Pharmaceutical composition

Abstract

The present invention provides a novel antisense oligonucleotide pharmaceutical composition that has higher RNA foci-removal activity than conventional pharmaceutical compositions. This pharmaceutical composition includes an oligonucleotide that has a nucleic acid sequence having one of the repetitive sequences wherein cytosine–adenine–guanine trinucleotides repeat 5–13 times at the 5'-end to the 3'-end. The oligonucleotide includes at least two types of RNase H inactive nucleotide analogs and has a mixmer structure. The present invention provides this pharmaceutical composition which is used for treating the symptoms of any of the myotonic dystrophy type 1 diseases.

Description

Pharmaceutical Composition

[0001] The present invention relates to pharmaceutical compositions, particularly to pharmaceutical compositions comprising antisense oligonucleotides, which can be used to treat any symptom of myotonic dystrophy type 1 disease.

[0002] Myotonic dystrophy type 1 (DM1) is one of the most prevalent muscular dystrophies. The prevalence of DM1 varies by country, with reports ranging from 1 in 2,000 to 1 in 8,000 people, and even as low as 1 in 500 in some regions (Non-Patent Document 1). DM1 is caused by an expansion of a CTG trinucleotide repeat sequence within the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase (DMPK) gene. The DMPK gene encodes a non-receptor serine / threonine kinase protein. DMPK protein is expressed ubiquitously throughout the body, with high expression in skeletal and cardiac muscles. The specific function of DMPK protein is unknown. Most cells in DM1 patients have 50-2,000 CTG repeats in the DMPK gene, while unaffected individuals have only 5-38 CTG repeats (Non-Patent Document 1). The number of CTG repeats roughly correlates with the severity and onset of the disease. DMPK RNA with expanded CUG repeats forms RNA aggregates (foci) in the cell nuclei of affected individuals. It has been reported that RNA aggregates adsorb and deplete RNA-binding molecules, including CUG-binding protein 1 (CUG-BP1) and muscleblind-like protein 1 (MBNL1), from the nucleus, causing disruptions in many cellular mechanisms, such as alternative splicing, transcription, translation, and post-translational regulation (Non-Patent Document 2). In addition to DM1, numerous other diseases with expanded trinucleotide repeats have been reported, collectively referred to as triplet diseases.

[0003] Antisense oligonucleotide (ASO) treatment is considered a promising therapeutic strategy for diseases caused by abnormal RNA in cells and / or nuclei, including mRNA and non-coding RNA, such as triplet disease (Non-Patent Documents 3 and 4). ASOs cause cleavage of target RNA strands by RNase H in the nucleus and cytoplasm, or sterically hinder the association of the target RNA with RNA-binding molecules. ASOs containing various nucleotide analogs have been developed to increase the stability and target binding affinity of single-stranded DNA consisting only of deoxyribonucleotides. Modifications of nucleotides used in ASO include phosphorothioate (PS) modification of the phosphodiester backbone (hereinafter also referred to as "S modification"), methylation of the 5' position of the pyrimidine ring of the base moiety of cytidine (hereinafter referred to as "5mC"), substitution of the sugar moiety of any nucleotide with a morpholino ring, substitution of the 2'-position hydroxyl group of the ribose of the sugar moiety of any nucleotide with a fluoro group (hereinafter referred to as "2'-F"), modification with a methoxy group (hereinafter referred to as "2'-OMe"), modification with an O-methoxyethyl group (hereinafter referred to as "MOE"), and cross-linking modification of the 2'-position oxygen atom and the 4'-position carbon atom of ribose (for example, locked nucleic acid in which the conformation of the furanose ring is fixed to the C3'-endo type by bridging the 2'-position oxygen atom and the 4'-position carbon atom of the ribose ring with a methylene bridge). acid (hereinafter referred to as "LNA"), 2'-O,4'-C-ethylene-bridged nucleic acid (2'-O,4'-C-Ethylene-bridged Nucleic Acid (hereinafter referred to as "ENA") in which the oxygen atom at the 2' position and the carbon atom at the 4' position of ribose are ethylene-bridged, and 2',4'-(S)-constrained ethyl nucleotide analogs (hereinafter referred to as "cEt") in which the oxygen atom at the 2' position and the carbon atom at the 4' position of ribose are methyl (methyleneoxy)-bridged (4'-CH(CH3)-O-2').Although nucleotide analogs may cause adverse side effects when administered to the body, they have the advantage that smaller doses of nucleotide analogs can be administered than unmodified deoxyribonucleotides because they have a higher binding affinity to complementary RNA and are often more resistant to nucleases than unmodified deoxyribonucleotides.

[0004] When a heteroduplex nucleic acid formed by pairing an oligonucleotide consisting of a specific type of nucleotide analog with a complementary RNA to the oligonucleotide is degraded when reacted with RNase H under specific conditions, the specific type of nucleotide analog is referred to as an RNase H-active nucleotide. Conversely, when a heteroduplex nucleic acid formed by pairing an oligonucleotide consisting of a specific type of nucleotide analog with a complementary RNA to the oligonucleotide is not degraded when reacted with RNase H under specific conditions, the specific type of nucleotide analog is referred to as an RNase H-inactive nucleotide. Examples of RNase H-active nucleotides contained in the oligonucleotides of the present invention include, but are not limited to, unmodified deoxyribonucleotides and PS-modified deoxyribonucleotide analogs. Examples of RNase H-inactive nucleotides contained in the oligonucleotides of the present invention include, but are not limited to, morpholino ring-substituted nucleotide analogs, 2'-OMe-nucleotide analogs, MOE-nucleotide analogs, 2'-F nucleotide analogs, LNA, ENA, and cEt.

[0005] In designing ASOs, it is known that even ASOs with the same base sequence may have different properties depending on the arrangement of RNase H-active nucleotides and RNase H-inactive nucleotides. A gapmer structure is a structure in which an oligonucleotide consisting only of RNase H-active nucleotides (hereinafter referred to as a "gap region") is linked to the 5'-end and 3'-end of an oligonucleotide consisting only of RNase H-inactive nucleotides (hereinafter referred to as a "wing region"). The wing region has high binding affinity with complementary RNA, forming a stable double-stranded chain with the target RNA. Since the gap region contains consecutive RNase H-active nucleotides, the double-stranded chain with the target RNA is degraded by RNase H. Therefore, it is preferable to adopt a gapmer structure for ASOs that exert their therapeutic effects by degrading the double-stranded chain with RNA. Conversely, for ASOs that exert their therapeutic effects by not degrading the double-stranded chain with RNA, it is preferable to use an oligonucleotide in which RNase H-inactive nucleotides are distributed throughout the entire length of the ASO and there is no clear gap region. In the present invention, the mixmer structure may be either a structure consisting only of the RNase H inactive nucleotides, or a structure in which RNase H active nucleotides and RNase H inactive nucleotides are mixed, but the RNase H inactive nucleotides are positioned near the RNase H active nucleotides, so that the double strand between the ASO and the target RNA is not degraded by RNase H. The RNase H inactive nucleotides contained in the mixmer of the present invention may include an oligonucleotide in which all nucleotides have the same modified sugar and all internucleotide bonds are the same modified bond, or may include a plurality of nucleotide derivatives with different modified sugars and / or modified bonds. The gapmer and mixmer of the present invention refer to oligonucleotides having a gapmer structure and a mixmer structure, respectively.

[0006] Several types of MOE / DNA gapmer ASOs have been reported to be effective in reducing DMPK RNA with expanded CUG repeat sequences in in vitro and in vivo DM1 disease models (Non-Patent Documents 5 and 6). 2'-OMe-modified ASOs (Non-Patent Documents 7 and 8) and morpholino-substituted ASOs (Non-Patent Documents 8 and 9) have also been reported to alleviate the effects of DMPK RNA with expanded CUG repeat sequences in DM disease models. Some of these MOE / DNA gapmer ASOs have progressed to clinical trials, but they have not been able to deliver ASOs to muscle at concentrations sufficient to exert therapeutic effects in vitro (Non-Patent Document 4). Therefore, there is a need to develop ASOs that exert their therapeutic effects at lower concentrations.

[0007] Although only a few of the previous experiments investigating the effects of ASOs for treating DM1 used fibroblasts, muscle satellite cells, or myoblasts derived from DM1 patients, most of them used transgenic mice into which genomic DNA of a human DMPK gene with an expanded CTG repeat sequence derived from DM1 patients had been introduced, or myoblasts or myotubes derived from such mice. However, the CTG repeat sequence of the human DMPK gene introduced into the transgenic mice had a repeat count of only a few hundred, which was insufficient as a model of human DM1 disease, which has a repeat count of more than a thousand, and sometimes even several thousand.

[0008] Patient-derived induced pluripotent stem cells (hereinafter referred to as "iPSCs" or "iPS cells") resources are useful for drug discovery and development. Several iPSCs have been described that were generated from primary cultures of fibroblasts, myoblasts, urine-derived cells, or immortalized lymphoblastic cells from myotonic dystrophy patients (Non-Patent Documents 10-18). Nuclear RNA aggregates have been observed as a pathological phenotype in iPSCs derived from myotonic dystrophy patients and in skeletal muscle cells and cardiomyocytes differentiated from iPSCs derived from myotonic dystrophy patients (Non-Patent Documents 16-18). However, the effects of ASOs for the treatment of DM1 have not been investigated using iPSCs derived from DM1 patients or cells differentiated from such iPSCs. Therefore, it is necessary to establish iPSCs derived from multiple DM1 patients with different numbers of CTG repeats and evaluate ASOs for the treatment of DM1 in a disease model cell system that more faithfully reproduces the pathology of human DM1 disease.

[0009] Furthermore, among the nucleotide analogs described above, ENA is a thermodynamically stable nucleotide analog with high resistance to nuclease degradation (Non-Patent Documents 8, 19, and 20). ASOs incorporating ENA have high affinity for complementary RNA strands (Non-Patent Documents 8, 19, and 20). ENA / 2'-OMe ribonucleotide analog mixmer ASOs, which can induce exon skipping through steric hindrance, are currently being developed as therapeutic agents for Duchenne muscular dystrophy (DMD) (Non-Patent Documents 20-22). ENA / deoxyribonucleotide gapmer and ENA / 2'-OMe ribonucleotide analog mixmer ASOs can reduce RNA aggregates in iPS cells derived from patients with spinocerebellar ataxia type 36 (SCA36) and in neurons derived from these iPS cells (Non-Patent Document 23). Thus, ENA-incorporated ASOs hold particular promise as therapeutic agents with a steric hindrance-mediated mechanism of action, and therefore, there is a need to develop ENA-incorporated oligonucleotides as ASOs for the treatment of DM1.

[0010] Furthermore, DM1 is a multisystem disease that affects not only skeletal and smooth muscles, but also the eyes, heart, endocrine system, and central nervous system. Clinical symptoms range from mild to severe, and although there is some overlap, it is classified into three types: mild, classic, and congenital. Mild DM1 is characterized by cataracts and mild myotonia (prolonged muscle contraction), and has a normal life prognosis. Classic DM1 is characterized by muscle weakness and atrophy, myotonia, and cataracts, and is often accompanied by cardiac conduction disorders. Physical function declines in adulthood, and life prognosis may be shortened. Congenital DM1 is characterized by hypotonia and marked generalized muscle weakness at birth, often leading to respiratory failure and early death. Intellectual disability is often present. DM1 has also been reported to cause muscle weakness / atrophy, myocardial damage, cardiac conduction disorders, cataracts, retinal degeneration, gastrointestinal symptoms such as dysphagia, constipation, and diarrhea associated with smooth muscle disorders, higher brain dysfunction, endocrine disorders such as hyperinsulinemia, diabetes, testicular atrophy, and abnormal growth hormone secretion, and skin symptoms such as pilomatricoma and epithelioma. Therefore, depending on the patient's clinical symptoms, treatment of organs other than muscle may be necessary. Therefore, it is also necessary to develop ASOs for treating DM1 that target not only skeletal muscle, but also satellite cells, which are skeletal muscle precursor cells resident in muscle tissue, and tissues other than muscle, such as ocular tissues (e.g., lens cells that cause cataracts, retinal cells that cause retinal degeneration), nerves, skin, the endocrine system, and other tissues.

[0011] Chakraborty, S. et al., Current Protocols in Human Genetics, 91: 9.29.1 - 9.29.19. (2016) Pettersson, O. J. et al., Nucleic Acids Research, 43: 2433 - 2441. (2015) Crooke, S. T. et al., Cell Metabolism, 27: 714 - 739. (2018) Overby, S. J. et al., Drug Discovery Today 23: 2013 - 2022. (2018) Wheeler, T. M. et al., Nature, 488: 111 - 115. (2012) Jauvin, D. et al., Mol Ther Nucleic Acids, 7: 465 - 474. (2017) Mulders, S. A. et al., Proc Natl Acad Sci U S A, 106: 13915 - 13920. (2009) Gonzalez - Barriga, A. et al., Mol Ther Nucleic Acids, 2: e81. (2013) Klein, A. F. et al., J Clin Invest, 129: 4739 - 4744. (2019) Du, J. et al., Hum Mol Genet, 22: 5276 - 5287. (2013) Xia, G. et al., Cellular reprogramming, 15: 166 - 177. (2013) Xia, G. et al., Stem Cells, 33: 1829 - 1838. (2015) Gao, Y et al., Mol Ther, 24: 1378 - 1387. (2016) Ueki, J. et al., Sci Rep, 7: 42522. (2017) Martineau, L. et al., Stem Cell Res, 26: 103 - 106. (2018) Mondragon - Gonzalez, R. and Perlingeiro, R. C. R., Dis Model Mech, 11: dmm034728. (2018) Dastidar, S. et al., Nucleic Acids Res., 46: 8275 - 8298. (2018) Kim, E. Y. et al., JCI Insight, 4: e122686. (2019) Morita,K. et al., Bioorganic & Medicinal Chemistry, 11: 2211-2226. (2003) Lee, T. et al., Genes, 8: 67. (2017) Malueka, R. G. et al., Nucleic Acid Therapeutics, 21: 347-353. (2011) Shoji, E. et al., Sci Rep, 5: 12831. (2015) Matsuzono, K. et al., Mol Ther Nucleic Acids, 8: 211-219. (2017),

[0012] An object of the present invention is to provide a novel antisense oligonucleotide therapeutic agent for myotonic dystrophy type 1 disease which has higher activity than conventional agents in removing RNA aggregates.

[0013] The present inventors have conducted further studies to achieve the above-mentioned object and have discovered that a disease model system using iPS cells derived from patients with myotonic dystrophy type 1 is suitable for screening antisense oligonucleotides with high activity in removing RNA aggregates, and have completed the present invention using this assay system.

[0014] The present invention provides a pharmaceutical composition comprising an oligonucleotide having any of the base sequences of repeating sequences in which cytosine-adenine-guanine trinucleotides are repeated 5 to 13 times from the 5' to 3' end, and the oligonucleotide has a mixmer structure containing at least two types of RNase H-inactivating nucleotide analogs.

[0015] In the pharmaceutical composition of the present invention, the oligonucleotide may comprise: (1) an oligonucleotide consisting of a base sequence of any one of SEQ ID NOS: 1 to 9; (2) an oligonucleotide consisting of a base sequence consisting of adenine-guanine, a base sequence of any one of SEQ ID NOS: 1 to 9, and cytosine from the 5' to 3' end; or (3) an oligonucleotide consisting of a base sequence consisting of guanine, a base sequence of any one of SEQ ID NOS: 1 to 9, and cytosine-adenine from the 5' to 3' end.

[0016] In the pharmaceutical composition of the present invention, the RNase H inactivating nucleotide analogue may be a nucleotide analogue in which the 2'-position of the ribose is modified and / or a nucleotide analogue in which a bridge is modified between the 2'- and 4'-positions of the ribose.

[0017] In the pharmaceutical composition of the present invention, the nucleotide analogue in which the oxygen atom at the 2'-position of ribose is modified can be a 2'-OMe-nucleotide analogue and / or an MOE-nucleotide analogue.

[0018] In the pharmaceutical composition of the present invention, the nucleotide analogue in which the 2'- and 4'-positions of the ribose are bridged can be β-D-oxy-L-LNA, β-D-ENA, and / or R-type cEt.

[0019] In the pharmaceutical composition of the present invention, all of the nucleotide analogues modified at the 2'-position of the ribose may have the same modified sugar.

[0020] In the pharmaceutical composition of the present invention, the nucleotide analogue modified at the 2'-position of ribose may have at least two different modified sugars.

[0021] In the pharmaceutical composition of the present invention, all of the nucleotide analogues in which the 2'- and 4'-positions of the ribose are bridged may have the same modified sugar.

[0022] In the pharmaceutical composition of the present invention, the nucleotide analogue having a bridged modification between the 2'-position and the 4'-position of the ribose may have at least two different modified sugars.

[0023] In the pharmaceutical composition of the present invention, the oligonucleotide may comprise β-D-ENA-cytidine, 2′-OMe-adenosine and 2′-OMe-guanosine.

[0024] In the pharmaceutical composition of the present invention, all cytidines of the oligonucleotide can be β-D-ENA-cytidines, all adenosines can be 2′-OMe-adenosines, and all guanosines can be 2′-OMe-guanosines.

[0025] In the pharmaceutical composition of the present invention, the oligonucleotide may contain at least one type of nucleotide linkage selected from the group consisting of phosphorothioate, phosphorodithioate, and boranophosphate.

[0026] The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier or diluent.

[0027] In the pharmaceutical composition of the present invention, the carrier may include a cell membrane-permeating carrier and / or a nuclear delivery carrier.

[0028] The pharmaceutical compositions of the present invention can be delivered parenterally by administration selected from the group consisting of transdermal, intraocular, intralental, intramucosal, submucosal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intracranial and intrathecal administration.

[0029] The pharmaceutical compositions of the present invention can be used for the treatment of any symptom of myotonic dystrophy type 1 disease.

[0030] The present invention provides a method for treating myotonic dystrophy type 1 disease by administering to a patient in need thereof an oligonucleotide comprising any one of a base sequence of a repeat sequence in which 5 to 13 repeats of cytosine-adenine-guanine trinucleotide are formed from the 5' end to the 3' end, the oligonucleotide having a mixmer structure containing at least two types of RNase H-inactivating nucleotide analogues.

[0031] The present invention provides an assay system for an antisense oligonucleotide therapeutic agent for myotonic dystrophy type 1, comprising cells derived from iPS cells established from a human suffering from myotonic dystrophy type 1. The iPS cell-derived cells include undifferentiated pluripotent stem cells, cells differentiated into at least one cell type, and aggregates containing the differentiated cells.

[0032] This panel of micrographs shows the expression of undifferentiated pluripotent stem cell markers in iPS cells derived from DM1 patients (DM-1 to 3) and iPS cells derived from healthy individuals (HC-1 to 3). The top row is a phase-contrast micrograph of a monolayer culture of undifferentiated cells from each iPS cell line. The second row is a fluorescence micrograph of a monolayer culture of undifferentiated cells from each iPS cell line stained with an antibody against NANOG and counterstained with 4',6-diamidino-2-phenylindole (hereinafter referred to as "DAPI"). The third row is a fluorescence micrograph of a monolayer culture of undifferentiated cells from each iPS cell line stained with an antibody against OCT4 and counterstained with DAPI. The fourth row is a fluorescence micrograph of a monolayer culture of undifferentiated cells from each iPS cell line stained with an antibody against SSEA4 and counterstained with DAPI. The fifth row is a fluorescence micrograph of a monolayer culture of undifferentiated iPS cells stained with an antibody against TRA-1-60, with the cell nuclei counterstained with DAPI. A panel of micrographs showing the expression of three germ layer differentiation markers in cells differentiated from iPS cells derived from DM1 patients (DM-1 to 3) and iPS cells derived from healthy individuals (HC-1 to 3). The first row is a fluorescence micrograph of cells differentiated from each iPS cell into mesoderm, stained with an antibody against α-smooth muscle actin (SMA), with the cell nuclei counterstained with DAPI. The second row is a fluorescence micrograph of cells differentiated from each iPS cell into endoderm, stained with an antibody against SOX17, with the cell nuclei counterstained with DAPI. The third row is a fluorescence micrograph of cells differentiated from each iPS cell into ectoderm, stained with an antibody against βIII-tubulin, with the cell nuclei counterstained with DAPI. Capillary electrophoresis of the reaction products of triplet repeat prime PCR (hereinafter referred to as "TP-PCR") of iPS cells derived from DM1 patients (DM-1 to 3) and iPS cells derived from healthy individuals (HC-1 to 3). For iPS cells derived from DM1 patients (DM-3), Cy3-labeled (CAG) ６Panel of fluorescence micrographs of the results of fluorescence in situ hybridization (hereinafter referred to as "FISH") using a -CA DNA / LNA probe. The upper left or lower left is the result of FISH performed without RNase A treatment, and the upper right and lower right are the results of FISH performed after RNase A treatment. The upper left and upper right are fluorescence micrographs in which the cell nuclei were counterstained with DAPI after FISH, and the lower left and lower right are fluorescence micrographs in which FISH was performed only without DAPI staining. Cy3-labeled (CAG) cells were used for undifferentiated cells of iPS cells derived from DM1 patients (DM-1 to 3) and iPS cells derived from healthy individuals (HC-1 to 3). ６ 3B is a panel of fluorescence micrographs showing the results of FISH using the -CA DNA / LNA probe. The upper right corner of each photograph is a high-magnification fluorescence micrograph of a single nucleus. The left side of Figure 3B is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results of Figure 3A. The right side of Figure 3B is a bar graph showing the percentage of RNA aggregate-positive cell nuclei, as detected by DAPI staining, out of all cell nuclei, calculated from the FISH results of Figure 3A. Schematic diagram showing the relative positions of the complementary sequences of the ASOs of the present examples on DMPK-pre mRNA, as well as the structural types of the ASOs. ASO-1 to ASO-3 are complementary to the sequence of the non-coding region of exon 15 on DMPK-mRNA, ASO-4 is complementary to the repeat sequence of the CUG sequence in the non-coding region of exon 15 on DMPK-mRNA, and ASO-5 is complementary to the sequence 3'-terminal of the repeat sequence in the non-coding region of exon 15 on DMPK-mRNA. ASO-4 is a mixmer consisting of ENA and 2'-OMe-nucleotide analogs, while the other ASO-1 to ASO-3 and ASO-5 are gapmers (hereinafter referred to as "ENA / DNA gapmers") consisting of ENA in the wing region and unmodified deoxyribonucleotides in the gap region. 48 hours after administration of ASO-C or ASO-1 to ASO-5 to iPS cells (DM-1) derived from a DM1 patient, Cy3-labeled (CAG) ６Fluorescence micrographs obtained by FISH using a -CA DNA / LNA probe and DAPI counterstaining. A bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-C and any of ASO-1 to 5. A bar graph showing the percentage of RNA aggregate-positive cell nuclei, detected by DAPI staining, among all cell nuclei, calculated from the FISH results of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-C and any of ASO-1 to 5. A bar graph showing the proliferation and survival status of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-C and any of ASO-1 to 5. The vertical axis shows the percentage of cell number, with the number of ASO-C cells set to 1.0. Schematic diagram showing the relative positions of complementary sequences on DMPK-pre mRNA for ASO-2 and ASO-4 in the present examples and their corresponding control ASO-A and ASO-B, as well as the structural types of ASOs. ASO-2 and ASO-A are complementary to the same sequences in the coding region of exon 15 on DMPK-mRNA, while ASO-4 and ASO-B are complementary to the same sequences in the CUG repeat sequence in the non-coding region of exon 15 on DMPK-mRNA. ASO-2 is an ENA / DNA gapmer, while ASO-A is a gapmer (hereinafter referred to as "MOE / DNA gapmer") in which the wing regions are composed of MOE-nucleotide analogs and the gap region is composed of unmodified deoxyribonucleotides. ASO-4 is a mixmer composed of ENA and 2'-OMe-nucleotide analogs, while ASO-B is composed of all nucleotides consisting of 2'-OMe-nucleotide analogs and all internucleotide linkages are phosphorothioates. iPS cells derived from DM1 patients (DM-1) were administered ASO-2 or ASO-A and then labeled with Cy3 (CAG). ６Fluorescence micrographs of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-2, ASO-A, or ASO-C by FISH and DAPI counterstaining were obtained using a 5-CA DNA / LNA probe. The left side of Figure 5C shows a bar graph representing the average number of RNA aggregates per cell nucleus, calculated from the results of FISH for iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-2, ASO-A, or ASO-C. The middle part of Figure 5C shows a bar graph representing the percentage of RNA aggregate-positive cell nuclei among all cell nuclei, as detected by DAPI staining, calculated from the results of FISH for iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-2, ASO-A, or ASO-C. The right side of Figure 5C shows a bar graph representing the proliferation and survival status of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-2, ASO-A, or ASO-C. The vertical axis represents the percentage of the number of cells, with the number of ASO-C cells set at 1.0. iPS cells derived from DM1 patient (DM-1) were administered with ASO-4 or ASO-B and then labeled with Cy3 (CAG). ６Fluorescence micrographs of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-4, ASO-B, or ASO-C, and then subjected to FISH analysis using a 5 nM or 10 nM ASO-4 DNA / LNA probe, followed by DAPI counterstaining. The left side of Figure 5E shows a bar graph representing the average number of RNA aggregates per cell nucleus, calculated from the FISH results of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-4, ASO-B, or ASO-C. The middle part of Figure 5E shows a bar graph representing the percentage of RNA aggregate-positive cell nuclei among all cell nuclei, calculated from the FISH results of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-4, ASO-B, or ASO-C. The right side of Figure 5E shows a bar graph representing the proliferation and survival status of iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-4, ASO-B, or ASO-C. The vertical axis represents the percentage of cells, with the number of ASO-C cells set at 1.0. This is a conceptual diagram showing the procedure for an experimental system for differentiation of undifferentiated iPS cells (DM1 patient-derived iPS cells, DM-1 to 3) and iPS cells (HC-1 to 3) derived from healthy individuals into muscle cells using a tetracycline-inducible MyoD expression system. "StemFit" represents the period of culture in medium for undifferentiated iPS cells, "KSR / α-MEM" represents the period of culture in medium for differentiated muscle cells, and "KSR / α-MEM+Dox" represents the period of culture in medium for muscle cells supplemented with doxycycline as a differentiation inducer. Undifferentiated iPS cells were transfected with the tetracycline-inducible MyoD expression system on day 0. On day 2, the medium was changed to medium for muscle cells supplemented with a differentiation inducer to initiate differentiation. On day 8, the medium was changed to medium for muscle cells without a differentiation inducer to terminate differentiation induction. Analysis was performed on day 12. A panel of fluorescence micrographs showing the expression of muscle differentiation markers in cells induced to differentiate from undifferentiated HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. The top panel shows fluorescence micrographs stained with an antibody against myosin heavy chain (MHC) and counterstained with DAPI for the cell nuclei. The middle panel shows fluorescence micrographs stained with an antibody against α-actinin and counterstained with DAPI for the cell nuclei. The bottom panel shows fluorescence micrographs stained with an antibody against myogenin (MyoG) and counterstained with DAPI for the cell nuclei. A bar graph showing the percentage of MHC-expressing cells among muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A.The vertical axis represents the percentage of cell nuclei expressing the muscle differentiation marker MHC (MHC / DAPI) among DAPI-stained positive cell nuclei induced to differentiate from undifferentiated cells. "N.S." indicates that no significant difference was observed between HC-1 to 3 cells and DM-1 to 3 cells. A bar graph showing the percentage of α-actinin-expressing cells among muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. The vertical axis represents the percentage of cell nuclei expressing the muscle differentiation marker α-actinin (α-actinin / DAPI) among DAPI-stained positive cell nuclei induced to differentiate from undifferentiated cells. "N.S." indicates that no significant difference was observed between HC-1 to 3 cells and DM-1 to 3 cells. A bar graph showing the percentage of myogenin-expressing cells among muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. The vertical axis represents the percentage of cell nuclei expressing the muscle differentiation marker myogenin (myogenin / DAPI) among DAPI-stained cell nuclei induced to differentiate from undifferentiated cells. "ns" indicates that no significant difference was observed between HC-1 to 3 cells and DM-1 to 3 cells. Muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A were labeled with Cy3 (CAG). ６ Panels of fluorescence micrographs showing the results of FISH using the -CA DNA / LNA probe and DAPI counterstaining. The left side of Figure 6G is a bar graph showing the average number of RNA aggregates per cell nucleus calculated from the FISH results for each of the muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. The right side of Figure 6G is a bar graph showing the percentage of RNA aggregate-positive cell nuclei detected by DAPI staining, calculated from the FISH results for each of the muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. After administration of 10 nM ASO-C or ASO-4 to muscle cells derived from DM1 patient iPS cells (DM-1 to 3), Cy3-labeled (CAG) ６A panel of fluorescence micrographs of FISH using a -CA DNA / LNA probe and DAPI counterstaining. A bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results of muscle cells derived from DM-1 to 3 cells administered 10 nM ASO-C or ASO-4. A bar graph showing the percentage of RNA aggregate-positive cell nuclei among all cell nuclei detected by DAPI staining, calculated from the FISH results of muscle cells derived from DM-1 to 3 cells administered 10 nM ASO-C or ASO-4. A bar graph showing the proliferation and survival status of muscle cells derived from DM-1 to 3 cells administered 10 nM ASO-C or ASO-4. In both bar graphs, the vertical axis represents the percentage of cell number, with the number of cells in cultures not administered ASO (Untreated) set to 1.0. A schematic diagram of the protocol for generating neuromuscular organoids from iPS cells. On the left, phase-contrast micrographs of neuromuscular organoids derived from iPS cells from a healthy individual (HC, top) and neuromuscular organoids derived from iPS cells from a DM1 patient (DM, bottom) are shown at day 5 and day 50, respectively (scale bar 200 μm). On the right, a composite image of multicolor fluorescence micrographs of a section of a neuromuscular organoid at day 50 (scale bar 20 μm) is shown. On the left side of the panel, neuromuscular organoids were stained with anti-myosin heavy chain (MHC) antibody, TUJ1 antibody (anti-β-tubulin III antibody), and DAPI. They are divided into a region rich in skeletal muscle cells (muscle compartment) and a region rich in nerve cells (nerve compartment). On the right side of the panel, neuromuscular organoids were stained with anti-TITIN antibody and DAPI. The striations of skeletal muscle cells were stained with anti-TITIN antibody. In both cases, the upper row shows neuromuscular organoids (HC) derived from iPS cells of healthy individuals, and the lower row shows neuromuscular organoids (DM) derived from iPS cells of DM1 patients. The upper row shows a composite image of muscle compartment fluorescence micrographs of neuromuscular organoids multicolor stained with anti-desmin antibody, anti-PAX7 antibody, and DAPI. The lower row shows Cy3-labeled (CAG) ６Composite images of fluorescence micrographs of the muscle compartment of neuromuscular organoids were subjected to FISH using a β-CA DNA / LNA probe and DAPI counterstaining. In both the upper and lower panels, the left panel shows a neuromuscular organoid derived from iPSCs of a healthy donor (HC), the center panel shows a neuromuscular organoid derived from iPSCs of a DM1 patient (DM), and the right panel shows a neuromuscular organoid derived from iPSCs of a DM1 patient treated with ASO-4M (same as ASO-4) (DM ASO-4M). Scale bars represent 20 μm. Bar graphs show the percentage of cells with RNA aggregates expressing desmin and PAX7. Values ​​are the mean ± standard deviation (SD) obtained from five organoids. "***" indicates a significant difference between HC and DM, and between DM and DM + ASO4M (p<0.001, unpaired two-tailed t-test). The top row shows a composite image of muscle compartment fluorescence micrographs of neuromuscular organoids stained with anti-MYOD antibody, anti-PAX7 antibody, and DAPI. The bottom row shows a composite image of muscle compartment fluorescence micrographs of neuromuscular organoids stained with anti-Ki67 antibody, anti-PAX7 antibody, and DAPI. In both the top and bottom rows, the left shows neuromuscular organoids derived from iPS cells of a healthy individual (HC), the center shows neuromuscular organoids derived from iPS cells of a DM1 patient (DM), and the right shows neuromuscular organoids derived from iPS cells of a DM1 patient treated with ASO-4M (same as ASO-4) (DM ASO-4M). The top left shows a bar graph representing the percentage of cells expressing PAX7 among all cells. The top right shows a bar graph representing the percentage of cells expressing MYOD among all cells. The bottom left shows a bar graph representing the percentage of cells expressing PAX7 among cells expressing MYOD. The bottom right shows a bar graph representing the percentage of Ki67-expressing cells among PAX7-expressing cells. Values ​​are the mean ± standard deviation (SD) obtained from five organoids. "***" indicates a significant difference between HC and DM, and between DM and DM + ASO4M (paired two-tailed t-test, p<0.001). The left shows phase-contrast micrographs of lens epithelial cells and lens fiber cells differentiated from iPSCs of a healthy individual (HC). The right shows phase-contrast micrographs of lens epithelial cells and lens fiber cells differentiated from iPSCs of a DM1 patient (DM1). The scale bar represents 100 μm.The left image shows a fluorescence micrograph of lens epithelial cells and lens fiber cells differentiated from iPS cells from a healthy individual stained with an anti-αA crystallin antibody (HC). The right image shows a fluorescence micrograph of lens epithelial cells and lens fiber cells differentiated from iPS cells from a DM1 patient stained with an anti-αA crystallin antibody (DM1). The scale bar represents 200 μm. The left image shows lens epithelial cells and lens fiber cells differentiated from iPS cells from a healthy individual labeled with Cy3 (CAG). ６ Fluorescence micrographs (HC) of lens epithelial cells and lens fiber cells differentiated from iPS cells of a DM1 patient (CAG) were labeled with Cy3 after FISH using the -CA DNA / LNA probe and counterstained with DAPI. ６ Fluorescence micrographs (DM1) of lens epithelial cells and lens fiber cells differentiated from iPSCs of a DM1 patient treated with ASO-4M (same as ASO-4) were labeled with Cy3 (CAG). ６ Fluorescence micrographs (DM1+ASO-4M) of FISH using the -CA DNA / LNA probe and DAPI counterstaining. On the left, a bar graph showing the average number of RNA aggregates per cell nucleus ± standard deviation calculated from the FISH results for HC, DM1, and DM1+ASO-4M in Figure 9C. On the right, a bar graph showing the average percentage of RNA aggregates in total cells ± standard deviation calculated from the FISH results for HC, DM1, and DM1+ASO-4M in Figure 9C. On the left, a brightfield micrograph (HC) of a skin organoid derived from iPS cells of a healthy individual after 4 months of culture under differentiation conditions. On the right, a brightfield micrograph (DM1) of a skin organoid derived from iPS cells of a DM1 patient. The scale bar represents 500 μm. On the left, a Cy3-labeled skin organoid derived from iPS cells of a healthy individual (CAG). ６ Fluorescence micrographs (HC) of dermal organoids derived from iPSCs of a DM1 patient (CAG) were subjected to FISH using the -CA DNA / LNA probe and counterstained with DAPI. ６Fluorescence micrographs (DM1) of skin organoids derived from iPSCs of a DM1 patient treated with ASO-4M (same as ASO-4) and Cy3-labeled (CAG) were obtained by FISH using the -CA DNA / LNA probe and counterstained with DAPI. ６ Fluorescence micrographs (DM1+ASO-4M) of cells subjected to FISH using a -CA DNA / LNA probe and counterstained with DAPI. The scale bar represents 20 μm. A bar graph showing the mean ± standard deviation of the percentage of cells in which RNA aggregates were detected, calculated from the FISH results for HC, DM1, and DM1+ASO-4M in Figure 10B. A schematic diagram showing the procedure for an experimental system for differentiating undifferentiated iPS cells (DM-1 to 3) derived from DM1 patients and iPS cells (HC-1 to 3) derived from healthy individuals into neurons using a tetracycline-inducible NGN2 expression system. "B27 / N2 / NB+DOX" and "B27 / NB+DOX" represent the period of culture in a neuronal induction medium supplemented with doxycycline as a differentiation inducer, while "B27 / NB" represents the period of culture in a differentiated neuronal medium. Undifferentiated iPS cells transfected with a tetracycline-inducible NGN2 expression system were cultured in a neuronal induction medium, and differentiation induction was initiated on day 0. On day 8, the medium was changed to a neuronal induction medium containing no differentiation inducer, completing the differentiation induction. Analysis was performed on day 11. Fluorescence micrographs of neurons differentiated from iPS cells derived from DM1 patients, stained with an anti-tubulin βIII antibody and counterstained with DAPI. The scale bar represents 100 μm. Composite images of fluorescence micrographs of neurons differentiated from iPS cells derived from healthy individuals (HC-1 to 3) and DM1 patients (DM-1 to 3), stained with an anti-tubulin βIII antibody (top row), an anti-MAP2 antibody (middle row), or an anti-tubulin βIII antibody and an anti-Tbr1 antibody, and counterstained with DAPI. Neurons differentiated from iPS cells derived from healthy individuals (HC-1 to 3) and iPS cells derived from DM1 patients (DM-1 to 3) were labeled with Cy3 (CAG). ６Fluorescence micrographs of FISH using the α-CA DNA / LNA probe and DAPI counterstaining. The left bar graph shows the average number of RNA aggregates per cell nucleus ± standard deviation calculated from the FISH results for each of HC-1 to 3 and DM-1 to 3 in Figure 11C. The right bar graph shows the average percentage of RNA aggregates in all cells ± standard deviation calculated from the FISH results for each of HC-1 to 3 and DM-1 to 3 in Figure 11C. The top left bar graph shows Cy3-labeled neurons derived from DM1 patient iPS cells treated with control ASO (CAG). ６ A composite image of fluorescence micrographs (Control) of neurons derived from iPSCs from a DM1 patient treated with ASO-4M (same as ASO-4) and Cy3-labeled neurons (CAG) was obtained by FISH using a -CA DNA / LNA probe, followed by staining with an anti-tubulin βIII antibody and counterstaining with DAPI. ６ A composite image of fluorescence micrographs (ASO-4M) obtained by FISH using a -CA DNA / LNA probe, staining with an anti-tubulin βIII antibody, and counterstaining with DAPI. The bottom left bar graph shows the mean number of RNA aggregates per cell nucleus ± standard deviation of neurons derived from DM1 patient iPSCs treated with ASO-4M or control ASO, or without ASO treatment, calculated from the FISH results shown in the top of Figure 11F (ASO-4M, Control ASO, or Untreated, respectively). The bottom right bar graph shows the mean ± standard deviation of the percentage of RNA aggregates in total cells of neurons derived from DM1 patient iPSCs treated with ASO-4M or control ASO, or without ASO treatment, calculated from the FISH results shown in the top of Figure 11F (ASO-4M, Control ASO, or Untreated, respectively).

[0033] One embodiment of the present invention is a pharmaceutical composition comprising an oligonucleotide having any base sequence of a repeat sequence in which 5 to 13 cytosine-adenine-guanine trinucleotides are repeated from the 5' to 3' end, and the oligonucleotide has an RNase H-inactivating nucleotide analog and / or a mixmer structure containing at least two types of internucleotide linkages.

[0034] The base sequences of the oligonucleotides described herein are set forth in SEQ ID NOs: 1 to 23 in the Sequence Listing attached to the present specification. Among these sequences, the base sequences of repeating sequences in which cytosine-adenine-guanine trinucleotides are repeated 5 to 13 times from the 5' end to the 3' end, and the correspondence between these sequences and the SEQ ID NOs in the Sequence Listing attached to the present specification are shown in Table 1.

[0035]

[0036] In Table 1, "(CAG) ｎ " refers to an oligonucleotide having n trinucleotide repeats in which cytidine, adenosine, and guanosine are linked in this order from the 5' end to the 3' end, where n is any integer from 5 to 13.

[0037] The antisense oligonucleotides administered to iPS cells in the Examples herein are ASO-1 to 5, and the ASOs used as controls are ASO-A to C. The nucleotide sequences of ASO-1 to 3, 4, and 5 are listed as SEQ ID NOS: 11 to 13, 3, and 14, respectively, in the Sequence Listing attached to this specification. Their nucleotide sequences and structural features are shown in Table 2.

[0038]

[0039] In the structure of each ASO in Table 2, "A," "G," and "C" represent adenosine, guanosine, and cytidine, respectively; "dA," "dG," "dC," and "dT" represent deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine, respectively; adjacent nucleotides are linked by a phosphodiester bond; the nucleotide to the right of the subscript "E" represents a β-D-ENA nucleic acid (hereinafter referred to as "ENA"); and the nucleotide to the right of the subscript "m" represents a 2'-O-methyl (hereinafter referred to as "2'-OMe") nucleotide analog. Nucleotide analogs that can be used in the ASO of the present invention are described in detail below.

[0040] Among the ASOs, ASO-C, ASO-1 to ASO-3, and ASO-5 have a gapmer structure in which the nucleosides constituting the wing regions are ENA and the nucleosides constituting the gap segment are DNA (hereinafter referred to as "ENA / DNA gapmers"). In ASO-1 to ASO-3 and ASO-5, all nucleotides of the gapmer pair with the RNA of the DMPK gene of myotonic dystrophy type 1 patients, and the nucleotides constituting the gap segment are DNA. Therefore, when the gapmer pairs with the RNA of the DMPK gene, the gap segment becomes a heteroduplex of DNA and RNA, which is degraded by RNase H. On the other hand, ASO-4 has a mixmer structure of ENA and 2'-O-methylribonucleotides (hereinafter referred to as "ENA / 2'-OMe ribonucleotide mixmer"), in which β-D-ENA-modified cytidine, 2'-OMe-adenosine, and 2'-OMe-guanosine are repeated tandemly seven times from 5' to 3' in this order. The entire length of this mixmer pairs with the CUG trinucleotide repeat sequence in the 3' UTR of the RNA of the DMPK gene of myotonic dystrophy type 1 patients, but because ASO-4 does not contain DNA, it is not degraded by RNase H. The ASO-C of the present invention has the same base sequence as the oligonucleotide consisting of a nonspecific sequence used in an inhibition experiment using an antisense oligonucleotide in another study (Hagemann, TL et al., Ann Neurol, 83:27-39 (2018)), and the gap segment of the gapmer is also deoxyribonucleotides. However, the wing region of the gapmer in the other study is a 2'-O-methoxyethyl (hereinafter referred to as "MOE") nucleotide analog, whereas the wing region of the ASO-C used as a control in this specification is an ENA.

[0041] Table 3 below compares the structures of ASOs in the examples of the present invention with those in the related prior art. Because the ASOs in the examples of the present invention have the same nucleotide sequences as those in the related prior art, the nucleotide sequences of ASO-1 and PS115 are listed as SEQ ID NO: 11 in the sequence listing attached hereto. The nucleotide sequences of ASO-2 and ASO-A (ISIS 445569) are listed as SEQ ID NO: 12. The nucleotide sequences of ASO-3 and PS116 are listed as SEQ ID NO: 13. The nucleotide sequences of ASO-4 and ASO-B (PS58) are listed as SEQ ID NO: 3. The nucleotide sequences of ASO-5 and ISIS 486178 are listed as SEQ ID NO: 14. The nucleotide sequence of the control ASO is listed as SEQ ID NO: 23.

[0042]

[0043] In the structure of each ASO in Table 3, "A," "G," and "C" represent adenosine, guanosine, and cytidine, respectively; "dA," "dG," "dC," and "dT" represent deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine, respectively; and the nucleotide to the right of the subscript "k" is 6'-(S)-CH ３ "cEt" represents a bicyclic nucleotide analog; "e" represents a 2'-O-methoxyethyl (MOE) nucleotide analog; "m" represents a 2'-OMe nucleotide analog; "s" represents a phosphorothioate (PT) bond between the nucleoside to its left and the nucleotide to its right, rather than a phosphodiester bond; "mC" represents a 5-methylcytosine nucleoside.

[0044] In Table 3, "uniformly modified" means that all nucleotides of the oligonucleotide have the same modified sugar and all internucleotide bonds are the same modified bond.

[0045] A prior art related to ASO-1 is PS115, described in Non-Patent Document 7. PS115 shares the same base sequence as ASO-1. However, they differ in that all internucleoside bonds are phosphodiester bonds in ASO-1, whereas PS115 are PT bonds, and that the wing regions of ASO-1 are gapmers consisting of ENA, whereas PS115 is entirely deoxyribonucleotides.

[0046] Prior art related to ASO-2 is ISIS 445569 (hereinafter also referred to as "ASO-A"), described in Non-Patent Documents 5 and 6. ISIS 445569 and ASO-2 share the same base sequence and gapmer structure with wing regions of the same length. However, they differ in that all internucleoside linkages are phosphodiester bonds in ASO-2 but PT linkages in ISIS 445569, the gapmer wing regions are ENA in ASO-2 but MOE-nucleotide analogs in ISIS 445569, and the 5'- and 3'-terminal nucleotides are cytidine in ASO-2 but 5-methylcytidine in ISIS 445569.

[0047] A prior art related to ASO-3 is PS116, described in Non-Patent Document 7. PS116 and ASO-3 share the same base sequence. However, they differ in that all internucleoside bonds are phosphodiester bonds in ASO-3, whereas PS116 are PT bonds, and that the wing regions of ASO-3 are gapmers consisting of ENA, whereas PS115 is entirely deoxyribonucleotides.

[0048] Prior art related to ASO-4 is PS58, described in Non-Patent Documents 7 and 8. PS58 (hereinafter also referred to as "ASO-B") has the same base sequence as ASO-4, and they share the same point that all adenosine nucleotides and guanosine nucleotides are OMe nucleotide analogs. However, they differ in that the cytidine nucleotides in PS58 are also OMe nucleotide analogs, whereas in ASO-4, the cytidine nucleotides are ENA. They also differ in that all internucleotide bonds in PS58 are PT bonds, whereas all internucleotide bonds in ASO-4 are phosphodiester bonds.

[0049] Prior art related to ASO-5 is ISIS 486178, described in Non-Patent Document 6. ISIS 486178 and ASO-5 have the same base sequence and a gapmer structure with wing regions of the same length. However, while ASO-5 is an ENA / DNA gapmer, ISIS 486178 has a wing region with 6'-(S)-CH ３ It differs in that it is a gapmer (hereinafter referred to as "cEt / DNA gapmer") in that the gap region is composed of bicyclic (cEt) nucleotides and unmodified deoxyribonucleotides.

[0050] The primer sets used in triplet repeat prime PCR (hereinafter referred to as "TP-PCR") analysis are shown in Table 4. The nucleotide sequences of each primer are listed as SEQ ID NOS: 15 to 22 in the sequence listing attached hereto.

[0051]

[0052] "FAM-" in the sequence of primer FAM-P1-F in Table 4 means that 6-carboxyfluorescein is covalently bound to the 5' end of the primer oligonucleotide, and "HEX-" in the sequence of primer HEX-P2-R in Table 4 means that hexachlorofluorescein is covalently bound to the 5' end of the primer oligonucleotide.

[0053] The oligonucleotide according to the present invention has a mixmer structure that is not cleaved by RNase H. Here, the term "mixmer structure" refers to a structure of a single-stranded oligonucleotide containing a mixture of RNase H-active nucleotides and RNase H-inactive nucleotides, and the heteroduplex nucleic acid formed by pairing the single-stranded oligonucleotide with a complementary RNA is not degraded by RNase H. Monia, B.P. (J. Biological. Chem., 268: 14514 (1993)), the RNase H degradation activity of a heteroduplex nucleic acid consisting of a gapmer ASO, in which a gap region of at least five consecutive RNase H-active nucleotides is sandwiched between wing regions of RNase H-inactive nucleotides, and an RNA complementary to the ASO, exceeded 75%, whereas the RNase H degradation activity of a heteroduplex nucleic acid consisting of a gapmer ASO, in which a gap region of four or fewer consecutive RNase H-active nucleotides is sandwiched between wing regions of RNase H-inactive nucleotides, and an RNA complementary to the ASO, was less than 20%. Thus, the oligonucleotide having a mixmer structure of the present invention may have five or four or fewer consecutive RNase H-active nucleotides, for example, only five, four, three, or two. "Mixmer ASO" or "mixmer ASO" refers to an ASO having a mixmer structure.

[0054] In the present invention, whether a certain 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 certain type is flanked by wing regions consisting of known RNase H active nucleotides, for example, five 2'-OMe-nucleotide analogs. A heteroduplex is prepared by pairing 300 pmoles of the gapmer oligonucleotide with 1500 pmoles of RNA that pairs with the oligonucleotide. The heteroduplex complex and 3.0 units of E. coli RNase H are added to a reaction solution for RNase H, for example, 50 mM Tris-HCl (pH 8.0), 75 mM KCl, 3 mM MgCl.２ and 10 mM dithiothreitol to make the total volume 150 μL, and the reaction is carried out at 37° C. for 60 minutes, and the reaction product is analyzed by agarose gel electrophoresis.

[0055] Alternatively, one skilled in the art can determine whether a particular type of nucleotide analog is RNase H active or RNase H inactive. For example, EP 1 222 309 provides an in vitro method for determining RNase H activity that can be used to determine the ability to recruit RNase H.

[0056] The nucleoside monomers of the oligonucleotides contained in the pharmaceutical compositions of the present invention are linked together via internucleoside linking groups. Optionally, each monomer is linked to the 3' adjacent monomer via a linking group. The terminal 5' monomer of the oligonucleotide may or may not contain a 5' terminal group or a linking group for conjugation, but does not contain a 5' linking group.

[0057] The term "linking group" or "internucleotide linkage" refers to a group capable of covalently linking two nucleosides to form a dinucleotide. Natural nucleotides are linked by phosphodiester bonds via phosphate groups. Non-natural, i.e., modified nucleotide linkages include, but are not limited to, phosphorothioate, phosphorodithioate, and boranophosphate groups. The term "internucleoside linkage" may be used interchangeably with "internucleotide linkage." In general, oligonucleotides containing modified nucleotide linkages have higher membrane permeability and higher resistance to intracellular and extracellular nucleases than oligonucleotides linked solely by phosphodiester linkages. On the other hand, heteroduplex nucleic acids formed by intracellular hybridization of an oligonucleotide containing modified nucleotide linkages with RNA are degraded by RNase H, similar to heteroduplex nucleic acids formed by intracellular hybridization of an oligonucleotide linked solely by phosphodiester linkages with RNA. Modified nucleotide bonds, such as phosphorothioate groups, have stereoisomers with respect to the sulfur atom. The oligonucleotides contained in the pharmaceutical compositions of the present invention may be synthesized so that a specific stereoisomer of the modified nucleotide bond is used at a specific internucleotide linkage of the oligonucleotide, or may be synthesized so that some or all of the internucleotide linkages are racemic.

[0058] In one embodiment of the pharmaceutical composition of the present invention, the oligonucleotide may comprise: (1) an oligonucleotide consisting of a base sequence of any one of SEQ ID NOS: 1 to 9; (2) an oligonucleotide consisting of a base sequence consisting of adenine-guanine, a base sequence of any one of SEQ ID NOS: 1 to 9, and cytosine from the 5' to 3' end; or (3) an oligonucleotide consisting of a base sequence consisting of guanine, a base sequence of any one of SEQ ID NOS: 1 to 9, and cytosine-adenine from the 5' to 3' end.

[0059] The first oligonucleotide according to the above embodiment of the pharmaceutical composition of the present invention, i.e., "an oligonucleotide consisting of a base sequence of any one of SEQ ID NOs: 1 to 9", is an oligonucleotide consisting of a base sequence in which the number of trinucleotide repeats in which cytidine, adenosine, and guanosine are linked in this order from the 5' end to the 3' end is 5 to 13.

[0060] The second oligonucleotide according to the above embodiment of the pharmaceutical composition of the present invention, i.e., "an oligonucleotide comprising, from the 5'-end to the 3'-end, a base sequence consisting of adenine-guanine, a base sequence of any one of SEQ ID NOs: 1 to 9, and cytosine," is an oligonucleotide comprising a base sequence with 6 to 14 trinucleotide repeats in which adenosine, guanosine, and cytidine are linked in this order from the 5'-end to the 3'-end.

[0061] The third oligonucleotide according to the above embodiment of the pharmaceutical composition of the present invention, i.e., "an oligonucleotide comprising, from the 5'-end to the 3'-end, a base sequence consisting of guanosine, any of the base sequences of SEQ ID NOs: 1 to 9, and cytosine-adenine," is an oligonucleotide comprising a base sequence in which guanosine, cytidine, and adenosine are linked in this order from the 5'-end to the 3'-end, with a trinucleotide repeat count of 6 to 14.

[0062] In one embodiment of the pharmaceutical composition of the present invention, the RNase H inactivating nucleotide analogue may include a nucleotide analogue in which the 2'-position of ribose is modified and / or a nucleotide analogue in which a bridge is modified between the 2'- and 4'-positions of ribose.

[0063] Nucleotide analogs modified at the 2'-position of ribose contained in the oligonucleotide 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.

[0064] The nucleotide analogue modified at the 2'-position of ribose can be, for example, a nucleotide analogue represented by the following chemical formula (1): ＊ are independently selected from an internucleotide linkage, R, a terminal group, or a protecting group; B is selected from a natural or non-natural nucleotide base moiety (nucleobase); R is hydrogen, a hydroxyl group, C １－４ Alkyl group and C １－４ an alkoxy group].

[0065]

[0066] In chemical formula (1), a nucleotide analogue in which the substituent X attached to the 2'-position of ribose is a fluoro group is a 2'-F nucleotide analogue, a nucleotide analogue in which the substituent X attached to the 2'-position of ribose is a methoxy group is a 2'-OMe-nucleotide analogue, a nucleotide analogue in which the substituent X attached to the 2'-position of ribose is an O-methoxyethyl group is an MOE-nucleotide analogue, a nucleotide analogue in which the substituent X attached to the 2'-position of ribose is a 2'-(3-hydroxy)propyl group is a 2'-(3-hydroxy)propyl nucleotide analogue, and a nucleotide analogue in which the substituent X attached to the 2'-position of ribose is a 2'-(3-amino)propyl group is a 2'-AP nucleotide analogue.

[0067] The nucleotide analogues modified with a bridge between the 2'-position and the 4'-position of ribose contained in the oligonucleotide of the above embodiment include, but are not limited to, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE.

[0068] Nucleotide analogs modified with a bridge between the 2' and 4' positions of the ribose are called locked nucleic acids (LNA) because the conformation between the 2' and 4' positions is locked. They are also called bicyclic nucleic acids (BNA) because they are bridged. Nucleotide analogs modified with a bridge between the 2' and 4' positions of the ribose 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 β-D-configuration and α-L-configuration stereoisomers, and cEt and cMOE have R- and S-configuration stereoisomers with the carbon atom bridging the 2' and 4' positions of the ribose as an asymmetric center.

[0069] The nucleotide analogues in which the 2'-position and the 4'-position of the ribose are bridged are, for example, those represented by the following chemical formulas (2) and (3), in which the divalent atomic group "-Y-" bonded to the 2'-position of the ribose is replaced with "-O-", "-S-", or "-CH ２ -S-”, “-N(H)-”, “-N(R ａ )-", "-CH ２ -N(H)-" and "-CH ２ -N(R ａ )-" and Z and Z ＊ is the internucleotide linkage, R ｂ , a terminal group, or a protecting group; B is selected from a natural or non-natural nucleotide base moiety (nucleobase); R ａ is hydrogen and C １－４ alkyl; R ｂ is hydrogen, hydroxyl group, C １－４ Alkyl group and C １－４ It has a structure selected from alkoxy groups.

[0070] Oxy-LNA, thio-LNA, and amino-LNA in the β-D-configuration are represented by general formula (2), and oxy-LNA, thio-LNA, and amino-LNA in the α-L-configuration are represented by general formula (3). In chemical formulas (2) and (3), a nucleotide analog in which the divalent atomic group "-Y-" bonded to the 2'-position of ribose is "-O-" is oxy-LNA, and in which the divalent atomic group "-Y-" bonded to the 2'-position of ribose is "-S-" or "-CH ２ The nucleotide analogue of -S- is thio-LNA, in which the divalent atomic group "-Y-" attached to the 2' position of ribose is "-N(H)-", "-N(R ａ )-", "-CH ２ -N(H)-" and "-CH ２ -N(R ａ )-" (here R ａ is C １－４ The nucleotide analogue selected from the group consisting of nucleotides selected from alkyl, nucleotides selected from aryl ...

[0071]

[0072]

[0073] 5'-methyl-LNA in the β-D-configuration is represented by the following chemical formula (4), and 5'-methyl-LNA in the α-L-configuration is represented by the following chemical formula (5).

[0074]

[0075]

[0076] The β-D-configuration ENA is represented by the following chemical formula (6), and the α-L-configuration ENA is represented by the following chemical formula (7).

[0077]

[0078]

[0079] The R-type cEt and cMOE are represented by the following general formula (8), and the S-type cEt and cMOE are represented by the following general formula (9). In the chemical formulas (8) and (9), W is selected from a methyl group and a methoxymethyl group, and Z and Z ＊ is the internucleotide linkage, R (R is hydrogen, hydroxyl group, C１－４ Alkyl group and C １－４ an alkoxy group), a terminal group, or a protecting group; and B has a structure selected from a natural or unnatural nucleotide base moiety (nucleobase).

[0080]

[0081]

[0082] In chemical formulas (8) and (9), the nucleotide analogue in which W is a methyl group is cEt, and the nucleotide analogue in which W is a methoxymethyl group is cMOE.

[0083] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogue in which the oxygen atom at the 2'-position of ribose is modified may include a 2'-OMe-nucleotide analogue and / or an MOE-nucleotide analogue.

[0084] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogue having a bridge modified between the 2'- and 4'-positions of ribose may include β-D-oxy-L-LNA, β-D-ENA, and / or R-type cEt.

[0085] In one embodiment of the pharmaceutical composition of the present invention, all of the nucleotide analogues modified at the 2'-position of the ribose can have the same modified sugar.

[0086] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogue modified at the 2'-position of ribose can have at least two different modified sugars.

[0087] In one embodiment of the pharmaceutical composition of the present invention, all of the nucleotide analogues modified with a bridge between the 2'- and 4'-positions of the ribose can have the same modified sugar.

[0088] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogue bridged between the 2'- and 4'-positions of the ribose can have at least two different modified sugars.

[0089] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogue in which the oxygen atom at the 2'-position of ribose contained in the oligonucleotide is modified can be a 2'-OMe-nucleotide analogue and / or an MOE-nucleotide analogue.

[0090] The 2'-OMe-nucleotide analogue refers to a nucleotide analogue in which the substituent X attached to the 2'-position of ribose in the chemical formula (1) is a methoxy group and the base B is adenine, guanine, thymine, or cytosine. The MOE-nucleotide analogue refers to a nucleotide analogue in which the substituent X attached to the 2'-position of ribose in the chemical formula (1) is an O-methoxyethyl group and the base B is adenine, guanine, thymine, or cytosine.

[0091] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogue bridged between the 2'-position and the 4'-position of ribose contained in the oligonucleotide can be LNA, ENA and / or cEt.

[0092] In one embodiment of the pharmaceutical composition of the present invention, all of the nucleotide analogues modified at the 2'-position of ribose may have the same modified sugar, i.e., the substituent bonded to the 2'-position of ribose in the nucleotide analogues modified at the 2'-position of ribose contained in the oligonucleotide may be any one of a fluoro group, a methoxy group, and an O-methoxyethyl group.

[0093] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogue modified at the 2'-position of ribose can have at least two different modified sugars. That is, the nucleotide analogue modified at the 2'-position of ribose contained in the oligonucleotide can have two or three types of substituents attached to the 2'-position of ribose selected from the group consisting of a fluoro group, a methoxy group, and an O-methoxyethyl group.

[0094] In one embodiment of the pharmaceutical composition of the present invention, all of the nucleotide analogues bridged between the 2'- and 4'-positions of ribose may have the same modified sugar, i.e., the nucleotide analogues bridged between the 2'- and 4'-positions of ribose contained in the oligonucleotide may be any one of LNA, ENA, and cEt.

[0095] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analogues bridged between the 2'- and 4'-positions of ribose can have at least two different modified sugars, i.e., the nucleotide analogues bridged between the 2'- and 4'-positions of ribose contained in the oligonucleotide can be two or three types selected from the group consisting of LNA, ENA, and cEt.

[0096] In one embodiment of the pharmaceutical composition of the present invention, the oligonucleotide may comprise β-D-ENA-cytidine, 2′-OMe-adenosine and 2′-OMe-guanosine.

[0097] In one embodiment of the pharmaceutical composition of the present invention, all of the cytidines in the oligonucleotide can be β-D-ENA-cytidines, all of the adenosines can be 2'-OMe-adenosines, and all of the guanosines can be 2'-OMe-guanosines.

[0098] In one embodiment of the pharmaceutical composition of the present invention, the oligonucleotide contained in the pharmaceutical composition of the present invention may contain at least one type of nucleotide linkage selected from the group consisting of phosphorothioate, phosphorodithioate, and boranophosphate.

[0099] In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent. Those skilled in the art can appropriately select a carrier appropriate for the situation as a carrier that can be included in the pharmaceutical composition 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 sodium monohydrogen phosphate, citric acid, and sodium citrate; tonicity adjusters 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 function, but can also be used for the purpose of exerting multiple functions. Diluents that can be included in the pharmaceutical composition of the present invention include, but are not limited to, liquids such as phosphate-buffered saline.

[0100] In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention may comprise a carrier for cell membrane permeation and / or nuclear delivery, and the carrier for cell membrane permeation and / or nuclear delivery may be bound to or associated with an oligonucleotide according to the pharmaceutical composition of the present invention.

[0101] The cell membrane permeation and / or nuclear delivery carrier may comprise a peptide. Examples of peptides serving as cell membrane permeation carriers include, but are not limited to, cell membrane permeation peptides such as oligoarginines such as octaarginine, basic peptides derived from the HIV-1 Tat protein, and basic helix peptides derived from the Drosophila Antennapedia homeodomain protein. Examples of peptides serving as nuclear delivery carriers include, but are not limited to, nuclear localization signal peptides that specifically bind to importin, such as those derived from the SV40 virus large T antigen, c-myc, and nucleoplasmin.

[0102] The cell membrane permeation and / or intranuclear delivery carrier may include a peptide mimetic. A peptide mimetic as a cell membrane permeation and / or intranuclear delivery carrier refers to a synthetic compound that mimics the structure of the peptide as the cell membrane permeation and / or intranuclear delivery carrier, and binds to or associates with the oligonucleotide of the pharmaceutical composition of the present invention to promote cell membrane permeation and / or intranuclear delivery. A peptide mimetic as a cell membrane permeation and / or intranuclear delivery carrier is a compound in which all or part of the peptide as the cell membrane permeation and / or intranuclear delivery carrier is substituted with a compound other than a natural peptide, such as a heterocyclic or cyclic compound, an unnatural amino acid, a D-amino acid, or an equivalent of various functional groups.

[0103] Carriers for cell membrane penetration and / or nuclear delivery may include cationic polymers of DEAE-dextran, polyethyleneimine (PEI) and polypropyleneimine (PPI).

[0104] The cell membrane permeation and / or nuclear delivery carrier may contain a lipid. The lipid as the cell membrane permeation and / or nuclear delivery carrier may be 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-SN-glycero-3-phosphatidylcholine (DSPC), 1-palmitoyl-2-myristo ... lecithin (PMPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphatidylcholine (SMPC), hydrogenated soybean lecithin (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 ... Glycero-3-phosphoglycerol (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-(l-glycerol) (DMPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (D The lipids may include, but are not limited to, phospholipids such as 1,5-O-dihexadecyl-N-succinyl-L-glutamic acid, anionic lipids such as 1,5-O-dihexadecyl-N-succinyl-L-glutamic acid, cholesterol, PEG-conjugated 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).

[0105] The cell membrane permeation and / or nuclear delivery carrier may include a lipitoid. The lipitoid as a cell membrane permeation and / or nuclear delivery carrier refers to a synthetic compound in which a peptide and / or a peptidomimetic as the cell membrane permeation and / or nuclear delivery carrier and a lipid as the cell membrane permeation and / or nuclear delivery carrier are covalently linked.

[0106] The cell membrane permeation and / or intranuclear delivery carrier may comprise a liposome. The liposome as the cell membrane permeation and / or intranuclear delivery carrier refers to a lipid membrane structure dispersed in an aqueous solvent, encapsulating the aqueous solvent inside. The lipid membrane of the liposome may be a lipid bilayer or lipid multilayer having one or more layers. The liposome may be particulate or reticular. The lipid component of the liposome may comprise a lipid as the cell membrane permeation and / or intranuclear delivery carrier, but is not limited thereto. The lipid component of the liposome may comprise a lipitoid as the cell membrane permeation and / or intranuclear delivery carrier. The liposome as the cell membrane permeation and / or intranuclear delivery carrier may encapsulate the oligonucleotide of the pharmaceutical composition of the present invention therein.

[0107] The cell membrane permeation and / or intranuclear delivery carrier may comprise a lipid nanoparticle (LNP). The lipid nanoparticle as a cell membrane permeation and / or intranuclear delivery carrier is composed of the same lipid as the lipid component of the liposome and the oligonucleotide according to the pharmaceutical composition of the present invention. A pH-sensitive lipid that is positively charged in a low pH environment, such as 1,2-dioleyl-3-dimethylammonium propane (DODAP), may be used for the lipid nanoparticle. Unlike liposomes, lipid nanoparticles have a single lipid membrane and a core structure filled with lipids rather than enclosing an aqueous solvent inside. The lipid nanoparticle as a cell membrane permeation and / or intranuclear delivery carrier may contain the oligonucleotide according to the pharmaceutical composition of the present invention in the core structure.

[0108] In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention can be delivered by at least one parenteral administration selected from the group consisting of subcutaneous administration, intravenous administration, intraarterial administration, intramuscular administration, intraperitoneal administration, intracranial administration, and intrathecal administration. The administration may be continuous or long-term, or short-term or intermittent. The pharmaceutical compositions of the present invention result in downregulation of CUG-RNA aggregate formation and / or a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% reduction in intracellular levels of CUG-RNA or DMPK protein 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.

[0109] The pharmaceutical composition of the present invention may be administered orally in the form of, for example, tablets, capsules, granules, powders, or syrups. These preparations may contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha 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 cellulose derivatives such as calcium sulfate). Examples of suitable excipients include inorganic excipients such as sulfates, lubricants (for example, metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as Veegum and Gay's wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; sodium salts of fatty acids; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and hydrated silicic acid; and the above-mentioned starch derivatives. Examples of suitable additives include: binders (for example, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the above-mentioned excipients); disintegrants (for example, cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally crosslinked sodium carboxymethyl cellulose; and chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone); stabilizers (for example, parahydroxybenzoates 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 agents (for example, commonly used sweeteners, acidulants, fragrances, etc.).) and other additives by well-known methods.

[0110] The active ingredient contained in the pharmaceutical composition of the present invention is the oligonucleotide (when the oligonucleotide is bound to or associated with a carrier for cell membrane penetration and / or intranuclear delivery, the oligonucleotide bound to or associated with the carrier for cell membrane penetration and / or intranuclear delivery). The proportion of the active ingredient contained in the pharmaceutical composition of the present invention can be appropriately set within a range that can achieve 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.

[0111] The dosage of the pharmaceutical composition of the present invention must be carefully adjusted, taking into account the age, weight, and condition of the individual being treated, as well as the route, dosage form, and method of administration. The exact dosage must be determined by a physician. The actual dosage is within the physician's discretion and may vary depending on the specific circumstances of the present invention to achieve the desired therapeutic effect. However, the dosage of the pharmaceutical composition of the present invention is not necessarily limited by the type of active ingredient, the weight, age, and symptoms of the patient. For example, in the case of parenteral administration, the lower limit is 0.001 mg / kg body weight (preferably 0.01 mg / kg body weight) and the upper limit is 100 mg / kg body weight (preferably 10 mg / kg body weight) per dose. In the case of oral administration, the lower limit is 0.01 mg / kg body weight (preferably 0.1 mg / kg body weight) and the upper limit is 1000 mg / kg body weight (preferably 100 mg / kg body weight) per dose, and is preferably administered once or several times per day depending on the symptoms.

[0112] The pharmaceutical composition of the present invention is administered to patients with myotonic dystrophy type 1. In the examples of the present invention, the effect of eliminating RNA aggregates was more pronounced in DM-1 and 3 cells than in DM-2 cells, and therefore patients with a large number of CTG repeats in the DMPK gene are preferred as recipients of the pharmaceutical composition of the present invention.

[0113] The most important desirable therapeutic effect used to determine the active ingredient, carrier, administration dose, and administration method of the pharmaceutical composition of the present invention is the inhibition and / or alleviation of the progression of clinical symptoms of myotonic dystrophy type 1 disease (gastrointestinal symptoms such as myotonia (prolonged muscle contraction), muscle weakness / atrophy, cataracts, retinal degeneration, cardiac conduction disorders, myocardial disorders, swallowing associated with smooth muscle disorders, constipation, and diarrhea, higher brain dysfunction, impaired glucose tolerance, hyperinsulinemia, diabetes, testicular atrophy, and endocrine disorders such as abnormal growth hormone secretion, hyperlipidemia, and skin symptoms such as pilomatricoma and epithelioma). However, pharmacodynamic biomarkers (formation of DMPK RNA aggregates and / or reduction and / or elimination of deregulation of RNA-binding molecules including CUG-BP1 and MBNL1) can also be used as surrogate endpoints preceding clinical symptoms to determine the active ingredient, carrier, administration dose, and administration method of the pharmaceutical composition of the present invention.

[0114] The pharmaceutical composition of the present invention can be delivered by any one or more parenteral routes selected from the group consisting of transdermal administration, intraocular administration, intralental administration, gastrointestinal intramucosal administration, gastrointestinal submucosal administration, subcutaneous administration, intravenous administration, intraarterial administration, intramuscular administration, intraperitoneal administration, intracranial administration, and intrathecal administration. Furthermore, the pharmaceutical composition of the present invention can be administered to target tissues, including, but not limited to, ocular tissues including skeletal muscle, cardiac muscle, smooth muscle, satellite cells, lens, retina, etc., the gastrointestinal tract from the esophagus to the colon, nerves of the central nervous system and peripheral nervous system, pancreas, adrenal gland, pituitary gland and endocrine system tissues involved in the control of these, skin tissue, etc., depending on the clinical symptoms of the patient.

[0115] In one embodiment of the assay system for the pharmaceutical composition of the present invention, the assay system comprises undifferentiated iPS cells established from a human suffering from myotonic dystrophy type 1, and a compound to be evaluated. The compound to be evaluated comprises an oligonucleotide that targets the RNA of myotonic dystrophy protein kinase in myotonic dystrophy type 1.

[0116] In this specification, the conjunction "about" used to modify a numerical value means a numerical range of 90% or more and 110% or less of the numerical value. For example, "about 40 bases" refers to a numerical range of 36 bases or more and 44 bases or less.

[0117] All documents mentioned herein are incorporated by reference in their entirety.

[0118] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention.

[0119] Example 1: Establishment of iPSCs from Myotonic Dystrophy Patients (1.1) Materials and Methods. iPSCs were generated by reprogramming peripheral blood mononuclear cells (PBMCs) collected from three myotonic dystrophy type 1 patients (experimental group) and two healthy control subjects, as well as skin fibroblasts collected from one healthy control subject, with an episomal vector encoding SOX2, KLF4, OCT4, L-MYC, LIN28, a p53 carboxyl-terminal dominant-negative fragment, and EBNA1 (Okita, K. et al., Stem Cells, 31:458-466 (2013)). Authenticity of the cell lines was confirmed by STR analysis using the PowerPlex® 16 system (Promega, Madison, Wisconsin, USA). Karyotype and Southern blot analysis were performed by LSI Medience Corporation (Tokyo, Japan). iPS cells were cultured and maintained on culture vessels coated with iMatrix-511 (892012, Nippi Corporation, Tokyo, Japan) in StemFit® medium (AK02N, Ajinomoto Co., Inc., Tokyo, Japan) as described by Nakagawa, M. et al. (Sci Rep, 4: 3594 (2014)).

[0120] TP-PCR analysis in the Examples herein was carried out as follows. Genomic DNA from iPS cells was extracted using a PureLink™ Genomic DNA Mini Kit (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and subjected to TP-PCR analysis as described in Singh, S. et al. (Front Genet, 5: 94 (2014)) and Non-Patent Document 1 (Chakraborty, S. et al., Current Protocols in Human Genetics, 91: 9.29.1-9.29.19. (2016)). The base sequences of the primers used in TP-PCR analysis are shown in Table 4 above.

[0121] Immunocytochemical analysis in the Examples herein was performed as follows. Cells were fixed in PBS containing 4% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) for 20 minutes at room temperature and then incubated in blocking buffer (PBS containing 5% Blocking One (Nacalai Tesque, Kyoto, Japan) and 0.2% Triton-X100 (Nacalai Tesque, Kyoto, Japan)). The sources and dilutions of the primary and secondary antibodies used in the Examples herein are listed in Table 5 below. The primary antibodies were incubated with the cells in the blocking buffer at 4°C for 16 hours, and the secondary antibodies were incubated at room temperature for 1 hour. Nuclei were stained with DAPI. Images were acquired using an Opera Phoenix™ High Content Screening System (PerkinElmer, Waltham, MA) or a BZ-X710 microscope (Keyence, Osaka, Japan).

[0122]

[0123] Embryoid body formation assays were performed to differentiate iPS cells into the three germ layers (Suga, M. et al., Stem Cell Res. 36: 101406 (2019)). Embryoid bodies were formed by culturing iPS cells (9,000 cells per embryoid body) dissociated with 0.5x TrypLE Select in a low-adhesion, V-bottom 96-well plate for 11 days in DMEM / F12 medium supplemented with 20% KSR, 2 mM L-glutamine, 0.1 mM NEAA, 0.1 mM 2-mercaptoethanol (all Gibco (Thermo Fisher Scientific)), and 10 μM Y-27632 (Nacalai Tesque)). They were then cultured for 7 days in DMEM supplemented with 10% FBS (Gibco (Thermo Fisher Scientific)) on tissue culture plates coated with Matrigel (BD Biosciences). The differentiation potential into mesoderm was assessed by determining whether smooth muscle cells expressing smooth muscle actin differentiated. The differentiation potential into endoderm was assessed by determining whether definitive endoderm cells expressing SOX17 differentiated. The differentiation potential into ectoderm was assessed by determining whether motor neurons expressing βIII-tubulin differentiated.

[0124] The cell types of differentiated cells derived from iPS cells used to examine the effect of ASO treatment on the formation of nuclear RNA aggregates, the culture method for differentiated cells, the method for inducing differentiated cells from iPS cells, and the ASO treatment method are outlined in Table 6. Details of each method are provided in the examples using the respective differentiated cells.

[0125]

[0126] The bibliographic information of the references listed in Table 6 is as follows: Kokubu, Y. et al. (2019) Stem Cells Transl Med, DOI: 10.1002 / sctm.18-0280 Faustino Martins, J. M. et al. (2020) Cell Stem Cell, 26: 172-186 e176. Imamura, K. et al. (2016) Sci Rep., 6:34904. Fu, Q. et al. (2017) Invest Ophthalmol Vis Sci. 58: 517-527. Lee, J. et al. (2020) Nature, 582: 399-404.

[0127] Cy3-labeled (CAG) RNA aggregates were used to detect nuclear RNA aggregates. ６ Fluorescence in situ hybridization (hereinafter referred to as "FISH") was performed using the -CA DNA / LNA probe. Cells were cultured on a clear-bottom microplate (CellCarrier-96 Ultra, PerkinElmer), fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 70% ethanol at -30°C for at least 2 hours, rehydrated with 1x SSC prepared with diethyl dicarbonate (DEPC) water, and incubated at 55°C for 2 hours in hybridization pretreatment buffer [2x SSC prepared with DEPC water and 50% formamide]. The cells were then hybridized with hybridization buffer [2x SSC, 50% formamide, 10% dextrin sulfate, and 0.1 ng / μL Cy3-labeled (CAG) ６The cells were incubated overnight at 55°C with a 1xSSC (C16-CA DNA / LNA probe (Gene Design, Osaka)) (de Mezer, M. et al., Nucleic Acids Res, 39:3852 (2011)). The cells were washed twice with the hybridization pretreatment buffer at 55°C for 20 minutes and twice with 1xSSC prepared in DEPC water at room temperature for 5 minutes. The cells were further incubated at 37°C for 60 minutes with 1xSSC prepared in DEPC water and washed twice with 1xSSC prepared in DEPC water. The cells were stained with 1 μg / mL 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, Thermo Fisher Scientific) in PBS for 10 minutes at room temperature and then washed with PBS. Images were analyzed on an Opera Phoenix™ high content screening system (PerkinElmer) using a 40x water immersion objective.

[0128] (1.2) Results iPS cells derived from three DM1 patients in the experimental group and three healthy individuals in the control group were designated DM-1 to 3 and HC-1 to 3, respectively. No mycoplasma contamination was detected in the cell culture. Table 7 shows the starting cells for DM-1 to 3 and HC-1 to 3, the donor's gender and age, the number of CTG repeats analyzed by Southern blotting, and the vector used to induce pluripotent stem cells. Among the starting cells in Table 7, PBMC refers to peripheral blood mononuclear cells.

[0129]

[0130] Figure 1A is a panel of micrographs showing the expression of undifferentiated pluripotent stem cell markers in DM-1 to 3 and HC-1 to 3. The top row is a phase-contrast micrograph of a monolayer culture of undifferentiated iPS cells. The second row is a fluorescence micrograph of a monolayer culture of undifferentiated iPS cells stained with an antibody against NANOG and counterstained with DAPI for the cell nuclei. The third row is a fluorescence micrograph of a monolayer culture of undifferentiated iPS cells stained with an antibody against OCT4 and counterstained with DAPI for the cell nuclei. The fourth row is a fluorescence micrograph of a monolayer culture of undifferentiated iPS cells stained with an antibody against SSEA4 and counterstained with DAPI for the cell nuclei. The fifth row is a fluorescence micrograph of a monolayer culture of undifferentiated iPS cells stained with an antibody against TRA-1-60 and counterstained with DAPI for the cell nuclei. The scale bar in the lower right corner of each micrograph represents 200 μm.

[0131] As shown in Figure 1A, DM-1 to 3 and HC-1 to 3 all exhibited a morphology resembling that of human undifferentiated embryonic stem cells, with cells tightly adhered together and the boundaries between individual cells indistinguishable under a phase-contrast microscope. Furthermore, DM-1 to 3 and HC-1 to 3 all expressed NANOG, OCT4, SSEA4, and TRA-1-60, which are markers of undifferentiated human iPS cells, and thus possessed the characteristics of undifferentiated human iPS cells.

[0132] Figure 1B is a panel of micrographs showing the expression of three germ layer differentiation markers in cells differentiated from DM-1 to 3 and HC-1 to 3. The first row is a fluorescent micrograph in which cells differentiated from each iPS cell into mesoderm were stained with an antibody against α-smooth muscle actin (SMA) and the cell nuclei were counterstained with DAPI. The second row is a fluorescent micrograph in which cells differentiated from each iPS cell into endoderm were stained with an antibody against SOX17 and the cell nuclei were counterstained with DAPI. The third row is a fluorescent micrograph in which cells differentiated from each iPS cell into ectoderm were stained with an antibody against βIII-tubulin and the cell nuclei were counterstained with DAPI. The scale bar in the lower right corner of each fluorescent micrograph represents 50 μm.

[0133] As shown in Figure 1B, DM-1 to 3 and HC-1 to 3 all exhibited the ability to differentiate into mesodermal cells (α-smooth muscle actin), endodermal cells (SOX17), and ectodermal cells (βIII-tubulin), demonstrating that iPS-1 to 3 and HC-1 to 3 are all undifferentiated human iPS cells with pluripotency.

[0134] Figure 1C is a capillary electrophoresis image of the TPPCR reaction products of DM-1 to 3 and HC-1 to 3. As shown in Figure 1C, the repeat numbers of the CTG repeat sequences of the DMPK genes of DM-1 to 3 and HC-1 to 3 did not differ from the repeat numbers of the donor genomic DNA analyzed by Southern blotting shown in Table 7.

[0135] Figure 2 shows the Cy3-labeled (CAG) iPS cells (DM-3) derived from a DM1 patient. ６ This panel shows fluorescent micrographs of the results of fluorescent in situ hybridization (hereinafter referred to as "FISH") using a -CA DNA / LNA probe. The upper left or lower left panels show the results of FISH performed without RNase A treatment, while the upper right and lower right panels show the results of FISH performed after RNase A treatment. The upper left and upper right panels are fluorescent micrographs in which cell nuclei were counterstained with DAPI after FISH, and the lower left and lower right panels are fluorescent micrographs in which FISH was performed only without DAPI staining.

[0136] As shown in Figure 2, bright spots observed when FISH was performed without RNase A treatment were no longer observed when FISH was performed after RNase A treatment. These experimental results indicated that the bright spots were signals from CUG-RNA aggregates (CUG-RNA foci) that had hybridized with the probe.

[0137] Figure 3A shows the Cy3-labeled (CAG) undifferentiated cells of DM-1 to 3 and HC-1 to 3. ６ This panel shows fluorescence micrographs of the results of FISH using the -CA DNA / LNA probe. The upper right corner of each photograph shows a high-magnification fluorescence micrograph of a single nucleus. The scale bar at the bottom right of each fluorescence micrograph represents 10 μm.

[0138] Next, based on the FISH results in Figure 3A, image analysis of RNA aggregates was performed using the Opera Phoenix™ High Content Screening System (PerkinElmer).

[0139] The left side of Figure 3B is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results in Figure 3A. The right side of Figure 3B is a bar graph showing the percentage of RNA aggregate-positive cell nuclei, as detected by DAPI staining, among all cell nuclei, calculated from the FISH results in Figure 3A. "**" indicates that the values ​​for DM-1 to 3 are significantly different from the values ​​for HC-1 to 3 (p<0.01).

[0140] Image analysis results shown in Figure 3B revealed that the number of RNA aggregates per cell nucleus and the number of cells with CUG-RNA aggregates were significantly increased in iPSCs derived from DM1 patients compared to iPSCs derived from healthy individuals. Furthermore, among iPSCs derived from DM1 patients, DM-1 (over 1000 repeats) and DM-3 (over 2300 repeats), which have a high number of CTG repeats (Table 7), had a high number of RNA aggregates (3.32 ± 0.23 (mean ± standard deviation) for DM-1 and 1.87 ± 0.23 for DM-3, as shown in Figure 3B. In contrast, DM-2, which has a low number of CTG repeats (over 150 repeats), also had a significantly lower number of RNA aggregates (0.078 ± 0.009) (p < 0.01).

[0141] These results demonstrate that the iPS cells derived from DM1 patients produced in this study possess the basic characteristics of pluripotent stem cells and reproduce the phenotype of cells from myotonic dystrophy type 1 patients.

[0142] Example 2 Evaluation of ASOs Using iPS Cells Derived from DM1 Patients (1) Undifferentiated Cells (2.1) Materials and Methods Figure 4A is a schematic diagram showing the relative positions of the complementary sequences of the ASOs in this example on DMPK-pre mRNA, as well as the structural types of the ASOs. ASO-1 to 3 are complementary to the sequence of the non-coding region of exon 15 on DMPK-pre mRNA, ASO-4 is complementary to the CUG repeat sequence in the non-coding region of exon 15 on DMPK-mRNA, and ASO-5 is complementary to the sequence 3'-terminal to the repeat sequence in the non-coding region of exon 15 on pre-DMPK-mRNA. ASO-4 is a mixmer composed of ENA and 2'-OMe-nucleotide analogs, while the other ASO-1 to 3 and 5 are gapmers with ENA wing regions and unmodified deoxyribonucleotides in the gap region (hereinafter referred to as "ENA / DNA gapmers"). ASO-C, used as a negative control in the Examples herein, is an ENA / DNA gapmer that targets the transcription product of a gene completely different from DMPK.

[0143] 5A is a schematic diagram showing the relative positions of complementary sequences on DMPK pre-mRNA for ASO-2 and ASO-4 of the present example and their corresponding control ASO-A and ASO-B, as well as the structural types of the ASOs. ASO-2 and ASO-A are complementary to the same sequences in the coding region of exon 15 on DMPK mRNA, while ASO-4 and ASO-B are complementary to the same sequences in the CUG repeat sequence in the non-coding region of exon 15 on DMPK mRNA. ASO-2 is an ENA / DNA gapmer, while ASO-A is a gapmer with wing regions consisting of MOE-nucleotide analogs and a gap region consisting of unmodified deoxyribonucleotides (hereinafter referred to as "MOE / DNA gapmer"). ASO-4 is a mixmer consisting of ENA and 2'-O-Me-nucleotide analogs, whereas all nucleotides in ASO-B are 2'-O-Me-nucleotide analogs (RNase H-inactivating nucleotides).

[0144] The nucleotide sequences and structural features of ASO-1 to 5 and ASO-A to C are shown in Tables 2 and 3 in the detailed description of the invention herein. The nucleotide sequences of ASO-1 to 4 and 5 are listed as SEQ ID NOs: 11 to 14 and 3, respectively, in the sequence listing attached hereto. The nucleotide sequence of ASO-A is listed as SEQ ID NO: 10. ASO-A and B have the same nucleotide sequences as ASO-1 and 5, respectively. ENA was obtained from Kobe Natural Products Chemical Co., Ltd. (Kobe) and oligonucleotides were synthesized. ASO was administered to cultured iPS cells at a final concentration of 5 or 10 nM with DharmaFECT1 (Horizon Discovery, Cambridge, UK) according to the manufacturer's instructions. 48 hours after administration of ASO to cultured iPS cells, Cy3-labeled (CAG) was used. ６ FISH was performed using a -CA DNA / LNA probe.

[0145] (2.2) Results Figure 4B shows the results of Cy3-labeled (CAG) iPSCs 48 hours after administration of ASO-C or ASO-1 to ASO-5 to undifferentiated iPSCs (DM-1) derived from a DM1 patient. ６ Fluorescence micrographs were obtained by FISH using the -CA DNA / LNA probe and counterstained with DAPI. The scale bar at the bottom right of each fluorescence micrograph represents 10 μm.

[0146] Figure 4C is a bar graph showing the average number of RNA aggregates per cell nucleus calculated from the FISH results of undifferentiated iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-C and any of ASO-1 to ASO-5.

[0147] Figure 4D is a bar graph showing the percentage of RNA aggregate-positive nuclei detected by DAPI staining among all cell nuclei, calculated from the FISH results of undifferentiated iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-C and any of ASO-1 to ASO-5.

[0148] As shown in Figure 4B–D, all of ASO-1–5 significantly reduced RNA aggregates in undifferentiated iPSCs derived from DM1 patients. Among the ENA / DNA gapmers ASO-1–3 and 5, ASO-5 most strongly suppressed RNA aggregate formation (Figure 4C and D). When iPSCs were treated with ASO-4, a mixmer consisting of ENAs targeting CUG repeats and 2'-OMe-nucleotide analogs, RNA aggregates completely disappeared (Figure 4B–D).

[0149] 4E is a bar graph showing the proliferation and survival of undifferentiated iPS cells (DM-1) derived from a DM1 patient administered 5 nM or 10 nM ASO-C and any of ASO-1 to 5. The vertical axis represents the percentage of the number of cells, with the number of ASO-C cells taken as 1.0.

[0150] As shown in Figure 4E, ASO-1, 4, and 5 did not affect the proliferation or survival of undifferentiated iPS cells at either 5 nM or 10 nM, whereas administration of ASO-2 and 3 resulted in a decrease in cell number even at low doses.

[0151] As shown in Figure 5A, ASO-2 and ASO-A target the same sequence on DMPK-mRNA. However, they differ in that ASO-2 is an ENA / DNA gapmer, while ASO-A is an MOE / DNA gapmer. ASO-A was reported under the code name ISIS 445569 (Wheeler, T.M. et al., Nature, 488, 111 (2012)). Similarly, ASO-4 and ASO-B target the same sequence on DMPK-mRNA. However, they differ in that ASO-4 is a mixmer consisting of ENA and 2'-OMe-nucleotide analogs, while ASO-B is a mixmer in which all nucleotides are 2'-OMe-nucleotide analogs. ASO-B was reported under the code name PS58 (Mulders, S.A. et al., Proc Natl Acad Sci USA, 106:13915 (2009)). Therefore, ASO-A and B were prepared in this study and used as positive controls for ASO-2 and ASO-4, respectively.

[0152] Figure 5B shows the results of Cy3-labeled iPS cells (CAG) after administration of ASO-2 or ASO-A to undifferentiated iPS cells (DM-1) derived from a DM1 patient. ６ Fluorescence micrographs were obtained by FISH using the -CA DNA / LNA probe and counterstained with DAPI. The scale bar at the bottom right of each fluorescence micrograph represents 10 μm.

[0153] The left side of Figure 5C is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results of undifferentiated iPS cells (DM-1) derived from DM1 patients administered 5 nM or 10 nM ASO-2, ASO-A, or ASO-C. The middle side of Figure 5C is a bar graph showing the percentage of RNA aggregate-positive cell nuclei among all cell nuclei, as detected by DAPI staining, calculated from the FISH results of undifferentiated iPS cells (DM-1) derived from DM1 patients administered 5 nM or 10 nM ASO-2, ASO-A, or ASO-C. The right side of Figure 5C is a bar graph showing the proliferation and survival status of undifferentiated iPS cells (DM-1) derived from DM1 patients administered 5 nM or 10 nM ASO-2, ASO-A, or ASO-C. The vertical axis represents the percentage of cell number, where the number of ASO-C cells is set to 1.0.

[0154] Figure 5D shows the results of Cy3-labeled iPS cells (CAG) after administration of ASO-4 or ASO-B to undifferentiated iPS cells (DM-1) derived from a DM1 patient. ６ Fluorescence micrographs were obtained by FISH using the -CA DNA / LNA probe and counterstained with DAPI. The scale bar at the bottom right of each fluorescence micrograph represents 10 μm.

[0155] The left side of Figure 5E is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results of undifferentiated iPS cells (DM-1) derived from DM1 patients administered 5 nM or 10 nM ASO-4, ASO-B, or ASO-C. The middle side of Figure 5E is a bar graph showing the percentage of RNA aggregate-positive cell nuclei among all cell nuclei, calculated from the FISH results of undifferentiated iPS cells (DM-1) derived from DM1 patients administered 5 nM or 10 nM ASO-4, ASO-B, or ASO-C. The right side of Figure 5E is a bar graph showing the proliferation and survival status of undifferentiated iPS cells (DM-1) derived from DM1 patients administered 5 nM or 10 nM ASO-4, ASO-B, or ASO-C. The vertical axis represents the percentage of cell number, with the number of ASO-C cells set to 1.0.

[0156] As shown in Figure 5C, both 5 nM and 10 nM ASO-A reduced RNA aggregates without reducing the number of undifferentiated iPS cells, whereas ASO-2 reduced RNA aggregates but also reduced the number of undifferentiated iPS cells, suggesting that the effect of ASO-2 is due to cytotoxicity.

[0157] In contrast, as shown in Figure 5E, both ASO-4 and ASO-B reduced RNA aggregates at both 5 nM and 10 nM without reducing the number of undifferentiated iPS cells, suggesting that the effects of ASO-4 and ASO-B are not due to cytotoxicity. Furthermore, ASO-4 almost completely eliminated RNA aggregates, demonstrating a significantly superior RNA aggregation inhibitory effect compared to ASO-B, which retained more than half of the RNA aggregates. The difference between ASO-B and ASO-4 is that ASO-4 is a mixmer composed of ENA and 2'-OMe-nucleotide analogs, whereas ASO-B consists entirely of 2'-OMe-nucleotide analogs and all internucleotide linkages are phosphorothioates. Therefore, it is believed that the significantly superior RNA aggregation inhibitory effect of ASO-4 is due to the mixmer structure containing at least two types of RNase H-inactivating nucleotide analogs and / or internucleotide linkages.

[0158] Example 3 Differentiation of Muscle Cells from iPSCs Derived from a Myotonic Dystrophy Patient (3.1) Materials and Methods The method for differentiating muscle cells from the undifferentiated iPSCs established in this example of the present invention is as follows. A Tet-On inducible MyoD expression system (Non-Patent Document 22, Shoji, E. et al., Sci Rep, 5, 12831 (2015)) and a puromycin resistance marker were incorporated into the piggyBac vector KW110_PB_TA_ERN (AddGene, Watertown, Massachusetts, USA). The constructed vector was co-transfected with the pCyL43 vector encoding a transposase into undifferentiated iPSCs derived from a DM1 patient (DM-1 to 3) and iPSCs derived from a healthy individual (HC-1 to 3) using Lipofectamine LTX (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. iPS cell clones were selected using puromycin (Nacalai Tesque). iPS cells were seeded onto Matrigel-coated cell culture vessels in StemFit medium supplemented with 10 μM Rock inhibitor Y27632 (Nacalai Tesque). Cultures were then cultured for 2 days. The medium was then switched to α-MEM (Nacalai Tesque) supplemented with 5% KnockOut™ Serum Replacement (KSR, Thermo Fisher Scientific), 200 μM 2-mercaptoethanol (Thermo Fisher Scientific), 100 U / mL penicillin / streptomycin (Thermo Fisher Scientific), and 1 μg / mL doxycycline (Clontech, Takara Bio, Shiga). Doxycycline was removed on day 8. The medium was replaced every other day.

[0159] Figure 6A is a conceptual diagram showing the procedure for an experimental system for differentiation of undifferentiated iPS cells (DM1 patient-derived iPS cells, DM-1 to 3) and iPS cells (HC-1 to 3) derived from healthy individuals into muscle cells using a tetracycline-inducible MyoD expression system. "StemFit" represents the period of culture in medium for undifferentiated iPS cells, "KSR / α-MEM" represents the period of culture in medium for differentiated muscle cells, and "KSR / α-MEM+Dox" represents the period of culture in medium for muscle cells supplemented with doxycycline as a differentiation inducer. Undifferentiated iPS cells were transfected with the tetracycline-inducible MyoD expression system on day 0, and differentiation induction was initiated by changing the medium to medium for muscle cells supplemented with a differentiation inducer on day 2. Differentiation induction was terminated by changing the medium to medium for muscle cells without a differentiation inducer on day 8. Analysis was performed on day 12.

[0160] The cells differentiated from undifferentiated cells, DM-1 to 3 and HC-1 to 3 cells, into muscle cells were analyzed using the immunocytochemical technique and the method for detecting nuclear RNA aggregates described in Example 1.

[0161] (3.2) Results Figure 6B is a panel of fluorescence micrographs showing the expression of muscle differentiation markers in cells induced to differentiate from undifferentiated iPS cells (HC-1 to 3) derived from healthy individuals and iPS cells (DM-1 to 3) derived from DM1 patients according to the procedure shown in Figure 6A. The top panel shows fluorescence micrographs stained with an antibody against myosin heavy chain (MHC), with the cell nuclei counterstained with DAPI. The middle panel shows fluorescence micrographs stained with an antibody against α-actinin, with the cell nuclei counterstained with DAPI. The bottom panel shows fluorescence micrographs stained with an antibody against myogenin (MyoG), with the cell nuclei counterstained with DAPI.

[0162] As shown in Figure 6B, for both HC-1 to 3 and DM-1 to 3, differentiation into skeletal muscle cells expressing myosin heavy chain (MHC), α-actinin, and myogenin could be induced using the tetracycline-inducible MyoD expression system.

[0163] Figure 6C is a bar graph showing the percentage of MHC-expressing cell nuclei (MHC / DAPI) among muscle cells differentiated from undifferentiated HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. "ns" indicates that no significant difference was observed between HC-1 to 3 and DM-1 to 3 cells.

[0164] Figure 6D is a bar graph showing the percentage of cell nuclei expressing the muscle differentiation marker α-actinin (α-actinin / DAPI) among DAPI-stained positive cell nuclei of muscle cells differentiated from undifferentiated HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. "n.s." indicates that no significant difference was observed between HC-1 to 3 cells and DM-1 to 3 cells.

[0165] Figure 6E is a bar graph showing the percentage of cell nuclei expressing the muscle differentiation marker myogenin (myogenin / DAPI) among DAPI-stained cell nuclei of muscle cells induced to differentiate from undifferentiated HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. "ns" indicates that no significant difference was observed between HC-1 to 3 cells and DM-1 to 3 cells.

[0166] Figure 6F shows Cy3-labeled myocytes (CAG) differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. ６ This is a panel of fluorescence micrographs showing the results of FISH using the -CA DNA / LNA probe. The scale bar at the bottom right of each fluorescence micrograph represents 10 μm.

[0167] The left side of Figure 6G is a bar graph showing the average number of RNA aggregates per cell nucleus calculated from the FISH results for muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. The right side of Figure 6G is a bar graph showing the percentage of RNA aggregate-positive cell nuclei among all cell nuclei detected by DAPI staining, calculated from the FISH results for muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. "**" indicates that the values ​​for DM-1 to 3 are significantly different from the values ​​for HC-1 to 3 (p<0.01).

[0168] As shown in Figures 6B-D, more than 70% of myocytes derived from HC-1-3 and DM-1-3 cells expressed myosin heavy chain (MHC) and α-actinin. As shown in Figure 6E, in myocytes derived from HC-1 and DM-1 cells, 60-70% of DAPI-positive nuclei expressed myogenin. However, in myocytes derived from HC-2 and 3 cells and DM-2 and 3 cells, only 20-40% of DAPI-positive nuclei expressed myogenin.

[0169] Example 4 Evaluation of ASO using iPS cells derived from DM1 patients (2) Muscle cells (4.1) Materials and methods Muscle cells derived from HC-1 to 3 and DM-1 to 3 described in Example 3 were analyzed using the method for detecting nuclear RNA aggregates described in Example 1 and the method for administering ASO to cultured iPS cells described in Example 2.

[0170] (4.2) Results Figure 6F shows Cy3-labeled (CAG) myocytes differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. ６ This is a panel of fluorescence micrographs showing the results of FISH using the -CA DNA / LNA probe. The scale bar at the bottom right of each fluorescence micrograph represents 10 μm.

[0171] The left side of Figure 6G is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results for each of the muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. The right side of Figure 6G is a bar graph showing the percentage of RNA aggregate-positive cell nuclei, as detected by DAPI staining, among all cell nuclei, calculated from the FISH results for each of the muscle cells differentiated from HC-1 to 3 and DM-1 to 3 cells according to the procedure shown in Figure 6A. "**" indicates that the values ​​for DM-1 to 3 are significantly different from the values ​​for HC-1 to 3 (p<0.01).

[0172] As shown in Figures 6F and 6G, RNA aggregates were detected in muscle cells derived from iPS cells (DM-1 to 3) derived from DM1 patients. Comparing the graphs in Figures 3B and 6G, there was no significant difference in the percentage of RNA aggregate-positive nuclei among all nuclei between undifferentiated DM-1 to 3 cells and DM-1 to 3-derived muscle cells. However, there was a significant difference in the average number of RNA aggregates per nucleus between undifferentiated DM-1 and 3 cells (2 to 3.5) and DM-1 and 3-derived muscle cells (11 to 16). Furthermore, almost no RNA aggregates were detected in DM-2-derived muscle cells.

[0173] Figure 7A shows the results of the administration of 10 nM ASO-C or ASO-4 to DM-1 to DM-3 derived myocytes by lipofection followed by Cy3 labeling (CAG). ６ -CA DNA / LNA probe and DAPI counterstaining.

[0174] FIG. 7B is a bar graph showing the average number of RNA aggregates per cell nucleus calculated from the FISH results of myocytes derived from DM-1 to 3 cells administered 10 nM ASO-C or ASO-4.

[0175] Figure 7C is a bar graph showing the percentage of RNA aggregate-positive nuclei detected by DAPI staining among all nuclei (ASO-C-treated muscle cells set at 100%), calculated from the results of FISH of muscle cells derived from DM-1 to DM-3 cells treated with 10 nM ASO-C or ASO-4.

[0176] Figure 7D is a bar graph showing the proliferation and survival of muscle cells derived from DM-1 to DM-3 cells administered with 10 nM ASO-C or ASO-4. In each bar graph, the vertical axis represents the percentage of the number of cells in the ASO-C group, with the number of cells set to 1.0.

[0177] As shown in Figures 7A-D, ASO-4 administration significantly reduced the average number of RNA aggregates per nucleus in DM-1 and DM-3 cell-derived muscle cells compared with ASO-C. ASO-4 administration showed little change in the average number of RNA aggregates per nucleus in DM-2 cell-derived muscle cells. This may be related to the fact that DM-2 cells were derived from a patient with a low number of CTG repeats in the DMPK gene. As shown in Table 7 and Figure 1C, the donor of DM-2 cells had a maximum of 150 CTG repeats, which is significantly lower than the 1000-1150 and 2300-2850 CTG repeats of the donors of DM-1 and DM-3 cells, respectively. Therefore, it is believed that the effect of ASO-4 administration on reducing RNA aggregates is less pronounced in DM-2 cells than in DM-1 and DM-3 cells.

[0178] From these results, it was concluded that ASO-4 can reduce RNA aggregates in skeletal muscle cells derived from DM1 patient iPS cells.

[0179] Example 5 Differentiation of Neuromuscular Organoids from iPSCs Derived from Patients with Myotonic Dystrophy (5.1) Materials and Methods Neuromuscular organoids were generated using a protocol published by Faustino Martins, J. M. et al. (Cell Stem Cell, 26: 172-186 e176. (2020)) with minor modifications. As outlined in the schematic diagram in Figure 8A, 70% confluent iPSCs were dissociated into single cells and cultured at a density of 75,000 cells / cm in Neurobasal (NB) medium supplemented with 10 μM Y27632, 3 μM CHIR99021 (Tocris Bioscience), and 40 ng / mL FGF-2 (Wako). ２The cells were seeded at a density of 1000 μg / ml onto Matrigel-coated culture vessels. NB was a 1:1 mixture of Advanced Dulbecco's Modified Eagle Medium F12 (Gibco) supplemented with 1x N2 (Gibco) and Neurobasal Medium (Gibco) supplemented with 1x B27 (Gibco), 2 mM L-glutamine (Gibco), 75 μg / mL BSA Fraction V (Sigma), and 0.1 mM 2-mercaptoethanol (Gibco). The next day, Y27632 was removed, and the cells were allowed to differentiate into neuromesodermal progenitor cells (NMPs) for 2 days. On day 0 of organoid formation, mNMPs were dissociated using Accumax (Innovative Cell Technologies), and 4,500 to 7,200 NMPs suspended in 100 μL of NB medium supplemented with 50 μM Y27632, 10 ng / mL FGF-2, 2 ng / mL IGF, and 2 ng / mL HGF (Peprotech) were seeded into a PrimeSurface 96-well plate per well. On day 2, half of the culture medium was replaced with NB medium supplemented with 2 ng / mL IGF and 2 ng / mL HGF. From day 4 onward, organoids were maintained in NB medium. On day 10, organoids were transferred to a 60 mm culture dish, and on day 30, they were transferred to a 100 mm culture dish. Organoids were cultured with a rotation at 75 rpm and replaced with fresh NB medium twice a week. For ASO treatment, organoids were cultured with a rotation in NB medium containing 500 nM ASO.

[0180] Immunohistochemical analysis of organoids was performed as follows. Organoids were fixed in 4% paraformaldehyde at room temperature for 15 minutes, washed three times in PBS for 10 minutes, and then immersed overnight in 30% sucrose solution for settling. Organoids were embedded in OCT compound (Sakura Finetech Japan Co., Ltd., Tokyo) and flash-frozen in liquid nitrogen. Frozen organoids were cut into 12-μm-thick cryosections using a cryostat (CM1850, Leica Microsystems, Wetzlar, Germany) at -18°C to -20°C. For immunohistochemistry, sections were permeabilized in 0.5% Triton-X100 / PBS (0.5% PBST) at room temperature for 30 minutes and then incubated in Blocking One Histo (Nacalai Tesque) at room temperature for 2 hours. Sections were incubated with primary antibodies in blocking solution overnight at 4°C. After washing four times with 0.1% PBST for 15 minutes each time, samples were incubated with secondary antibodies at room temperature for 2 hours in the dark. Samples were then washed four times with 0.1% PBST for 15 minutes each time and mounted with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific). Data were acquired using a confocal laser scanning microscope (Nikon A1, Nikon, Tokyo). Images were analyzed using NIS-Element AR Analysis (version 5.11.0.1, Nikon).

[0181] (5.2) Results To verify whether the ASO of the present invention reduces or eliminates RNA aggregates not only in muscle cells but also in muscle-differentiating progenitor cells and skeletal muscle fibers, neuromuscular organoids (NMOs) were cultured in 3D for 50 days according to the method reported by Faustino Martins, J.M. et al. The 3D aggregates were formed from iPS cell-derived neuromuscular precursor cells (NMPs), which are destined to differentiate into neuroectodermal and mesodermal cells. As shown in Figure 8B, the 3D aggregates grew into organoids containing neural and mesodermal regions by day 5. By day 50, the NMOs exhibited a neural compartment rich in TUJ1-expressing neurons and a skeletal muscle compartment rich in MHC-expressing cells. As shown on the left side of the panel in Figure 8B, immunofluorescence analysis of muscle regions of NMOs on day 50 revealed expression of the sarcomeric protein TITIN in skeletal muscle fibers.

[0182] As shown in the upper panel of Figure 8C, PAX7-expressing muscle progenitor / satellite cells were observed along with desmin-expressing myocytes and myotubes in the skeletal muscle compartment of organoids. As shown in the lower panel of Figure 8C, RNA aggregates were detected in both PAX7-expressing and desmin-expressing cells in neuromuscular organoids derived from iPSCs of DM1 patients.

[0183] Example 6 Evaluation of ASO using iPS cells derived from DM1 patients (3) Neuromuscular organoids (6.1) Materials and methods Differentiation of neuromuscular organoids from iPS cells derived from healthy individuals and myotonic dystrophy patients and immunohistochemical examination of the organoids were performed in the same manner as in Example 5. Cy3-labeled (CAG) was used to detect intranuclear RNA aggregates. ６ FISH using the -CA DNA / LNA probe was performed in the same manner as in Example 1. For ASO treatment of organoids, organoids were cultured in NB medium containing 500 nM ASO for 7 days with rotation.

[0184] (6.2) Results As shown in the bottom panel of Figure 8C and Figure 8D, treatment of neuromuscular organoids derived from iPS cells of a DM1 patient with 500 nM ASO-4M reduced the number of RNA aggregates in both desmin-expressing muscle cells and PAX7-expressing muscle progenitor / satellite cells.

[0185] Furthermore, the upper photograph in Figure 8C revealed that the number of PAX7-expressing muscle precursor cells / satellite cells was reduced on day 50 of culture in the neuromuscular organoids derived from iPS cells from DM1 patients compared with that in the neuromuscular organoids derived from iPS cells from healthy individuals. Therefore, we investigated the ratio of the number of PAX7-expressing cells to the total number of cells (PAX7) in the skeletal muscle compartment of NMOs. ＋ / DAPI), and the ratio of the number of PAX7-expressing cells to the number of myogenic cells expressing MYOD (PAX7 ＋ / MYOD ＋ ) were analyzed (Figures 8E and 8F). Compared with neuromuscular organoids derived from healthy controls, the number of PAX7-expressing muscle progenitor cells / satellite cells was significantly reduced in neuromuscular organoids derived from DM1 patients. Conversely, the ratio of the number of MYOD-expressing myogenic cells to the total number of cells, and the ratio of the number of Ki67-expressing cells to the number of PAX7-expressing muscle progenitor cells / satellite cells, were unchanged between DM1 patients and healthy controls. These results suggest that satellite cells are reduced in neuromuscular organoids from DM1 patients, but that treatment with ASO-4M restored the number to the same level as in neuromuscular organoids from healthy controls.

[0186] The results of this example demonstrated that DM1-specific intranuclear RNA aggregate formation was detected in both desmin-expressing muscle cells and PAX7-expressing muscle progenitor / satellite cells. It was also revealed that PAX7-expressing cells were reduced in neuromuscular organoids from DM1 patients. These results suggest that the action of DMPK RNA with expanded CUG repeat sequences may affect the entire process of skeletal muscle differentiation, from satellite cell behavior to myofiber formation. Therefore, the 3D muscle organoid culture experimental system offers a new dimension as an in vitro disease model.

[0187] Example 7 Differentiation of Lens Epithelial Cells and Lens Fiber Cells from iPS Cells Derived from a Patient with Myotonic Dystrophy (7.1) Materials and Methods Lens epithelial cells and lens fiber cells were generated from undifferentiated iPS cells based on the method of Fu, Q. et al. (Invest Ophthalmol Vis Sci. 58: 517-527 (2017)), a reference listed in Table 6. First, approximately 80 iPS cell colonies, each consisting of approximately 20 colonies, were seeded and cultured in mTesR medium (Stemcell, Vancouver, Canada) on a Matrigel-coated culture dish. Four hours after plating, iPS cells were induced to differentiate into ectoderm / neurectoderm using 100 ng / ml of the BMP inhibitor noggin until the first epithelial-like cells appeared at the periphery of the colony on day 6 of culture. Approximately 50 differentiated iPS cells were then mechanically dissociated from the surrounding epithelial cells, and 30-50 differentiated iPS cell colonies were selected and replated on Matrigel-coated culture dishes. BMP signaling was then reactivated using its agonists, BMP4 and BMP7 (20 ng / ml). Fibroblast growth factor signaling was simultaneously activated with bFGF (100 ng / ml). Morphologically distinct from surrounding cells, cell clusters containing lens epithelial cells extending onto the culture substrate were observed on day 11 of culture. On day 15 of culture, BMP4 and BMP7 were replaced with Wnt3a (20 ng / ml) to activate the Wnt signal and induce differentiation of lens epithelial cells into lens fiber cells.

[0188] Differentiation into lens epithelial cells and lens fiber cells was confirmed using αA-crystallin as a marker. Immunocytochemical detection using anti-αA-crystallin antibodies was performed as in Example 1. In addition, iPS cell-derived cultures containing cells differentiated into lens epithelial cells and lens fiber cells were analyzed using the method for detecting nuclear RNA aggregates described in Example 1.

[0189] (7.2) Results Figure 9A shows phase-contrast micrographs of lens epithelial cells and lens fiber cells (HC and DM1, respectively) derived from iPS cells of a healthy subject and a DM1 patient. The scale bar in Figure 9A represents 100 μm. Figure 9B shows fluorescence micrographs of lens epithelial cells and lens fiber cells (HC and DM1, respectively) derived from iPS cells of a healthy subject and a DM1 patient, stained with an anti-αA-crystallin antibody, with the cell nuclei counterstained with DAPI. The scale bar in Figure 9B represents 200 μm. Figure 9B confirms that the flattened cells observed in Figure 9A express αA-crystallin and are therefore lens epithelial cells and lens fiber cells.

[0190] Figure 9C shows fluorescent micrographs of iPSC-derived lens epithelial cells and lens fiber cells from a healthy individual and a DM1 patient (HC and DM1, respectively) after FISH and DAPI counterstaining. RNA aggregates were not detected in HC, but were observed in DM1.

[0191] Example 8 Evaluation of ASO using iPS cells derived from DM1 patient (4) Lens epithelial cells and lens fiber cells (8.1) Materials and Methods 10 nM ASO was administered by lipofection to lens epithelial cells and lens fiber cells derived from iPS cells of the DM1 patient described in Example 7. 48 hours later, FISH was performed using a Cy3-labeled (CAG)6-CA DNA / LNA probe in the same manner as in Example 7.

[0192] (8.2) Results As shown in Figure 9C, RNA aggregates were detected in lens epithelial cells and lens fiber cells (DM1) derived from iPS cells of a DM1 patient, but treatment with ASO-4M almost completely eliminated the RNA aggregates.

[0193] The left graph in Figure 9D is a bar graph showing the mean number of RNA aggregates per cell nucleus ± standard deviation calculated from the FISH results for HC, DM1, and DM1 + ​​ASO-4M in Figure 9C. ASO-4M treatment significantly reduced the mean number of RNA aggregates per cell nucleus in lens epithelial and fiber cells derived from DM1 patient cells. The right graph in Figure 9D is a bar graph showing the mean ± standard deviation of the percentage of RNA aggregates in total cells calculated from the FISH results for HC, DM1, and DM1 + ​​ASO-4M in Figure 9C. ASO-4M treatment significantly reduced the percentage of cells in which RNA aggregates were detected relative to the total number of lens epithelial and fiber cells derived from DM1 patient cells. These results suggest that ASO-4M treatment can also reduce RNA aggregates in lens epithelial and fiber cells derived from DM1 patient cells.

[0194] Example 9 Differentiation of Skin Organoids from iPSCs Derived from Myotonic Dystrophy Patients (9.1) Materials and Methods Skin organoids were prepared based on the method of Lee, J. et al. (Nature, 582: 399-404 (2020)). iPSCs derived from DM1 patients and healthy individuals were dissociated using Accutase and suspended in Essential 8 Flex (Gibco) medium (hereinafter referred to as E8-20Y medium) supplemented with 20 μM Y27632 and 100 μg / ml Normocin. 3,500 live cells (confirmed by trypan blue staining) were seeded into a round-bottom 96-well plate in 100 μL of E8-20Y medium per well, and the plate was centrifuged at 100 rcf to promote aggregate formation. After 24 hours, 100 μL of Essential 8 Flex (Gibco) medium supplemented with 100 μg / ml Normocin (hereinafter referred to as E8Y medium) was added and further cultured. After 24 hours, the cell aggregates were individually collected and transferred to a new round-bottom 96-well plate with 100 μL of Essential 6 (Gibco) medium (hereinafter referred to as E6 medium) supplemented with 2% Matrigel, 10 μM SB (Stemgent), 4 ng / mL b-FGF, and 2.5 ng / mL BMP-4, along with 100 μL per well, to initiate differentiation into skin organoids. On day 3 of differentiation, to induce cranial neural crest cell formation, 25 μL of E6 medium supplemented with 200 ng / mL LDN (BMP inhibitor, Stemgent) and 50 μg / mL FGF was added per well, for a final volume of 125 μL. On day 6 of differentiation, 75 μL of fresh E6 medium was added to bring the final volume to 200 μL. Half of the medium was replaced on days 8 and 10. On day 12, to induce epidermal self-organization, all aggregates were transferred to individual wells of a 24-well low-attachment plate in 500 μL of organoid maturation medium (OMM) containing 1% Matrigel. To culture the aggregates in suspension and maintain constant medium circulation, the 24-well plate was placed on an orbital shaker at 65 rpm in a 37°C incubator containing 5% CO2. OMM was prepared by mixing Advanced DMEM / F12 (Gibco) and Neurobasal (Gibco) in a 1:1 ratio, supplemented with 1X GlutaMax.TM The medium was supplemented with 0.5X B-27 Minus Vitamin A (Gibco), 0.5X N2 (Gibco) supplements, 0.1 mM 2-mercaptoethanol (Gibco), and 100 μg / mL Normocin. On day 15 of differentiation, half of the spent medium was replenished with OMM containing 1% Matrigel. From day 18 onwards, half of the medium was replaced with fresh OMM medium without Matrigel every three days (from day 18 to day 45) or every other day (from day 45 to day 150 or thereafter), with complete medium changes performed weekly from day 45 onwards. As the aggregates matured and grew, it was sometimes necessary to increase the total volume of medium per well to 1 mL from day 80 onwards.

[0195] The differentiation of the skin organoids was confirmed by the formation of hair follicle structures.Frozen sections of the skin organoids were also analyzed using the method for detecting nuclear RNA aggregates as in Example 5.

[0196] (9.2) Results As shown in Figure 10A, after 4 months of culture under differentiation conditions, hair follicle structures were formed in the iPS cell-derived skin organoids of healthy individuals and DM1 patients (HC and DM1, respectively). As shown in Figures 10B and 10C, RNA aggregates were detected in sections of the iPS cell-derived skin organoids of DM1 patients after 4 months of culture, but not in sections of the iPS cell-derived skin organoids of healthy individuals.

[0197] Example 10 Evaluation of ASO using iPS cells derived from DM1 patients (5) Skin organoids (10.1) Materials and methods Differentiation of skin organoids from iPS cells derived from healthy individuals and myotonic dystrophy patients and Cy3-labeled (CAG) cells were used to detect intranuclear RNA aggregates. ６ FISH using the -CA DNA / LNA probe was performed in the same manner as in Example 9. For ASO treatment of organoids, organoids were cultured in a rotational manner in NB medium containing 500 nM ASO-4M.

[0198] (10.2) Results: RNA aggregates were reduced in the ASO-4M-treated skin organoids (DM1+ASO-4M) shown on the left in Figure 10B. Figure 10C is a bar graph showing the mean ± standard deviation of the percentage of cells in which RNA aggregates were detected among all cells, calculated from the FISH results for each of HC, DM1, and DM1+ASO-4M in Figure 10B. Figure 10C confirmed that ASO-4M treatment significantly reduced the formation of RNA aggregates in skin organoids derived from DM1 patient iPS cells.

[0199] Example 11 Differentiation of Neurons from iPS Cells Derived from Myotonic Dystrophy Patients (11.1) Materials and Methods Neurons differentiated from iPS cells derived from DM1 patients and healthy individuals were prepared based on the method described by Imamura, K. et al. (Sci Rep., 6:34904. (2016)). Figure 11A is a schematic diagram showing the procedure for an experimental system for differentiating undifferentiated iPS cells derived from DM1 patients (DM-1 to 3) and healthy individual-derived iPS cells (HC-1 to 3) into neurons using a tetracycline-inducible NGN2 expression system. As shown in Figure 11A, "B27 / N2 / NB + DOX" and "B27 / NB + DOX" represent the period of culture in a neuronal induction medium supplemented with doxycycline as a differentiation inducer, and "B27 / NB" represents the period of culture in a medium for differentiated neurons. The tetracycline-inducible NGN2 expression system was introduced into undifferentiated iPS cells by transfection, and differentiation induction was initiated on day 0 by changing the medium to a neuronal induction medium. On day 8, differentiation induction was terminated by changing the medium to a neuronal medium that did not contain a differentiation inducer, and analysis was performed on day 11.

[0200] (11.2) Results Figure 11B is a fluorescence micrograph of neurons differentiated from iPSCs derived from a DM1 patient, stained with an anti-tubulin βIII antibody and counterstained with DAPI. The scale bar represents 100 μm. As shown in Figure 11B, the procedure of this example resulted in the production of uniform neurons expressing tubulin βIII and extending axons. Figure 11C is a composite image of fluorescence micrographs of neurons differentiated from iPSCs derived from healthy individuals (HC-1 to 3) and from iPSCs derived from a DM1 patient (DM-1 to 3), stained with an anti-tubulin βIII antibody (top row), an anti-MAP2 antibody (middle row), or an anti-tubulin βIII antibody and an anti-Tbr1 antibody, and counterstained with DAPI. As shown in Figure 11C, iPSCs derived from a DM1 patient differentiate into neurons at a rate not significantly different from that of iPSCs derived from healthy individuals.

[0201] FIG. 11D shows Cy3-labeled neurons differentiated from iPS cells derived from healthy individuals (HC-1 to 3) and iPS cells derived from DM1 patients (DM-1 to 3). ６ 11D are fluorescence micrographs obtained by FISH using a β-CA DNA / LNA probe and DAPI counterstaining. Similar to Examples 2 and 3, Fig. 11D shows that, among iPS cells derived from DM1 patient, neurons derived from iPS cells DM-2 had significantly lower average numbers of RNA aggregates per cell nucleus and lower percentages of RNA aggregates in total cells than neurons derived from iPS cells DM-1 and DM-3. Unlike Examples 2 and 3, almost no difference was observed in the average numbers of RNA aggregates per cell nucleus or percentages of RNA aggregates in total cells between neurons derived from iPS cells DM-1 and DM-3.

[0202] Example 12 Evaluation of ASO using iPS cells derived from DM1 patients (5) Neurons (12.1) Materials and Methods Neurons derived from iPS cells from DM1 patients described in Example 11 were administered 10 nM ASO by lipofection on day 8 of differentiation induction, and on day 11, they were labeled with Cy3 (CAG) in the same manner as in Example 7. ６ FISH was performed using a -CA DNA / LNA probe.

[0203] (12.2) Results. The upper left panel of Figure 11F shows Cy3-labeled iPS cells (DM-1) derived from a DM1 patient treated with control ASO (CAG). ６ The upper right image of Fig. 11F shows a composite image (CAG) of fluorescent micrographs obtained by FISH using a -CA DNA / LNA probe, staining with an anti-tubulin βIII antibody, and counterstaining with DAPI. The upper right image of Fig. 11F shows Cy3-labeled iPS cells (DM-1) derived from a DM1 patient treated with ASO-4M (same as ASO-4). ６ Figure 11F shows a composite image of fluorescence micrographs (ASO-4M) obtained by FISH using the -CA DNA / LNA probe, staining with anti-tubulin βIII antibody, and counterstaining with DAPI. As shown in Figure 11F, ASO-4M treatment significantly reduced RNA aggregates in neurons.

[0204] As shown in the examples herein, the ASO treatment of the present invention reduced nuclear RNA aggregates in undifferentiated iPS cells derived from DM1 patients, and in skeletal muscle cells, lens epithelial cells, lens fiber cells, neurons, neuromuscular organoids, and skin organoids differentiated from the iPS cells. Furthermore, ASO treatment restored the satellite cells reduced in neuromuscular organoids derived from DM1 patients to the same level as in neuromuscular organoids derived from healthy individuals. Therefore, the pharmaceutical composition of the present invention is useful for treating diseases, including myotonic dystrophy type 1. Furthermore, the iPS cells of the present invention or cells and organoids differentiated from the iPS cells are useful as an assay system for ASO treatment for not only myotonic dystrophy type 1, but also all other triplet diseases for which ASO treatment is effective.

[0205] This application is based on Patent Application No. 2020-213000 filed in Japan on December 22, 2020, the entire contents of which are incorporated herein by reference.

Claims

1. An oligonucleotide comprising a repeat sequence in which cytosine-adenine-guanine trinucleotide is repeated 5 to 13 times from the 5' end to the 3' end, An oligonucleotide having a mixmer structure in which all cytidines are β-D-ENA-cytidines, all adenosines are 2'-OMe-adenosines, and all guanosines are 2'-OMe-guanosines.

2. The oligonucleotide comprises: (1) an oligonucleotide consisting of any one of the base sequences of SEQ ID NOs: 1 to 9; (2) an oligonucleotide having a base sequence consisting of adenine-guanine, any one of the base sequences of SEQ ID NOs: 1 to 9, and cytosine from the 5' end to the 3' end; or (3) An oligonucleotide having a base sequence consisting of guanine, any one of the base sequences of SEQ ID NOs: 1 to 9, and cytosine-adenine from the 5' end to the 3' end. The oligonucleotide of claim 1 ,

3. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one type of nucleotide linkage selected from the group consisting of phosphorothioate, phosphorodithioate and boranophosphate.

4. A pharmaceutical composition comprising an oligonucleotide described in any one of claims 1 to 3 and a pharma- ceutically acceptable carrier or diluent.

5. The pharmaceutical composition according to claim 4 , wherein the carrier comprises a cell membrane permeation carrier and / or a nuclear delivery carrier.

6. 6. The pharmaceutical composition of claim 4 or 5, which is delivered by parenteral administration selected from the group consisting of transdermal, intraocular, intralental, intramucosal, submucosal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intracranial and intrathecal administration.

7. A pharmaceutical composition described in any one of claims 4 to 6 for the treatment of triplet disease caused by CUG repeat expansion.

8. The pharmaceutical composition described in claim 7, wherein the triplet disease is myotonic dystrophy type 1 disease.