleotide analog.

[0101] One embodiment of the present invention is a preventive or therapeutic agent for BAFME comprising a pharmaceutically acceptable carrier or diluent. Those skilled in the art can appropriately select a carrier suitable for such a situation as the carrier that may be included in the preventive or therapeutic agent of the present invention. Selectable carriers include, but are not limited to, preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; pH adjusters such as sodium dihydrogen phosphate, anhydrous monohydrogen phosphate, citric acid, and sodium citrate; isotonic agents such as glucose, sodium chloride, and potassium chloride; and divalent ion adjusters such as calcium chloride and magnesium chloride. Furthermore, these carriers are not limited to being used for the purpose of exerting a single action, but can be used for the purpose of exerting multiple actions. Diluents that may be included in the preventive or therapeutic agent of the present invention include, but are not limited to, liquids such as phosphate-buffered saline.

[0102] The carrier may include a carrier for cell membrane permeability and / or intranuclear delivery, and the cell membrane permeability and / or intranuclear delivery carrier may bind to or associate with an oligonucleotide relating to the preventive or therapeutic agent of the present invention.

[0103] The cell membrane permeable and / or intranuclear delivery carrier may include peptides. Examples of cell membrane permeable carriers include, but are not limited to, oligoarginines such as octaarginine, basic peptides derived from the HIV-1 Tat protein, and basic helix peptides derived from the Antennapedia homeodomain protein of Drosophila. Examples of intranuclear delivery carriers include, but are not limited to, peptides that are nuclear localization signals that specifically bind to importin, derived from the large T antigen of the SV40 virus, c-myc, nucleoplasmin, etc.

[0104] The aforementioned cell membrane permeation and / or nuclear delivery carrier may include peptidomimetics. Peptidomimetics as cell membrane permeation and / or nuclear delivery carriers refer to synthetic compounds that mimic the structure of the peptide as the cell membrane permeation and / or nuclear delivery carrier, and that bind to or associate with oligonucleotides relating to the preventive or therapeutic agent of the present invention to promote cell membrane permeation and / or nuclear delivery. Peptidomimetics as cell membrane permeation and / or nuclear delivery carriers are compounds in which all or part of the peptide as the cell membrane permeation and / or nuclear delivery carrier is replaced with compounds other than natural peptides, such as heterocyclic or cyclic compounds, unnatural amino acids, D-amino acids, and equivalents of various functional groups.

[0105] The carrier for cell membrane permeation and / or intranuclear delivery may include cationic polymers of DEAE dextran, polyelenimine (PEI), and polypropyleneimine (PPI).

[0106] The carrier for cell membrane permeation and / or intranuclear delivery may contain lipids. Lipids used as carriers for cell membrane permeation and / or intranuclear delivery include 1,2-dimryristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphatidylcholine (PMPC), and 1-stearoyl-2-myristoyl Stearoyl-sn-glycero-3-phosphatidylcholine (SMPC), hydrogenated lecithin derived from soybeans (HSPC), 1-stearoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (SOPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoglycero Examples include phosphates such as (DOPG), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG), and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). It may include, but is not limited to, phospholipids, anionic lipids such as 1,5-O-dihexadecyl-N-succinyl-L-glutamic acid, cholesterol, PEG-bound lipids such as N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and pH-sensitive lipids such as 1,2-dioleyl-3-dimethylammonium propane (DODAP).

[0107] The carrier for cell membrane permeation and / or intranuclear delivery may include a lipidoid. The lipidoid as the carrier for cell membrane permeation and / or intranuclear delivery refers to a synthetic compound in which a peptide and / or peptidomimetics as the carrier for cell membrane permeation and / or intranuclear delivery and a lipid as the carrier for cell membrane permeation and / or intranuclear delivery are covalently linked.

[0108] The carrier for cell membrane permeation and / or intranuclear delivery may include liposomes. The liposome as the carrier for cell membrane permeation and / or intranuclear delivery refers to a lipid membrane structure dispersed in an aqueous solvent, with the aqueous solvent encapsulated within it. The lipid membrane of the liposome may be a single-layer or multi-layer lipid bilayer or lipid multilayer. The liposome may be particulate or reticular. The lipid component of the liposome may include, but is not limited to, the lipid used as the carrier for cell membrane permeation and / or intranuclear delivery. The lipid component of the liposome may include the lipidoid used as the carrier for cell membrane permeation and / or intranuclear delivery. The liposome as the carrier for cell membrane permeation and / or intranuclear delivery can encapsulate oligonucleotides related to the preventive or therapeutic agent of the present invention within it.

[0109] The carrier for cell membrane permeation and / or intranuclear delivery may include lipid nanoparticles (LNPs). The lipid nanoparticles as the carrier for cell membrane permeation and / or intranuclear delivery are composed of the same lipids as the lipid components of the liposomes and oligonucleotides related to the preventive or therapeutic agent of the present invention. pH-sensitive lipids that become positively charged in a low pH environment, such as 1,2-dioleyl-3-dimethylammonium propane (DODAP), may be used for the lipid nanoparticles. Unlike liposomes, lipid nanoparticles have a single lipid membrane and a core structure filled with lipids rather than encapsulating an aqueous solvent inside. The lipid nanoparticles as the carrier for cell membrane permeation and / or intranuclear delivery may contain oligonucleotides related to the preventive or therapeutic agent of the present invention in the core structure.

[0110] One embodiment of the present invention provides a prophylactic or therapeutic agent for BAFME delivered by at least one parenteral administration selected from the group consisting of subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, intracranial, and intrathecal administration. The administration may be continuous or long-term, or short-term or intermittent. The prophylactic or therapeutic agent of the present invention results in downregulation of SAMD12, TNRC6A, and PAPGEF2 RNA aggregate formation for at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, at least 115 days, at least 120 days, or at least 1 year after administration.

[0111] The preventive or therapeutic agent of the present invention may be delivered by oral administration, for example, in the form of tablets, capsules, granules, powders, or syrups. These formulations may contain excipients (for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, and dextrin; cellulose derivatives such as crystalline cellulose; organic excipients such as gum arabic, dextran, and pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and calcium sulfate. Examples of inorganic excipients include stearates (such as stearates), lubricants (for example, metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as beegum and gypsum wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycols; fumaric acid; sodium benzoate; DL-leucine; sodium fatty acid salts; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; and the above starch derivatives). Examples of excipients include: ), binders (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and compounds similar to those described above), disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxylated methylcellulose, carboxylated methylcellulose calcium, and internally crosslinked carboxylated methylcellulose sodium; chemically modified starches and celluloses such as carboxylated methyl starch, carboxylated methyl starch sodium, and crosslinked polyvinylpyrrolidone), stabilizers (e.g., para-hydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), and flavoring and deodorizing agents (e.g., commonly used sweeteners, acidulants, flavorings, etc.).It is manufactured using well-known methods with additives such as ).

[0112] The active ingredient contained in the preventive or therapeutic agent of the present invention is the oligonucleotide (or, if a cell membrane permeability and / or nuclear delivery carrier is bound to or associated with the oligonucleotide, the oligonucleotide to which the cell membrane permeability and / or nuclear delivery carrier is bound or associated). The proportion of the active ingredient contained in the preventive or therapeutic agent of the present invention can be appropriately set within a range that can produce the desired effect, but is usually 0.01 to 100% by weight, preferably 0.1 to 99.9% by weight, and more preferably 0.5 to 99.5% by weight.

[0113] The dosage of the preventive or therapeutic agent of the present invention must be carefully adjusted considering the age, weight, and condition of the individual being treated, as well as the route of administration, form of administration, and method of administration, and the exact dosage must be determined by a physician. The actual dosage is within the physician's discretion and may vary by setting the dosage for the specific circumstances of the present invention in order to obtain the desired therapeutic effect. However, the dosage of the preventive or therapeutic agent of the present invention is not determined by general criteria, as it depends on the type of active ingredient, the weight and age of the recipient, and the symptoms. For example, in the case of parenteral administration, it is desirable to administer a minimum of 0.001 mg / kg body weight (preferably 0.01 mg / kg body weight) and a maximum of 100 mg / kg body weight (preferably 10 mg / kg body weight) per dose, and in the case of oral administration, a minimum of 0.01 mg / kg body weight (preferably 0.1 mg / kg body weight) and a maximum of 1000 mg / kg body weight (preferably 100 mg / kg body weight) per dose, once to several times a day depending on the symptoms.

[0114] The target population for the preventive or therapeutic agent of the present invention is patients with BAFME. The most important desirable therapeutic effect for determining the selection of the active ingredient, carrier, dosage, and method of administration of the preventive or therapeutic agent of the present invention is the suppression and / or reduction of the progression of clinical symptoms of BAFME (myoclonus and / or epileptic seizures). However, as a surrogate endpoint preceding clinical symptoms, pharmacodynamic biomarkers (formation of RNA aggregates of SAMD12, TNRC6A, and PAPGEF2, and / or reduction and / or disappearance of disregard of RNA-binding molecules sequestered in said RNA aggregates) can also be used to determine the selection of the active ingredient, carrier, dosage, and method of administration of the preventive or therapeutic agent of the present invention.

[0115] In one embodiment of the assay system for the preventive or therapeutic agent of the present invention, the assay system comprises undifferentiated iPS cells or differentiated cells of any cell type established from a human suffering from BAFME, and the compound to be evaluated. The compound to be evaluated comprises an ASO that targets RNA, which is the transcript of at least one gene selected from the group consisting of SAMD12, TNRC6A, and PAPGEF2.

[0116] One embodiment of the present invention is a method for preventing or treating BAFME, comprising administering the preventive or therapeutic agent of the present invention. In the method for preventing or treating BAFME of the present invention, the administration of the preventive or therapeutic agent of the present invention may be by directly administering an ASO having at least one nucleotide sequence of SEQ ID NOs: 1 to 4 from outside the body, or by administering a vector containing AAV or other viruses that has been modified to produce oligonucleotides containing at least one nucleotide sequence of SEQ ID NOs: 1 to 4.

[0117] In this specification, the adjective "approximately" modifying a numerical value means a numerical range of 90% or more and 110% or less of the numerical value. For example, "approximately 40 bases" refers to a numerical range of bases between 36 and 44 bases.

[0118] All references made herein are incorporated herein by citation in their entirety.

[0119] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0120] Example 1 Screening of candidate ASO prophylaxis or therapeutic agents for BAFME using patient-derived iPS cells (1) Materials and methods iPS cells were generated by reprogramming peripheral blood mononuclear cells (PBMCs) collected from four BAFME patients with episodic vectors encoding SOX2, KLF4, OCT4, L-MYC, LIN28, p53 carboxyl-terminal dominant-negative fragment, and EBNA1 (Okita, K. et al., Stem Cells, 31, 458-466 (2013)). Cell line authentication was confirmed by STR analysis using the PowerPlex® 16 system (Promega, Madison, Wisconsin, USA). Karyotype analysis was performed by LSI Medience Corporation (Tokyo). iPS cells were cultured and maintained in StemFit® medium (AK02N, Ajinomoto Co., Inc., Tokyo) as described by Nakagawa, M. et al. (Sci Rep, 4, 3594 (2014)) on culture vessels coated with iMatrix-511 (892012, Nippi Corporation, Tokyo). In the following examples, undifferentiated cells of HPS3905, a BAFME-iPSC strain in which RNA aggregates can be detected, were used for ASO lipofection and detection of RNA aggregates by FISH.

[0121] To detect nuclear RNA aggregates, FISH was performed using a Cy3-labeled DNA / LNA probe targeting UUUCA (hereinafter referred to as "Cy3") and a Cy5-labeled DNA / LNA probe targeting UUUUA (hereinafter referred to as "Cy5"). Cells were cultured on a clear-bottomed microplate (CellCarrier-96 Ultra, PerkinElmer), fixed with 4% paraformaldehyde at room temperature for 30 minutes, permeabilized with 70% ethanol at -30°C for at least 2 hours, rehydrated with 1×SSC solution prepared with diethyl dicarbonate (DEPC) water, and incubated in hybridization pretreatment buffer [2×SSC and 50% formamide prepared with DEPC water] at 37°C for 2 hours. The cells were then incubated overnight at 37°C in a hybridization buffer [2×SSC, 50% formamide, 10% dextrin sulfate, and 0.1 ng / μL of Cy3 and Cy5 probes (Gene Design Co., Ltd., Osaka)]. The cells were washed twice with the hybridization pretreatment buffer at 37°C for 20 minutes, and then washed twice at room temperature for 5 minutes with 1×SSC solution prepared in DEPC water. The cells were further incubated at 37°C for 60 minutes with 1×SSC prepared in DEPC water, and then washed twice with 1×SSC prepared in DEPC water. The cells were stained with 1 μg / mL of 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, Thermo Fisher Scientific Inc.) in PBS at room temperature for 10 minutes, and then washed with PBS. Images were analyzed using an Opera Phenix® high-content screening system (PerkinElmer) with a 40x water immersion objective lens.

[0122] Figure 1 is a schematic diagram showing the relative positional relationship of the target sites of the ASOs of this invention in the pre-mamma of the SAMD12 gene, and the classification of the ASOs as either RNase H active or inactive. Table 3 shows the names of the ASOs, whether or not they have RNase H activity, their structure, and their target sites in the fourth intron of the SAMD12 gene. Among the ASOs, 3009-1M to 3009-3M and 3009-4G to 3009-6G, which target the TTTTA / TTTCA repeat sequence, target not only the SAMD12 gene, which is the causative gene for BAFME1, but also the TTTTA / TTTCA repeat sequences of the TNRC6A gene and RAPGEF2 gene, which are the causative genes for BAFME6 and BAFME7, respectively. Since the fourth intron of the SAMD12 gene is quite long at approximately 180,000 base pairs, target candidate sequences for SAMD12-G1 to SAMD12-G4 and SAMD12-M1 to SAMD12-M4 were designed using Sfold software, as described by Ding, Y. et al. (Nucleic Acids Research, Vol. 32, Web Server issue W135-W141 (2004)), within a range of 1,000 base pairs upstream and downstream of the TTTTA / TTTCA repeat sequence.

[0123] In the structures of each ASO in Table 3, the underlined nucleotides are ENA, obtained from Kobe Natural Products Chemicals Co., Ltd. (Kobe). In RNase H-active ASOs, the ununderlined nucleotides are deoxyribonucleotides. In RNase H-inactive ASOs, the ununderlined nucleotides are 2'-OMe-nucleotide analogs. The ASOs were synthesized according to conventional methods. Administration of ASOs to cultured iPS cells was performed by administering 10 nM ASOs together with DharmaFECT1 (Horizon Discovery, Cambridge, UK) according to the manufacturer's instructions. 48 hours after administration of ASOs to cultured iPS cells, FISH was performed with Cy3-labeled DNA / LNA probes targeting UUUCA and Cy5-labeled DNA / LNA probes targeting UUUUA (both with 21 base pairs). The results were obtained by observing the same field of view using a fluorescence filter optical system specific to the labeled fluorescent dye Cy3 or Cy5 and DAPI used for counterstaining cell nuclei. The number of cell nuclei per unit area and the number of nuclei positive for RNA aggregates that reacted with the Cy3 probe or Cy5 probe were measured.

[0124] (2) Results Figures 2A, B, and C are graphs showing the results for undifferentiated iPS cells (HPS3905) derived from BAFME patients that underwent lipofection treatment with ASO according to the present invention, undifferentiated HPS3905 cells that underwent lipofection treatment with a negative control ASO instead of ASO according to the present invention (Control Gapmer and Control Mixmer), undifferentiated HPS3905 cells that underwent lipofection treatment without ASO (Mock), and undifferentiated HPS3905 cells that were not lipofected (Untreated). The height of the bar graph in Figure 2A represents the percentage of iPS cell nuclei 48 hours after lipofection. The height of the bar graph in Figure 2B represents the number of iPS cell nuclei in which RNA aggregates were detected by the Cy3 probe 48 hours after lipofection. The height of the bar graph in Figure 2C represents the percentage of iPS cell nuclei 48 hours after lipofection in which RNA aggregates were detected using the Cy5 probe.

[0125] Among ASOs that target repeat sequences, 3009-2M and 3009-3M, and 3009-5G and 3009-6G reduced the percentage of cells in which RNA aggregates were detected (Figures 2B and C), but did not reduce the number of cells (Figure 2A). All of these ASOs react with RNA containing UUUCA. In contrast, 3009-1M is an RNase H-inactive ASO that targets UUUUA, and had such a strong effect that there were no cells in which RNA aggregates containing UUUUA were detected (Figure 2C), but it had almost no effect on the percentage of cells in which RNA aggregates containing UUUCA were detected (Figure 2B). 3009-4G is a gapmer-type ASO that targets UUUUA, but it only slightly reduced the number of cells in which RNA aggregates containing UUUUA were detected (Figure 2C), and had almost no effect on the percentage of cells in which RNA aggregates containing UUUCA were detected (Figure 2B).

[0126] Among ASOs targeting sites other than the TTTTA / TTTCA repeat sequence, SAMD12-M4 and SAMD12-G4, which target the area between 355 and 375 bases downstream of the TTTTA repeat sequence end site, both reduced the percentage of cells in which RNA aggregates were detected (Figures 2B and C), but did not reduce the number of cells (Figure 2A). SAMD12-G1, a gapmer-type ASO targeting the area between 71 and 51 bases upstream of the TTTTA repeat sequence start site, also reduced the percentage of cells in which RNA aggregates were detected (Figures 2B and C), but did not reduce the number of cells (Figure 2A). In contrast, SAMD12-M1, an RNase H-inactive ASO with the same target site, had almost no effect on the percentage of cells in which RNA aggregates were detected (Figures 2B and 2C). SAMD12-G2, which targets the area 80-100 bases downstream 3' from the TTTTA repeat termination site, and SAMD12-G3, which targets the area 258-278 bases downstream 3' from the TTTTA repeat termination site, resulted in a decrease in cell number (Figure 2A). SAMD12-M2 and SAMD12-M3, which target the same site, did not show a decrease in cell number (Figure 2A), but they also had little effect on the proportion of cells in which RNA aggregates were detected (Figures 2B and 2C).

[0127] Based on these results, we concluded that among the ASOs experimented with in this example, 3009-2M, 3009-3M, 3009-5G, 3009-6G, SAMD12-M4, SAMD12-G4, and SAMD12-G1 can reduce RNA aggregates, which are considered to be the cause of BAFME, in iPS cells derived from BAFME patients.

[0128] Example 2 Differentiation of cerebral organoids from patient-derived iPS cells (1) Materials and methods The cerebral organoids were created by modifying the method described by Velasco et al. (Nature 570, 523-527, 2019), as shown in the schematic diagram in Figure 3A. Briefly, 80% confluent iPS cell colonies were dissociated at 37°C for 5 minutes using Accutase (Innovative Cell Technologies, Funakoshi Co., Ltd.), suspended in StemFit® medium (AK02N, Ajinomoto Co., Inc.) supplemented with 10 μM Y-27632, and the cell number and viability were measured. iPS cells were suspended in cortical differentiation medium (Glasgow-MEM containing CDM1, 20% Knockout Serum Replacement™, 0.1 mM non-essential amino acid mixture (NEAA), 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 1% penicillin / streptomycin (all Gibco, Thermo Fisher Scientific K.K.)) supplemented with 4 μM IWR-1-endo (Carbiochem, Merck K.K.), 5 μM SB431542 (Cayman Chemical, Funakoshi Co., Ltd.), and 20 μM Y-27632, and reassembled in ultra-low-cell-adhesion V-bottom 96-well plates (Sumitomo Bakelite Co., Ltd.). From day 6 to day 18, cell aggregates were cultured in CDM1 medium supplemented with 3 μM IWRI and 5 μM SB431542. From day 18 onward, suspended aggregates were cultured in 60 mm culture dishes (Corning International Ltd.) at a speed of 60 rpm with rotational shaking, using CDM2 medium consisting of DMEM / F12 medium, 2 mM GlutaMAX™, 1% N2 Supplement, 1% Chemically Defined Lipid Concentrate, 0.25 μg / mL Amphotericin B, and 1% Penicillin / Streptomycin (all Gibco, Thermo Fisher Scientific K.K.).From day 35, the organoids were transferred to CDM3 medium consisting of CDM2 supplemented with 10% fetal bovine serum (FBS, Biosera, Fujifilm Wako Pure Chemical Corporation), 5 μg / mL heparan (Sigma, Merck KGaA), and 1% Matrigel (Growth Factor Reduced, Corning International Ltd.). From day 70, the organoids were cultured in CDM4 medium consisting of CDM3 supplemented with B27 Supplement (Gibco, Thermo Fisher Scientific K.K.) and 2% Matrigel. ASO administration was performed by transferring cerebral organoids to CDM4 medium containing 500 nM ASO but without Matrigel.

[0129] Cerebral organoids were fixed in 4% paraformaldehyde for 30 minutes at room temperature, washed three times with PBS for 10 minutes each, immersed overnight in 30% sucrose solution, embedded in OCT compound, and rapidly frozen in liquid nitrogen. 12 μm thick frozen sections were prepared in a cryostat at -18°C to -20°C. For FISH, permeabilization was performed in 0.5% Triton-X100 / PBS for 30 minutes at room temperature, and RNA aggregates were detected by FISH as in Example 1. For multiplex immunohistochemistry, anti-MAP2 antibody (Millipore, Merck KGaA), anti-PAX6 antibody (Biolegend, Tommy Digital Biology Co., Ltd.), and anti-SOX2 antibody (eBioscience, Thermo Fisher Scientific K.K.) were used as primary antibodies. Blocking was performed in Blocking One Histo (Nacalai Tesque Co., Ltd.) for 2 hours at room temperature. Sections were incubated overnight at 4°C with primary antibody diluted in PBS containing Blocking One Histo and 0.1% Triton-X100. After washing four times in 0.1% Triton-X100 / PBS, the sections were incubated with secondary antibody at room temperature for 2 hours and washed four times again in 0.1% Triton-X100 / PBS. Mounting was performed using a ProLong® Gold Antifade Mountant (Thermo Fisher Scientific Co., Ltd.). Image data was acquired using a fluorescence confocal microscope system (A1, Nikon Corporation) and analyzed using NIS-Element AR Analysis software (version 5.11.01, 64-bit, Nikon Corporation).

[0130] Figure 3B shows multiplex immunofluorescence microscopy images of cerebral organoid sections cultured for 70 days using the HPS3905 iPS cell line derived from a BAFME patient. The scale bar represents 50 μm. MAP2, PAX6, and SOX2 are markers for neurons, neural progenitor cells, and glial cells, respectively. Neural tube-like structures and nervous system cells were observed in Figure 3B.

[0131] Figure 3C shows fluorescence microscopy images of cerebral organoid sections cultured for 70 days using the HPS3905 iPS cell line derived from a BAFME patient, obtained by FISH with a Cy3 probe and DAPI counterstaining. The scale bar represents 10 μm. RNA aggregates were observed in the nucleus in Figure 3C.

[0132] Figure 3D is a graph showing the percentage of cell nuclei in which RNA aggregates were detected using a Cy3 probe in cerebral organoid sections cultured for 70 days using iPS cell line HPS3905 (BAFME1) derived from BAFME patients and iPS cell line 201B7 (HC) derived from healthy individuals. From Figure 3D, similar to undifferentiated iPS cells, RNA aggregates were detected in 40% of the cell nuclei derived from BAFME patients in the cerebral organoids, unlike the cell nuclei derived from healthy individuals.

[0133] Figure 3E is a graph showing the effect of the ASO of this invention (ASO-4M, same as 3009-2M) or the negative control ASO (Control ASO, same as Control Mixmer) on RNA aggregate formation in cerebral organoids created from iPS cells derived from BAFME patients. The graph shows the percentage of cell nuclei in which RNA aggregates were detected by a Cy3 probe among the cell nuclei of cerebral organoids cultured for 5 months from BAFME patient-derived iPS cell line HPS3905, which were administered ASO-4M or Control ASO by lipofection for 1 week and then fixed. From Figure 3E, administration of ASO-4M, a mixmer ASO that targets the UUUCA repeat, suppressed RNA aggregate formation in cell nuclei derived from BAFME patients. This suggests that administration of the ASO of this invention to BAFME patients may exhibit therapeutic efficacy by suppressing RNA aggregate formation.

[0134] This application is based on Japanese Patent Application No. 2021-009903, filed in Japan on January 25, 2021, and by reference herein, all of its contents are incorporated herein by reference. [Industrial applicability]

[0135] The ASO of the present invention is useful as a preventive or therapeutic agent for BAFME.

Claims

1. A prophylactic or therapeutic agent for benign adult-type familial myoclonic epilepsy (BAFME), comprising an antisense oligonucleotide containing at least one nucleotide sequence of SEQ ID NOs: 1 to 4 (provided that the antisense oligonucleotide containing the nucleotide sequence of SEQ ID NO: 4 is in the RNase H active form).

2. The preventive or therapeutic agent according to claim 1, wherein the antisense oligonucleotide, of which at least one base sequence of sequence numbers 1 to 4 comprises at least one RNase H inactive nucleotide analog.

3. The preventive or therapeutic agent according to claim 2, wherein the RNase H inactive nucleotide analog is at least one nucleotide analog selected from the group consisting of a nucleotide analog in which the 2' position of ribose is modified, and a nucleotide analog in which the 2' position and the 4' position of ribose are cross-linked.

4. The preventive or therapeutic agent according to claim 3, wherein the nucleotide analog modified at the 2' position of ribose is at least one nucleotide analog selected from the group consisting of 2'-OMe-nucleotide analogs and MOE-nucleotide analogs.

5. The preventive or therapeutic agent according to claim 3 or 4, wherein the nucleotide analog obtained by cross-linking the 2' and 4' positions of ribose is at least one nucleotide analog selected from the group consisting of β-D-oxy-L-LNA, β-D-ENA, and R-type cEt.

6. The preventive or therapeutic agent according to any one of claims 1 to 5, wherein at least two adjacent nucleotides of an oligonucleotide consisting of the base sequence of any one of sequence numbers 1 to 4 are linked by modified nucleoside bonds among the antisense oligonucleotides.

7. The preventive or therapeutic agent according to claim 6, wherein all adjacent nucleotides of an oligonucleotide consisting of any of the base sequences of SEQ ID NOs: 1 to 4 are linked by modified nucleoside bonds.

8. The preventive or therapeutic agent according to claim 6 or 7, wherein the modified nucleoside bond is at least one bond selected from the group consisting of phosphorothioate bonds, phosphorodithioate bonds, and boranophosphate bonds.

9. The preventive or therapeutic agent according to any one of claims 1 to 8, wherein the antisense oligonucleotide is in the RNase H active form.

10. The preventive or therapeutic agent according to claim 9, wherein, of an oligonucleotide consisting of any of the base sequences of Sequence ID No. 1 to 4, the nucleotides from the 1st to 5th position from the 5' end and the nucleotides from the 16th to 20th position from the 5' end are RNase H inactive nucleotide analogs, and the nucleotides from the 6th to 15th position from the 5' end are RNase H active nucleotides.

11. The antisense oligonucleotide comprising the oligonucleotide having the base sequence of SEQ ID NO: 1, 2, or 3 is in the RNase H inactive form, the preventive or therapeutic agent according to any one of claims 1 to 8.

12. All nucleotides containing thymine in the oligonucleotide consisting of the base sequence of SEQ ID NO: 1 or 2 are nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked. Among oligonucleotides consisting of the base sequence of SEQ ID NO: 1 or 2, the nucleotides containing adenine at the 5th, 10th, 15th, and 20th positions from the 5' end are nucleotide analogs in which the ribose is cross-linked between the 2' and 4' positions. The preventive or therapeutic agent according to claim 11, wherein all other nucleotides are nucleotide analogs in which the 2' position of ribose is modified.

13. The preventive or therapeutic agent according to claim 12, wherein all nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked are β-D-ENA, and all nucleotide analogs in which the 2' position of ribose is modified are 2'-OMe-nucleotide analogs.

14. All cytosine and thymine nucleotides in the oligonucleotide consisting of the base sequence of Sequence ID No. 3 are nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked. The preventive or therapeutic agent according to claim 11, wherein all other nucleotides are nucleotide analogs in which the 2' position of ribose is modified.

15. The preventive or therapeutic agent according to claim 14, wherein all nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked are β-D-ENA, and all nucleotide analogs in which the 2' position of ribose is modified are 2'-OMe-nucleotide analogs.

Images