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

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition, and more specifically, to a pharmaceutical composition comprising an antisense oligonucleotide. The pharmaceutical composition of the present invention can be used for the treatment of any symptom of myotonic dystrophy type 1. [Background technology]

[0002] Myotonic dystrophy type 1 (hereinafter referred to as "DM1") is one of the most prevalent muscular dystrophy diseases. The prevalence of DM1 varies by country, with reports indicating a prevalence of 1 in 2,000 to 1 in 8,000 people, and in some regions, 1 in 500 people (Non-Patent Literature 1). The cause of DM1 is the elongation of the CTG trinucleotide repeat sequence in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase (hereinafter referred to as "DMPK") gene. The DMPK gene encodes a non-receptor type serine / threonine kinase protein, and the DMPK protein is ubiquitously expressed throughout the body, with strong expression in skeletal muscle and cardiac muscle. The specific function of the DMPK protein is unknown. Most cells in DM1 patients have 50 to 2,000 CTG repeat sequences in the DMPK gene, while unaffected individuals have only 5 to 38 CTG repeat sequences (Non-Patent Literature 1). The number of CTG repeats roughly correlates with the severity and onset time of this disease. RNA from DMPK, in which the CUG repeat sequence is elongated, forms RNA foci in the cell nucleus of affected individuals. These RNA foci have been reported to disrupt many cellular mechanisms, such as alternative splicing, transcription, translation, and post-translational regulation, by adsorbing and depleting RNA-binding molecules, including CUG-binding protein 1 (CUG-BP1) and muscleblind-like protein 1 (MBNL1), from the nucleus (Non-Patent Literature 2). In addition to DM1, numerous other diseases involving the elongation of trinucleotide repeat sequences have been reported and are collectively referred to as triplet diseases.

[0003] Antisense oligonucleotide (hereinafter referred to as "ASO") treatment is considered one of the promising therapeutic strategies for diseases caused by abnormal RNA in cells and / or the nucleus, including mRNA and non-coding RNA, such as triplet disease (Non-Patent Literature 3 and 4). ASOs cause cleavage of target RNA strands by RNase H in the nucleus and cytoplasm, or sterically impair 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 composed solely of deoxyribonucleotides. Modifications of nucleotides used in ASO include phosphorothioate (PS) modification of the phosphodiester skeleton (hereinafter also referred to as "S modification"), methylation modification of the 5' position in the pyrimidine ring of the cytidine base (hereinafter referred to as "5mC"), substitution of the sugar moiety of any nucleotide with a morpholino ring, substitution of the 2' hydroxyl group of the ribose in 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 bridge modification of the oxygen atom at the 2' position and carbon atom at the 4' position of ribose (for example, a locked nucleic acid in which the oxygen atom at the 2' position and carbon atom at the 4' position of the ribose ring are bridged with methylene to fix the conformation of the furanose ring to the C3'-endo type). The present invention includes an 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 a 2',4'-(S)-constrained ethyl nucleotide analog (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').While nucleotide analogs can sometimes cause harmful side effects when administered into the body, they often have a higher binding affinity to complementary RNA and greater resistance to nucleases than unmodified deoxyribonucleotides. Therefore, nucleotide analogs have the advantage of requiring lower dosages than unmodified deoxyribonucleotides.

[0004] When a heteroduplex nucleic acid formed by pairing an oligonucleotide composed of a specific type of nucleotide analog with an RNA complementary to the oligonucleotide is degraded when reacted with RNase H under predetermined conditions, the specific type of nucleotide analog is called an RNase H-active nucleotide. Conversely, when a heteroduplex nucleic acid formed by pairing an oligonucleotide composed of a specific type of nucleotide analog with an RNA complementary to the oligonucleotide is not degraded when reacted with RNase H under predetermined conditions, the specific type of nucleotide analog is called an RNase H-inactive nucleotide. The RNase H-active nucleotides contained in the oligonucleotides according to the present invention include, but are not limited to, unmodified deoxyribonucleotides and PS-modified deoxyribonucleotide analogs. The RNase H-inactive nucleotides contained in the oligonucleotides according to 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 and RNase H-inactive nucleotides. A gapmer structure is a structure in which oligonucleotides consisting only of RNase H-inactive nucleotides (hereinafter referred to as "wing regions") are linked to the 5' and 3' ends of an oligonucleotide consisting only of RNase H-active nucleotides (hereinafter referred to as "gap region"). The wing region has a high binding affinity to complementary RNA, so a stable double helix with the target RNA is formed. In the gap region, RNase H-active nucleotides are continuous, so the double helix with the target RNA is degraded by RNase H. Therefore, it is preferable to adopt a gapmer structure for ASOs that exert their therapeutic effect by degrading the double helix with RNA. Conversely, for ASOs that exert their therapeutic effect by not degrading the double helix with RNA, it is preferable to use oligonucleotides 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 mixed-mer structure may be either a structure consisting only of 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-stranded ASO and target RNA are not degraded by RNase H. The RNase H-inactive nucleotides included in the mixed-mer of the present invention may include oligonucleotides in which all nucleotides have the same modified sugar and all bonds between nucleotides are the same modified bond, or they may include multiple nucleotide derivatives with different modified sugars and / or modified bonds. The gapmer and mixed-mer of the present invention refer to oligonucleotides having a gapmer structure and a mixed-mer structure, respectively.

[0006] Several types of MOE / DNA gapmer ASOs have been reported to be effective in reducing DMPK RNA with elongated CUG 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 mitigate the effects of DMPK RNA with elongated CUG sequences in DM disease models. Some of the aforementioned MOE / DNA gapmer ASOs have progressed to the clinical trial stage, but it was not possible to deliver ASOs at concentrations that exert therapeutic effects in vitro to the muscle (Non-Patent Document 4). Therefore, there is a need to develop ASOs that exert therapeutic effects at lower concentrations.

[0007] While some experiments investigating the efficacy of conventional ASOs for DM1 treatment have used fibroblasts, muscle satellite cells, or myoblasts derived from DM1 patients, they have primarily used transgenic mice into which genomic DNA of the human DMPK gene with an extended CTG repeat sequence derived from DM1 patients has been introduced, or myoblasts or myotubes derived from such mice. However, the CTG repeat sequence of the human DMPK gene introduced into these transgenic mice had only a few hundred repeats, which was insufficient as a model for human DM1 disease, which has thousands or even thousands of repeats.

[0008] Patient-derived induced pluripotent stem cells (hereinafter referred to as "iPSCs" or "iPS cells") are useful for drug discovery and development. Several iPSCs created from primary cultured cells of fibroblasts, myoblasts, urine-derived cells, or immortalized lymphoblastic cells from myotonic dystrophy patients have been described (Non-Patent Literature 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 Literature 16-18). However, the effects of ASOs for DM1 treatment have not been investigated using iPS cells derived from DM1 patients or cells differentiated from said iPS cells. Therefore, it is necessary to establish multiple iPS cells derived from DM1 patients with different CTG sequence repeat counts and evaluate ASOs for DM1 treatment in a disease model cell system that more faithfully reproduces the pathogenesis of human DM1 disease.

[0009] Furthermore, among the nucleotide analogs described earlier, 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 due to steric hindrance, are under development as a treatment for Duchenne muscular dystrophy (DMD) (Non-Patent Documents 20-22). ASOs of ENA / deoxyribonucleotide gapmers and ENA / 2'-OMe ribonucleotide analog mixmers can reduce RNA aggregates in iPS cells derived from patients with spinocerebellar degeneration type 36 (SCA36) and in nerve cells derived from said iPS cells (Non-Patent Document 23). Thus, ASOs incorporating ENA are particularly promising as therapeutic agents with a mechanism of action mediated by steric hindrance, and therefore, it is necessary to develop oligonucleotides incorporating ENA as ASOs for the treatment of DM1.

[0010] Furthermore, DM1 is a multi-organ 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, classical, and congenital. Mild DM1 is characterized by cataracts and mild myotonia (prolonged muscle contraction), and the prognosis is normal. Classical DM1 is characterized by muscle weakness and atrophy, myotonia, and cataracts, and is often accompanied by cardiac conduction disorders. In adults, physical function declines, and the prognosis may be shorter. 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. In DM1, symptoms such as muscle weakness and atrophy, myocardial damage, cardiac conduction disorders, cataracts, retinal degeneration, gastrointestinal symptoms such as swallowing, constipation, and diarrhea due to smooth muscle damage, higher brain dysfunction, hyperinsulinemia, diabetes, testicular atrophy, endocrine disorders such as growth hormone secretion abnormalities, and skin symptoms such as pilomatoma and epithelioma are also reported. Therefore, treatment of organs other than muscles may be necessary depending on the patient's clinical symptoms. For this reason, it is also necessary to develop ASOs for DM1 treatment that target not only skeletal muscle, but also satellite cells, which are precursor cells of skeletal muscle that reside in muscle tissue, as well as other tissues, such as ocular tissue (lens cells that cause cataracts, retinal cells that cause retinal degeneration, etc.), nerves, skin, endocrine system, and other tissues. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Chakraborty, S. et al., Current Protocols in Human Genetics, 91: 9.29.1-9.29.19. (2016) [Non-Patent Document 2] Pettersson, OJ et al., Nucleic Acids Research, 43: 2433-2441. (2015) [Non-Patent Document 3] Crooke, ST et al., Cell Metabolism, 27: 714-739. (2018) [Non-Patent Document 4] Overby, SJら, Drug Discovery Today 23: 2013-2022. (2018)

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Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

[0013] To achieve the above objective, the inventors conducted research and 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 RNA aggregate removal activity. Using this assay system, the inventors completed the present invention.

[0014] The present invention provides a pharmaceutical composition. The pharmaceutical composition of the present invention comprises an oligonucleotide having a base sequence of any of the repeat sequences in which the cytosine-adenine-guanine trinucleotide is repeated 5 to 13 times from the 5' end to the 3' end, wherein the oligonucleotide has a mixed-mer structure that includes at least two RNase H-inactive nucleotide analogs.

[0015] In the pharmaceutical composition of the present invention, the oligonucleotide is (1) Oligonucleotides consisting of any of the base sequences of SEQ ID NOs. 1 to 9, (2) An oligonucleotide consisting of adenine-guanine, one of the base sequences of SEQ ID NOs. 1-9, and cytosine, from the 5' end to the 3' end, or (3) Oligonucleotides consisting of guanine, one of the base sequences of SEQ ID NOs: 1-9, and cytosine-adenine, from the 5' end to the 3' end. It's okay to include it.

[0016] In the pharmaceutical composition of the present invention, the RNase H inactive nucleotide analog can be a nucleotide analog in which the 2' position of ribose is modified, and / or a nucleotide analog in which the 2' and 4' positions of ribose are cross-linked.

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

[0018] In the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' and 4' positions of ribose are cross-linked 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 nucleotide analogs in which the 2' position of ribose is modified may be the same modified sugar.

[0020] In the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' position of ribose is modified may be at least two different modified sugars.

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

[0022] In the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' and 4' positions of ribose are cross-linked may be at least two different modified sugars.

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

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

[0025] In the pharmaceutical composition of the present invention, the oligonucleotide may include at least one nucleotide linkage selected from the group consisting of phosphorothioates, phosphorodithioates, and boranophosphates.

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

[0027] In the pharmaceutical composition of the present invention, the carrier may include a carrier for cell membrane permeation and / or a carrier for intranuclear delivery.

[0028] The pharmaceutical composition of the present invention can be delivered by parenteral administration selected from the group consisting of transdermal administration, intraocular administration, intralenical administration, intracrystalline lens administration, intramucosal administration of the gastrointestinal tract, submucosal administration of the gastrointestinal tract, subcutaneous administration, intravenous administration, intra-arterial administration, intramuscular administration, intraperitoneal administration, intracranial administration, and intrathecal administration.

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

[0030] The present invention provides a method for treating myotonic dystrophy type 1 disease by administering an oligonucleotide having a nucleotide sequence in which a cytosine-adenine-guanine trinucleotide trinucleotide is repeated 5 to 13 times from the 5' end to the 3' end, wherein the oligonucleotide has a mixed-mer structure containing at least two RNase H-inactive nucleotide analogs, to a patient in need of the oligonucleotide.

[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 comprise undifferentiated pluripotent stem cells, cells differentiated into at least one cell type, and aggregates containing the differentiated cells. [Brief explanation of the drawing]

[0032] [Figure 1A]A panel of micrographs showing the expression of undifferentiated pluripotent stem cell markers in iPS cells derived from DM1 patients (DM-1 to DM-3) and iPS cells derived from healthy individuals (HC-1 to HC-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 section shows fluorescence micrographs of monolayer cultures of undifferentiated iPS cells, stained with an antibody against TRA-1-60, and then counterstained with DAPI to isolate the cell nuclei. [Figure 1B] 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 DM-3) and iPS cells derived from healthy individuals (HC-1 to HC-3). The first row shows fluorescence micrographs of cells differentiated into the mesoderm from each iPS cell type, stained with an antibody against α-smooth muscle actin (SMA), and then counterstained with DAPI for the cell nucleus. The second row shows fluorescence micrographs of cells differentiated into the endoderm from each iPS cell type, stained with an antibody against SOX17, and then counterstained with DAPI for the cell nucleus. The third row shows fluorescence micrographs of cells differentiated into the ectoderm from each iPS cell type, stained with an antibody against βIII-tubulin, and then counterstained with DAPI for the cell nucleus. [Figure 1C] Capillary electrophoresis of reaction products of triplet repeat prime PCR (hereinafter referred to as "TP-PCR") from iPS cells derived from DM1 patients (DM-1 to DM-3) and iPS cells derived from healthy individuals (HC-1 to HC-3). [Figure 2]This panel displays fluorescence micrographs of iPS cells (DM-3) derived from DM1 patients, obtained by fluorescence in situ hybridization (FISH) using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The upper left or lower left images show results from FISH without RNase A treatment, while the upper right, A, and lower right images show results from FISH after RNase A treatment. The upper left and upper right images are fluorescence micrographs of cells with counterstained nuclei with DAPI after FISH, while the lower left and lower right images are fluorescence micrographs of FISH alone without DAPI staining. [Figure 3A] A panel of fluorescence micrographs showing the results of FISH (Focused Intracellular Spectroscopy) performed on undifferentiated iPS cells from DM1 patients (DM-1 to DM-3) and healthy individuals (HC-1 to HC-3), using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The upper right corner of each image shows a high-magnification fluorescence micrograph of a single nucleus. [Figure 3B] Figure 3B (left) is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results in Figure 3A. Figure 3B (right) 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 in Figure 3A. [Figure 4A] This schematic diagram shows the relative positional relationship of the complementary sequences of the ASOs in the embodiment of this application on DMPK-pre mRNA, as well as the structural type of the ASO. ASO-1 to 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' end of the repeat sequence of the non-coding region of exon 15 on DMPK-mRNA. ASO-4 is a mixed mer consisting of ENA and a 2'-OMe-nucleotide analog, while the other ASO-1 to 3 and 5 are gapmers (hereinafter referred to as "ENA / DNA gapmers") in which the wing region is ENA and the gap region is an unmodified deoxyribonucleotide. [Figure 4B]Fluorescence micrographs taken 48 hours after administration of either ASO-C or ASO-1~5 to iPS cells derived from DM1 patients (DM1), using FISH with a Cy3-labeled (CAG)6-CA DNA / LNA probe and DAPI counterstaining. [Figure 4C] A bar graph showing the average number of RNA aggregates per cell nucleus, calculated from FISH results of iPS cells (DM-1) derived from DM1 patients who were administered 5nM or 10nM of ASO-C and / or ASO-1-5. [Figure 4D] A bar graph showing the percentage of RNA aggregate-positive cell nuclei detected by DAPI staining, calculated from FISH results of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-C and / or ASO-1-5. [Figure 4E] A bar graph showing the proliferation and survival status of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-C and one of ASO-1 to 5. The vertical axis represents the percentage of cells, with the number of ASO-C cells set to 1.0. [Figure 5A] This schematic diagram shows the relative positional relationship of the complementary sequences on DMPK-pre mRNA for ASO-2 and ASO-4 of the present invention, and their corresponding control ASO-A and ASO-B, as well as the structural type of ASO. ASO-2 and ASO-A are complementary to the same sequence in the coding region of exon 15 on DMPK-mRNA, while ASO-4 and ASO-B are complementary to the same sequence in the repeat sequence of the CUG 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") whose wing region consists of MOE-nucleotide analogs and whose gap region consists of unmodified deoxyribonucleotides. ASO-4 is a mixed-mer consisting of ENA and 2'-OMe-nucleotide analogs, while ASO-B consists entirely of 2'-OMe-nucleotide analogs, with all internucleotide bonds being phosphorothioates. [Figure 5B]Fluorescence micrographs of iPS cells derived from DM1 patients (DM1) after administration of ASO-2 or ASO-A, followed by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. [Figure 5C] Figure 5C (left) is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from FISH results of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-2, ASO-A, or ASO-C. Figure 5C (center) is a bar graph showing the percentage of RNA aggregate-positive cell nuclei detected by DAPI staining, calculated from FISH results of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-2, ASO-A, or ASO-C. Figure 5C (right) is a bar graph showing the proliferation and survival status of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-2, ASO-A, or ASO-C. The vertical axis represents the percentage of cell numbers with the number of ASO-C cells set to 1.0. [Figure 5D] Fluorescence micrographs of iPS cells derived from DM1 patients (DM1) after administration of ASO-4 or ASO-B, followed by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. [Figure 5E] Figure 5E (left) is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from FISH results of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-4, ASO-B, or ASO-C. Figure 5E (center) is a bar graph showing the percentage of RNA aggregate-positive cell nuclei in all cell nuclei, calculated from FISH results of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-4, ASO-B, or ASO-C. Figure 5E (right) is a bar graph showing the proliferation and survival status of iPS cells (DM-1) derived from DM1 patients administered 5nM or 10nM ASO-4, ASO-B, or ASO-C. The vertical axis represents the percentage of cell numbers with the number of ASO-C cells set to 1.0. [Figure 6A]This is a conceptual diagram illustrating the procedure for a differentiation experiment using a tetracycline-inducible MyoD expression system to differentiate undifferentiated cells into muscle cells from iPS cells derived from DM1 patients (DM-1~3) and healthy individuals (HC-1~3). "StemFit" represents the period of culture in a medium for undifferentiated iPS cells, "KSR / α-MEM" represents the period of culture in a medium for differentiated muscle cells, and "KSR / α-MEM+Dox" represents the period of culture in a muscle cell medium supplemented with doxycycline as a differentiation inducer. On day 0, undifferentiated iPS cells were transfected with the tetracycline-inducible MyoD expression system. On day 2, the culture medium was changed to a muscle cell medium supplemented with a differentiation inducer to start differentiation induction. On day 8, the culture medium was changed to a muscle cell medium without a differentiation inducer to end differentiation induction, and analysis was performed on day 12. [Figure 6B] A panel of fluorescence micrographs showing the expression of muscle differentiation markers in cells differentiated from undifferentiated HC-1~3 and DM-1~3 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 in the cell nuclei. The middle panel shows fluorescence micrographs stained with an antibody against α-actinin and counterstained with DAPI in the cell nuclei. The bottom panel shows fluorescence micrographs stained with an antibody against myogenin (MyoG) and counterstained with DAPI in the cell nuclei. [Figure 6C] A bar graph showing the percentage of MHC-expressing cells among muscle cells differentiated from HC-1~3 and DM-1~3 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 cell nuclei differentiated from undifferentiated cells. "ns" indicates that no significant difference was observed between HC-1~3 cells and DM-1~3 cells. [Figure 6D]A bar graph showing the percentage of α-actinin-expressing cells among muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. The vertical axis represents the percentage of DAPI-stained cell nuclei expressing the muscle differentiation marker α-actinin (α-Actinin / DAPI) among the DAPI-positive cell nuclei differentiated from undifferentiated cells. "ns" indicates that no significant difference was observed between HC-1~3 cells and DM-1~3 cells. [Figure 6E] A bar graph showing the percentage of myogenin-expressing cells among muscle cells differentiated from HC-1~3 and DM-1~3 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 differentiated from undifferentiated cells. "ns" indicates that no significant difference was observed between HC-1~3 cells and DM-1~3 cells. [Figure 6F] A panel of fluorescence micrographs showing the results of FISH and DAPI counterstaining using Cy3-labeled (CAG)6-CA DNA / LNA probe on muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. [Figure 6G] Figure 6G (left) is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from FISH results for muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. Figure 6G (right) is a bar graph showing the percentage of RNA aggregate-positive cell nuclei in all cell nuclei detected by DAPI staining, calculated from FISH results for muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. [Figure 7A] A panel of fluorescence micrographs obtained by administering 10 nM ASO-C or ASO-4 to muscle cells derived from DM1 patient iPS cells (DM-1~3), followed by FISH using a Cy3-labeled (CAG)6-CA DNA / LNA probe and DAPI counterstaining. [Figure 7B] A bar graph showing the average number of RNA aggregates per cell nucleus, calculated from FISH results of muscle cells derived from DM-1 to DM-3 cells administered with 10 nM ASO-C or ASO-4. [Figure 7C] A bar graph showing the percentage of RNA aggregate-positive cell nuclei detected by DAPI staining, calculated from FISH results of DM-1~3 cell-derived muscle cells administered with 10 nM ASO-C or ASO-4. [Figure 7D] This bar graph shows the proliferation and survival status of DM-1~3 cell-derived muscle cells administered with 10 nM ASO-C or ASO-4. In all bar graphs, the vertical axis represents the percentage of cells, with the number of cells in the untreated culture (without ASO administration) set to 1.0. [Figure 8A] A schematic diagram of the protocol for creating neuromuscular organoids from iPS cells. [Figure 8B] The left panel shows phase-contrast micrographs at day 5 and bright-field micrographs at day 50 (scale bar 200 μm) of neuromuscular organoids derived from healthy iPS cells (HC, top) and neuromuscular organoids derived from DM1 patient iPS cells (DM, bottom). The right panel shows a composite image (scale bar 20 μm) of multicolor fluorescence micrographs of a section of neuromuscular organoid at day 50. The left side of the panel shows neuromuscular organoids stained with anti-myosin heavy chain (MHC) antibody, TUJ1 antibody (anti-β-tubulin III antibody), and DAPI. The organoids are divided into regions with a high concentration of skeletal muscle cells (muscular compartment) and regions with a high concentration of nerve cells (neural compartment). The right side of the panel shows neuromuscular organoids stained with anti-TITIN antibody and DAPI. The striae of skeletal muscle cells are stained with anti-TITIN antibody. In both images, the top row shows neuromuscular organoids (HC) derived from iPS cells of healthy individuals, and the bottom row shows neuromuscular organoids (DM) derived from iPS cells of DM1 patients. [Figure 8C]The top panel is a composite image of fluorescence microscopy images of the muscle compartments of neuromuscular organoids stained with anti-desmin antibody, anti-PAX7 antibody, and DAPI. The bottom panel is a composite image of fluorescence microscopy images of the muscle compartments of neuromuscular organoids treated with FISH using a Cy3-labeled (CAG)6-CA DNA / LNA probe and DAPI counterstaining. In both the top and bottom panels, the left image shows neuromuscular organoids derived from healthy iPS cells (HC), the center image shows neuromuscular organoids derived from DM1 patient iPS cells (DM), and the right image shows neuromuscular organoids derived from DM1 patient iPS cells treated with ASO-4M (same as ASO-4) (DM ASO-4M). The scale bar represents 20 μm. [Figure 8D] Bar graph showing the percentage of cells containing RNA aggregates expressing desmin and PAX7. The values ​​are the mean ± standard deviation (SD) obtained from 5 organoids. "***" indicates a significant difference between HC and DM, and between DM and DM+ASO4M (independent two-tailed t-test, p<0.001). [Figure 8E] The top image is a composite image of fluorescence microscopy images of muscle compartments of neuromuscular organoids stained with anti-MYOD antibody, anti-PAX7 antibody, and DAPI. The bottom image is a composite image of fluorescence microscopy images of muscle compartments of neuromuscular organoids stained with anti-Ki67 antibody, anti-PAX7 antibody, and DAPI. In both the top and bottom images, the left image shows neuromuscular organoids derived from healthy iPS cells (HC), the center image shows neuromuscular organoids derived from DM1 patient iPS cells (DM), and the right image shows neuromuscular organoids derived from DM1 patient iPS cells treated with ASO-4M (same as ASO-4) (DM ASO-4M). [Figure 8F] The top left bar graph shows the percentage of cells expressing PAX7 in all cells. The top right bar graph shows the percentage of cells expressing MYOD in all cells. The bottom left bar graph shows the percentage of cells expressing PAX7 among cells expressing MYOD. The bottom right bar graph shows the percentage of cells expressing Ki67 among cells expressing PAX7. The 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 (independent two-tailed t-test, p<0.001). [Figure 9A] The image on the left is a phase-contrast micrograph (HC) of lens epithelial cells and lens fiber cells differentiated from iPS cells of a healthy individual. The image on the right is a phase-contrast micrograph (DM1) of lens epithelial cells and lens fiber cells differentiated from iPS cells of a DM1 patient. The scale bar represents 100 μm. [Figure 9B] The image on the left is a fluorescence micrograph (HC) of lens epithelial cells and lens fiber cells differentiated from iPS cells of a healthy individual, stained with anti-αA crystallin antibody. The image on the right is a fluorescence micrograph (DM1) of lens epithelial cells and lens fiber cells differentiated from iPS cells of a DM1 patient, stained with anti-αA crystallin antibody. The scale bar represents 200 μm. [Figure 9C] The image on the left is a fluorescence micrograph (HC) of lens epithelial cells and lens fiber cells differentiated from healthy iPS cells, performed using FISH and DAPI counterstaining with a Cy3-labeled (CAG)6-CA DNA / LNA probe. The image in the center is a fluorescence micrograph (DM1) of lens epithelial cells and lens fiber cells differentiated from DM1 patient iPS cells, performed using FISH and DAPI counterstaining with a Cy3-labeled (CAG)6-CA DNA / LNA probe. The image on the right is a fluorescence micrograph (DM1+ASO-4M) of lens epithelial cells and lens fiber cells differentiated from DM1 patient iPS cells treated with ASO-4M (same as ASO-4), performed using FISH and DAPI counterstaining with a Cy3-labeled (CAG)6-CA DNA / LNA probe. [Figure 9D] The bar graph on the left shows the mean number ± standard deviation of RNA aggregates per cell nucleus, calculated from the FISH results for HC, DM1, and DM1+ASO-4M in Figure 9C. The bar graph on the right shows the mean ± standard deviation of the percentage of RNA aggregates in all cells, calculated from the FISH results for HC, DM1, and DM1+ASO-4M in Figure 9C. [Figure 10A] The image on the left is a bright-field micrograph (HC) of skin organoids derived from healthy iPS cells after 4 months of culture under differentiation conditions. The image on the right is a bright-field micrograph (DM1) of skin organoids derived from iPS cells of a DM1 patient. The scale bar represents 500 μm. [Figure 10B]The image on the left is a fluorescence micrograph (HC) of skin organoids derived from healthy iPS cells, obtained by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The image in the center is a fluorescence micrograph (DM1) of skin organoids derived from DM1 patient iPS cells, obtained by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The image on the right is a fluorescence micrograph (DM1+ASO-4M) of skin organoids derived from DM1 patient iPS cells treated with ASO-4M (same as ASO-4), obtained by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The scale bar represents 20 μm. [Figure 10C] The bar graphs in Figure 10B show the mean ± standard deviation of the percentage of cells in which RNA aggregates were detected in all cells, calculated from the FISH results for HC, DM1, and DM1+ASO-4M, respectively. [Figure 11A] This is a conceptual diagram illustrating the procedure for a differentiation experiment using a tetracycline-inducible NGN2 expression system to differentiate undifferentiated cells into neurons from iPS cells derived from DM1 patients (DM-1~3) and healthy individuals (HC-1~3). "B27 / N2 / NB+DOX" and "B27 / NB+DOX" represent the period of culture in a neuronal cell induction medium supplemented with doxycycline as a differentiation inducer, while "B27 / NB" represents the period of culture in a differentiated neuronal cell medium. Undifferentiated iPS cells, transfected with the tetracycline-inducible NGN2 expression system, were cultured in a neuronal cell induction medium starting on day 0, and then switched back to a neuronal cell medium without a differentiation inducer on day 8 to terminate the differentiation process. Analysis was performed on day 11. [Figure 11B] Fluorescence micrograph of neurons differentiated from iPS cells derived from DM1 patients, stained with anti-tubulin βIII antibody and counterstained with DAPI. The scale bar represents 100 μm. [Figure 11C]A composite image of fluorescence microscopy images of neurons differentiated from iPS cells derived from healthy individuals (HC-1~3) and iPS cells derived from DM1 patients (DM-1~3), stained with anti-tubulin βIII antibody (top panel), anti-MAP2 antibody (middle panel), or anti-tubulin βIII antibody and anti-Tbr1 antibody, followed by DAPI counterstaining. [Figure 11D] Fluorescence micrographs of neurons differentiated from iPS cells (HC-1~3) derived from healthy individuals and iPS cells (DM-1~3) derived from DM1 patients, obtained by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. [Figure 11E] The bar graph on the left shows the mean number ± standard deviation of RNA aggregates per cell nucleus, calculated from the FISH results for HC-1~3 and DM-1~3 in Figure 11C. The bar graph on the right shows the mean ± standard deviation of the percentage of RNA aggregates in all cells, calculated from the FISH results for HC-1~3 and DM-1~3 in Figure 11C. [Figure 11F]The top left image is a composite image (Control) of fluorescence microscopy images of neurons derived from DM1 patient iPS cells treated with Control ASO, stained with anti-tubulin βIII antibody, and stained with DAPI counterstaining, based on FISH using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The top right image is a composite image (ASO-4M) of fluorescence microscopy images of neurons derived from DM1 patient iPS cells treated with ASO-4M (same as ASO-4), stained with anti-tubulin βIII antibody, and stained with DAPI counterstaining, based on FISH results from Figure 11F. The bottom left bar graph shows the mean number ± standard deviation of RNA aggregates per cell nucleus in neurons derived from DM1 patient iPS cells treated with ASO-4M, Control ASO, or untreated (ASO-4M, Control ASO, or Untreated, respectively), calculated from the FISH results shown above in Figure 11F. The bottom right bar graph shows the mean ± standard deviation of the percentage of RNA aggregates in all cells of neurons derived from iPS cells of DM1 patients, calculated from the FISH results shown in Figure 11F, for those treated with ASO-4M or Control ASO, or those not treated with ASO (ASO-4M, Control ASO, or Untreated, respectively). [Modes for carrying out the invention]

[0033] One embodiment of the present invention is a pharmaceutical composition. The pharmaceutical composition of the present invention comprises an oligonucleotide having a base sequence of any of the repeat sequences in which the cytosine-adenine-guanine trinucleotide is repeated 5 to 13 times from the 5' end to the 3' end, wherein the oligonucleotide has a mixed-mer structure that includes at least two types of RNase H-inactive nucleotide analogs and / or internucleotide bonds.

[0034] The nucleotide sequences of oligonucleotides described herein are shown in Sequence IDs 1 to 23 of the sequence listing attached herein. Table 1 shows the correspondence between the nucleotide sequences of repeat sequences in which the cytosine-adenine-guanine trinucleotide is repeated 5 to 13 times from the 5' end to the 3' end, and the Sequence IDs in the sequence listing attached herein.

[0035] [Table 1]

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

[0037] The antisense oligonucleotides administered to iPS cells in the examples described herein are ASO-1 to ASO-5, and the ASOs used as controls are ASO-A to ASO-C. The nucleotide sequences of ASO-1 to ASO-3, ASO-4, and ASO-5 are listed as SEQ ID NOs. 11 to ASO-13, ASO-3, and ASO-14, respectively, in the sequence listings attached to this specification. Their nucleotide sequences and structural characteristics are shown in Table 2.

[0038] [Table 2]

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

[0040] Of the aforementioned ASOs, ASO-C, ASO-1 to 3, and ASO-5 have a gapmer structure (hereinafter referred to as "ENA / DNA gapmer") in which the nucleosides constituting the wing region are ENA and the nucleosides constituting the gap segment are DNA. In ASO-1 to 3 and ASO-5, all nucleotides of the gapmer pair with the RNA of the DMPK gene of a patient with myotonic dystrophy type 1, 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 and is degraded by RNase H. On the other hand, ASO-4 has a mixed-mer structure of ENA and 2'-O-methylribonucleotides (hereinafter referred to as "ENA / 2'-OMe ribonucleotide mixed-mer") in which the β-D-ENA-modified cytidine, 2'-OMe-adenosine, and 2'-OMe-guanosine are repeated seven times in tandem from 5' to 3' in that order. The full length of this mixed-mer pairs with the repeat sequence of CUG trinucleotides in the 3' UTR of the DMPK gene RNA of patients with myotonic dystrophy type 1, but since ASO-4 does not contain DNA, it is not degraded by RNase H. The ASO-C of this invention has the same base sequence as the nonspecific oligonucleotide used in the antisense oligonucleotide inhibition experiment in another study (Hagemann, TL et al., Ann Neurol, 83:27-39 (2018)), and also shares the characteristic that the gap segment of the gapmer is a deoxyribonucleotide. However, while the wing region of the gapmer in that other study is a 2'-O-methoxyethyl (hereinafter referred to as "MOE") nucleotide analog, the wing region of ASO-C used as a control in this specification is ENA.

[0041] Table 3 below compares the structures of the ASOs in the embodiments of the present invention with those of related prior art ASOs. Since the nucleotide sequences of the ASOs in the embodiments of the present invention are the same as those of related prior art ASOs, the nucleotide sequences of ASO-1 and PS115 are listed in Sequence ID No. 11 of the sequence listing attached to this specification. The nucleotide sequences of ASO-2 and ASO-A (ISIS445569) are listed in Sequence ID No. 12. The nucleotide sequences of ASO-3 and PS116 are listed in Sequence ID No. 13. The nucleotide sequences of ASO-4 and ASO-B (PS58) are listed in Sequence ID No. 3. The nucleotide sequences of ASO-5 and ISIS486178 are listed in Sequence ID No. 14. The nucleotide sequence of the Control ASO is listed in Sequence ID No. 23.

[0042] [Table 3]

[0043] In the structure of each ASO in Table 3, "A", "G", and "C" represent adenosine, guanosine, and cytidine, respectively, while "dA", "dG", "dC", and "dT" represent deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine, respectively. The nucleotide to the right of the subscript "k" represents a 6'-(S)-CH3 bicyclic nucleotide analog (hereinafter referred to as "cEt"). The nucleotide to the right of the subscript "e" represents a 2'-O-methoxyethyl (hereinafter referred to as "MOE") nucleotide analog. The nucleotide to the right of the subscript "E" represents ENA. The nucleotide to the right of the subscript "m" represents a 2'-OMe nucleotide analog. The subscript "s" indicates that the bond between the nucleoside to its left and the nucleotide to its right is a phosphorothioate (PT) bond, not a phosphodiester bond. "mC" indicates a 5-methylcytosine nucleoside.

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

[0045] Prior art related to ASO-1 is PS115, described in Non-Patent Document 7. PS115 shares the same base sequence as ASO-1. However, it differs in that all nucleoside bonds in ASO-1 are phosphodiester bonds, while those in PS115 are PT bonds, and that the wing region of ASO-1 is a gapmer consisting of ENA, while in PS115 it consists entirely of 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 shares the same base sequence as ASO-2 and is a gapmer structure consisting of wing regions of the same length. However, it differs in that all nucleoside bonds are phosphodiester bonds in ASO-2, while they are PT bonds in ISIS 445569; the wing region of the gapmer is ENA in ASO-2, while it is an MOE-nucleotide analog in ISIS 445569; and the 5' and 3' terminal nucleotides are cytidine in ASO-2, while they are 5-methylcytidine in ISIS 445569.

[0047] Prior art related to ASO-3 is PS116, described in Non-Patent Document 7. PS116 shares the same base sequence as ASO-3. However, it differs in that all nucleoside bonds in ASO-3 are phosphodiester bonds, while those in PS116 are PT bonds, and that the wing region of ASO-3 is a gapmer consisting of ENA, while in PS115 it consists entirely of 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 all adenosine nucleotides and guanosine nucleotides are OMe nucleotide analogs. However, PS58 also has OMe nucleotide analogs for its cytidine nucleotide, whereas ASO-4 has ENA for its cytidine nucleotide. Furthermore, PS58 has all PT bonds between nucleotides, while ASO-4 has all phosphodiester bonds between nucleotides.

[0049] Prior art related to ASO-5 is ISIS 486178, described in Non-Patent Literature 6. ISIS 486178 and ASO-5 share the same base sequence and are both gapmer structures consisting of wing regions of the same length. However, ASO-5 is an ENA / DNA gapmer, whereas ISIS 486178 is a gapmer (hereinafter referred to as "cEt / DNA gapmer") in which the wing region consists of a 6'-(S)-CH3 bicyclic (cEt) nucleotide and the gap region consists of an unmodified deoxyribonucleotide.

[0050] Table 4 shows the primer sets used for triplet repeat prime PCR (hereinafter referred to as "TP-PCR") analysis. The nucleotide sequences of each primer are listed in sequence numbers 15-22 of the sequence listing attached to this specification.

[0051] [Table 4]

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

[0053] The oligonucleotide according to the present invention has a mixed structure that is not cleaved by RNase H. Here, "mixed structure" refers to a single-stranded oligonucleotide structure in which RNase H-active nucleotides and RNase H-inactive nucleotides are mixed, and the heteroduplex nucleic acid formed when the single-stranded oligonucleotide pairs with complementary RNA is not degraded by RNase H. According to Monia, BP et al. (J. Biological. Chem., 268: 14514 (1993)), the degradation activity by RNase H against heteroduplex nucleic acid of a gapmer ASO in which at least five consecutive RNase H-active nucleotides are sandwiched between wing regions of RNase H-inactive nucleotides and RNA complementary to the ASO exceeded 75%, but the degradation activity by RNase H against heteroduplex nucleic acid of a gapmer ASO in which four or fewer consecutive RNase H-active nucleotides are sandwiched between wing regions of RNase H-inactive nucleotides and RNA complementary to the ASO was less than 20%. Therefore, the oligonucleotide with the mixed-mer structure of the present invention may have five or fewer RNase H-active nucleotides, for example, only five, four, three, or two consecutive nucleotides. "Mixed-mer ASO" or "ASO of mixed-mers" refers to an ASO having a mixed-mer structure.

[0054] In the present invention, whether a particular type of nucleotide analog is RNase H active or RNase H inactive can be determined, for example, by the following procedure. An oligonucleotide with a gapmer structure is prepared in which a gap region consisting of 10 consecutive nucleotide analogs of the particular type is sandwiched between wing regions consisting of 5 known RNase H active nucleotides, such as 2'-OMe-nucleotide analogs. A heteroduplex is prepared by pairing 300 picomoles of the gapmer oligonucleotide with 1500 picomoles of RNA that pairs with the oligonucleotide. The heteroduplex and E. coli RNase H 3.0 units are added to a reaction solution for RNase H, for example, a reaction solution consisting of 50 mM Tris-HCl (pH 8.0), 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol, to a total volume of 150 μL, and the mixture is reacted at 37°C for 60 minutes. The reaction product is analyzed by agarose gel electrophoresis.

[0055] Furthermore, those 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, which can be used to determine the ability to recruit RNase H.

[0056] The nucleoside monomers of the oligonucleotides contained in the pharmaceutical composition of the present invention are linked together via internucleoside linking groups. Optionally, each monomer is linked to a 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 it does not contain a 5'-linking group.

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

[0058] In one embodiment of the pharmaceutical composition of the present invention, the oligonucleotide is (1) Oligonucleotides consisting of any of the base sequences of SEQ ID NOs. 1 to 9, (2) An oligonucleotide consisting of adenine-guanine, one of the base sequences of SEQ ID NOs. 1-9, and cytosine, from the 5' end to the 3' end, or (3) Oligonucleotides consisting of guanine, one of the base sequences of SEQ ID NOs: 1-9, and cytosine-adenine, from the 5' end to the 3' end. It's okay to include it.

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

[0060] The second oligonucleotide according to the above embodiment of the pharmaceutical composition of the present invention, namely, "an oligonucleotide comprising a base sequence consisting of adenine-guanine, one of the base sequences of SEQ ID NOs: 1 to 9, and cytosine from the 5' end to the 3' end," is an oligonucleotide comprising a base sequence with 6 to 14 trinucleotide repeats in which adenosine, guanosine, and cytidine are linked in that 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, namely, "an oligonucleotide comprising a base sequence consisting of guanine, one of the base sequences of SEQ ID NOs: 1 to 9, and cytosine-adenine from the 5' end to the 3' end," is an oligonucleotide comprising a base sequence in which guanosine, cytidine, and adenosine are linked in the 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 inactive nucleotide analog may include a nucleotide analog in which the 2' position of ribose is modified, and / or a nucleotide analog in which the 2' and 4' positions of ribose are cross-linked.

[0063] The nucleotide analogs in which the 2' position of ribose in the oligonucleotide of the above embodiment is modified 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 analogs in which the 2' position of ribose is modified are, for example, those shown in the following chemical formula (1) [wherein the formula, the substituent X attached to the 2' position of ribose is selected from a fluoro group, a methoxy group, an O-methoxyethyl group, a 2'-(3-hydroxy)propyl group and a 2'-(3-amino)propyl group, and Z and Z * B is independently selected from internucleotide links, R, terminal groups, or protecting groups, B is selected from natural or unnatural nucleotide base parts (nucleic acid bases), and R is hydrogen, a hydroxyl group, C 1-4 Alkyl and C 1-4 It is expressed as [selected from alkoxy groups].

[0065] [ka]

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

[0067] The nucleotide analogs in which the ribose between the 2' and 4' positions of the oligonucleotide in the above embodiment is cross-linked include, but are not limited to, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE.

[0068] Nucleotide analogs in which the 2'- and 4'-positions of the ribose are crosslinked and modified are called locked nucleic acids or LNAs because the conformation of the 2'- and 4'-positions is fixed (locked). Alternatively, they are also called BNAs or bicyclic nucleic acids because they are bridged. Nucleotide analogs in which the 2'- and 4'-positions of the ribose are crosslinked and modified include oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, ENA, cEt, and cMOE. Among these, oxy-LNA, thio-LNA, amino-LNA, 5'-methyl-LNA, and ENA have stereoisomers of β-D-configuration and α-L-configuration, and cEt and cMOE have R-type and S-type stereoisomers with the carbon atom bridging the 2'- and 4'-positions of the ribose as an asymmetric center.

[0069] The nucleotide analogs in which the 2'- and 4'-positions of the ribose are crosslinked and modified, for example, in the following chemical formulas (2) and (3), the divalent atomic group "-Y-" bonded to the 2'-position of the ribose is selected from "-O-", "-S-", "-CH2-S-", "-N(H)-", "-N(R a )-", "-CH2-N(H)-", and "-CH2-N(R a )-", Z and Z * are independently selected from among internucleotide linkages, R b , terminal groups, or protecting groups, B is selected from natural or unnatural nucleotide base moieties (nucleobases), R a is independently selected from hydrogen and C 1-4 alkyl, and R b has a structure selected from hydrogen, a hydroxyl group, a C 1-4 alkyl group, and a C 1-4 alkoxy group.

[0070] Oxy-LNA, thio-LNA, and amino-LNA with a β-D- configuration are represented by general formula (2), and oxy-LNA, thio-LNA, and amino-LNA with an α-L- configuration are represented by general formula (3). In chemical formulas (2) and (3), the nucleotide analog in which the divalent atomic group "-Y-" attached to the 2' position of ribose is "-O-" is oxy-LNA, the nucleotide analog in which the divalent atomic group "-Y-" attached to the 2' position of ribose is "-S-" or "-CH2-S-" is thio-LNA, and the divalent atomic group "-Y-" attached to the 2' position of ribose is "-N(H)-" or "-N(R) a )-", "-CH2-N(H)-" and "-CH2-N(R a )-" (R here a is C 1-4 The nucleotide analog selected from (selected from alkyl groups) is amino-LNA.

[0071] [ka]

[0072] [ka]

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

[0074] [ka]

[0075] [ka]

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

[0077] [ka]

[0078] [ka]

[0079] R-type cEt and cMOE are represented by the following general formula (8), and S-type cEt and cMOE are represented by the following general formula (9). In chemical formulas (8) and (9), W is selected from a methyl group and a methoxymethyl group, and Z and Z * R is a nucleotide linkage, where R is hydrogen, hydroxyl group, C 1-4 Alkyl and C 1-4 B is independently selected from among alkoxy groups, terminal groups, or protecting groups, and has a structure selected from natural or unnatural nucleotide base moieties (nucleic acid bases).

[0080] [ka]

[0081] [ka]

[0082] In chemical formulas (8) and (9), the nucleotide analog with W being a methyl group is cEt, and the nucleotide analog with W being a methoxymethyl group is cMOE.

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

[0084] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' and 4' positions of ribose are cross-linked 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 nucleotide analogs in which the 2' position of ribose is modified may have the same modified sugar.

[0086] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' position of ribose is modified may have at least two different modified sugars.

[0087] In one embodiment of the pharmaceutical composition of the present invention, all nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked can have the same modified sugar.

[0088] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' and 4' positions of ribose are cross-linked can have at least two different modified sugars.

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

[0090] The 2'-OMe-nucleotide analog refers to a nucleotide analog in the chemical formula (1) in which the substituent X bonded to the 2' position of ribose is a methoxy group and the base B is adenine, guanine, thymine, or cytosine. The MOE-nucleotide analog refers to a nucleotide analog in the chemical formula (1) in which the substituent X bonded to the 2' position of ribose 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 analog in which the 2' and 4' positions of the ribose contained in the oligonucleotide are cross-linked can be LNA, ENA and / or cEt.

[0092] In one embodiment of the pharmaceutical composition of the present invention, all nucleotide analogs with the 2' position of ribose modified can have the same modified sugar. That is, the nucleotide analogs with the 2' position of ribose modified in the oligonucleotide can have a substituent attached to the 2' position of ribose that is one of a fluoro group, a methoxy group, or an O-methoxyethyl group.

[0093] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' position of ribose is modified may have at least two different modified sugars. That is, the nucleotide analog in which the 2' position of ribose is modified in the oligonucleotide may have two or three 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 nucleotide analogs in which the 2' and 4' positions of ribose are cross-linked can have the same modified sugar. That is, the nucleotide analog in which the 2' and 4' positions of ribose contained in the oligonucleotide can be any one of LNA, ENA, or cEt.

[0095] In one embodiment of the pharmaceutical composition of the present invention, the nucleotide analog in which the 2' and 4' positions of ribose are cross-linked can have at least two different modified sugars. That is, the nucleotide analog in which 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 include β-D-ENA-cytidine, 2'-OMe-adenosine, and 2'-OMe-guanosine.

[0097] In one embodiment of the pharmaceutical composition of the present invention, all cytidines in the oligonucleotides are β-D-ENA-cytidines, all adenosines are 2'-OMe-adenosine, and all guanosines are 2'-OMe-guanosine.

[0098] In one embodiment of the pharmaceutical composition of the present invention, the oligonucleotide contained in the pharmaceutical composition of the present invention may include at least one nucleotide linkage selected from the group consisting of phosphorothioates, phosphorodithioates, and boranophosphates.

[0099] In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier or diluent. A person skilled in the art can appropriately select a carrier suitable for such a situation as the carrier that may 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 monohydrogen phosphate, citric acid, and sodium citrate; isotonic agents such as glucose, sodium chloride, and potassium chloride; and divalent ion adjusters such as calcium chloride and magnesium chloride. Furthermore, these carriers are not limited to being used for the purpose of exerting a single action, but can be used for the purpose of exerting multiple actions. Diluents that may be included in the 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 include a cell membrane permeability and / or intranuclear delivery carrier, the cell membrane permeability and / or intranuclear delivery carrier may bind to or associate with oligonucleotides relating to the pharmaceutical composition of the present invention.

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

[0102] The aforementioned cell membrane permeation and / or intranuclear delivery carrier may include peptidomimetics. Peptidomimetics as cell membrane permeation and / or intranuclear delivery carriers refer to synthetic compounds that mimic the structure of the peptide as the cell membrane permeation and / or intranuclear delivery carrier, and that bind to or associate with oligonucleotides related to the pharmaceutical composition of the present invention to promote cell membrane permeation and / or intranuclear delivery. Peptidomimetics as cell membrane permeation and / or intranuclear delivery carriers are compounds in which all or part of the peptide as the cell membrane permeation and / or intranuclear delivery carrier is substituted with compounds other than natural peptides, such as heterocyclic or cyclic compounds, unnatural amino acids, D-amino acids, and equivalents of various functional groups.

[0103] The carrier for cell membrane permeation and / or intranuclear delivery may include cationic polymers of DEAE dextran, polyelenimine (PEI), and polypropyleneimine (PPI).

[0104] The carrier for cell membrane permeation and / or intranuclear delivery may contain lipids. The lipids used as carriers for cell membrane permeation and / or intranuclear delivery are 1,2-dimryristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-SN-glycero-3-phosphatidylcholine (DSPC), and 1-palmitoyl-2-myristoyl-SN-glycero-3-phosphatidylcholine. (PMPC), 1-stearoyl-2-myristoyl-SN-glycero-3-phosphatidylcholine (SMPC), hydrogenated lecithin derived from soybeans (HSPC), 1-stearoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (SOPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-SN- Glycero-3-phosphoglycerol (DOPG), 1,2-dioleoyl-SN-glycero-3-phospho-L-serine (DOPS), 1,2-dioloyl-SN-glycero-3-phosphate (DOPA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (D It may contain, but is not limited to, phospholipids such as OPE, anionic lipids such as 1,5-O-dihexadecyl-N-succinyl-L-glutamic acid, cholesterol, PEG-bound lipids such as N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-SN-glycero-3-phosphoethanolamine, and pH-sensitive lipids such as 1,2-dioleyl-3-dimethylammonium propane (DODAP).

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

[0106] The carrier for cell membrane permeation and / or intranuclear delivery may include liposomes. The liposome as the carrier for cell membrane permeation and / or intranuclear delivery refers to a lipid membrane structure dispersed in an aqueous solvent, with the aqueous solvent encapsulated within it. The lipid membrane of the liposome may be a single-layer or multi-layer lipid bilayer or lipid multilayer. The liposome may be particulate or reticular. The lipid component of the liposome may include, but is not limited to, the lipid used as the carrier for cell membrane permeation and / or intranuclear delivery. The lipid component of the liposome may include the lipidoid used as the carrier for cell membrane permeation and / or intranuclear delivery. The liposome as the carrier for cell membrane permeation and / or intranuclear delivery can encapsulate oligonucleotides related to the pharmaceutical composition of the present invention within it.

[0107] The carrier for cell membrane permeation and / or intranuclear delivery may include lipid nanoparticles (LNPs). The lipid nanoparticles as the carrier for cell membrane permeation and / or intranuclear delivery are composed of the same lipids as the lipid components of the liposomes and oligonucleotides according to the pharmaceutical composition of the present invention. pH-sensitive lipids that become positively charged in a low pH environment, such as 1,2-dioleyl-3-dimethylammonium propane (DODAP), may be used for the lipid nanoparticles. Unlike liposomes, lipid nanoparticles have a single lipid membrane and a core structure filled with lipids rather than encapsulating an aqueous solvent inside. The lipid nanoparticles as the carrier for cell membrane permeation and / or intranuclear delivery may contain oligonucleotides 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, intravenous, intra-arterial, intramuscular, intraperitoneal, intracranial, 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 a downregulation of CUG-RNA aggregate formation and / or a reduction of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of 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 tablets, capsules, granules, powders, or syrups. These preparations may contain excipients (for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, and dextrin; cellulose derivatives such as crystalline cellulose; organic excipients such as gum arabic, dextran, and pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and calcium sulfate. Examples of inorganic excipients include stearates (such as stearates), lubricants (for example, metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as beegum and gypsum wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycols; fumaric acid; sodium benzoate; DL-leucine; sodium fatty acid salts; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; and the above starch derivatives). Examples of excipients include: ), binders (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and compounds similar to those described above), disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxylated methylcellulose, carboxylated methylcellulose calcium, and internally crosslinked carboxylated methylcellulose sodium; chemically modified starches and celluloses such as carboxylated methyl starch, carboxylated methyl starch sodium, and crosslinked polyvinylpyrrolidone), stabilizers (e.g., para-hydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), and flavoring and deodorizing agents (e.g., commonly used sweeteners, acidulants, flavorings, etc.).It is manufactured using well-known methods with additives such as ).

[0110] The active ingredient contained in the pharmaceutical composition of the present invention is the oligonucleotide (or, if a cell membrane permeability and / or nuclear delivery carrier is bound to or associated with the oligonucleotide, the oligonucleotide to which the cell membrane permeability and / or nuclear delivery carrier is bound or associated). The proportion of the active ingredient contained in the pharmaceutical composition of the present invention can be appropriately set within a range that can produce the desired effect, but is usually 0.01 to 100% by weight, preferably 0.1 to 99.9% by weight, and more preferably 0.5 to 99.5% by weight.

[0111] The dosage of the pharmaceutical composition of the present invention must be carefully adjusted considering the age, weight, and condition of the individual being treated, as well as the route of administration, form of administration, and method of administration, and the exact dosage must be determined by a physician. The actual dosage is within the physician's discretion and may vary by setting the dosage for the specific circumstances of the present invention in order to obtain the desired therapeutic effect. However, the dosage of the pharmaceutical composition of the present invention is not defined in general terms, depending on the type of active ingredient, the weight, age, and symptoms of the recipient, but for example, in the case of parenteral administration, it is desirable to administer one dose at a time, with a lower limit of 0.001 mg / kg body weight (preferably 0.01 mg / kg body weight) and an upper limit of 100 mg / kg body weight (preferably 10 mg / kg body weight) per dose, and in the case of oral administration, with a lower limit of 0.01 mg / kg body weight (preferably 0.1 mg / kg body weight) and an upper limit of 1000 mg / kg body weight (preferably 100 mg / kg body weight) per dose, once to several times a day depending on the symptoms.

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

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

[0114] The pharmaceutical composition of the present invention can be delivered by one or more parenteral administration methods selected from the group consisting of transdermal administration, intraocular administration, intralenical administration, intracrystalline lens administration, intramucosal administration of the gastrointestinal tract, submucosal administration of the gastrointestinal tract, subcutaneous administration, intravenous administration, intra-arterial administration, intramuscular administration, intraperitoneal administration, intracranial administration, and intrathecal administration. Furthermore, the pharmaceutical composition of the present invention can be administered to 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 tissues involved in their control, 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 with myotonic dystrophy type 1 disease and the 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 disease.

[0116] In this specification, the adjective "approximately" modifying a numerical value means a numerical range of 90% or more and 110% or less of the numerical value. For example, "approximately 40 bases" refers to a numerical range of bases between 36 and 44 bases.

[0117] All references made herein are incorporated herein by citation in their entirety.

[0118] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0119] Example 1: Establishment of iPS cells derived from a patient with myotonic dystrophy. (1.1) Materials and Methods iPS cells were generated by reprogramming episodic vectors (Okita, K. et al., Stem Cells, 31:458-466 (2013)) encoding SOX2, KLF4, OCT4, L-MYC, LIN28, p53 carboxyl-terminal dominant-negative fragment, and EBNA1, using peripheral blood mononuclear cells (PBMCs) collected from three patients with myotonic dystrophy type 1 in the experimental group and two healthy control individuals in the control group, as well as dermal fibroblasts collected from one healthy control individual in the control group. Cell line authentication 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). iPS cells were cultured and maintained in StemFit® medium (AK02N, Ajinomoto Co., Inc., Tokyo) as described by Nakagawa, M. et al. (Sci Rep, 4: 3594 (2014)) on culture vessels coated with iMatrix-511 (892012, Nippi Corporation, Tokyo).

[0120] The TP-PCR analysis in the examples described herein was performed as follows: Genomic DNA from iPS cells was extracted using the PureLink® Genomic DNA Mini-Kit (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and subjected to TP-PCR analysis as described by Singh, S. et al. (Front Genet, 5: 94 (2014)) and Non-Patent Literature 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 for TP-PCR analysis are shown in Table 4 above.

[0121] Immunocytochemical analysis in the examples described herein was performed as follows: Cells were fixed in PBS containing 4% paraformaldehyde (Nacalai Tesque Co., Ltd., Kyoto) at room temperature for 20 minutes and incubated in blocking buffer (PBS containing 5% Blocking One (Nacalai Tesque Co., Ltd.) and 0.2% Triton-X100 (Nacalai Tesque Co., Ltd.)). A list of the sources and dilutions of the primary and secondary antibodies used in the examples described herein is shown in Table 5 below. The primary antibody was reacted with the cells in the blocking buffer at 4°C for 16 hours, and the secondary antibody was reacted at room temperature for 1 hour. The nuclei were stained with DAPI. Images were acquired using an Opera Phenix® high-content screening system (PerkinElmer, Massachusetts, Waltham) or a BZ-X710 microscope (Keyence, Osaka, Japan).

[0122] [Table 5]

[0123] Embryoid body formation assays were performed to differentiate iPS cells into three germ layers (Suga, M. et al., Stem Cell Res. 36: 101406 (2019)). Embryoid bodies were formed by culturing 9000 iPS cells (9000 cells per embryoid body) dissociated with 0.5×TrypLE Select 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) for 11 days on a tissue culture plate coated with Matrigel (BD Bioscience) in DMEM supplemented with 10% FBS (Gibco (Thermo Fisher Scientific)). The ability to differentiate into mesoderm was tested by whether smooth muscle cells expressing smooth muscle actin differentiated. The ability to differentiate into endoderm was tested by whether endoderm cells expressing SOX17 differentiated. The ability to differentiate into ectoderm was tested by whether motor neurons expressing βIII-tubulin differentiated.

[0124] Table 6 below outlines the cell types of iPS cell-derived differentiated cells used to investigate the effect of ASO treatment on nuclear RNA aggregate formation, the culture method of the differentiated cells, the method of inducing differentiated cells from iPS cells, and the ASO treatment method. Individual details are shown in the examples using each differentiated cell type.

[0125] [Table 6]

[0126] The bibliographic information for 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, JM 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] To detect nuclear RNA aggregates, fluorescence in situ hybridization (FISH) was performed using a Cy3-labeled (CAG)6-CA DNA / LNA probe. Cells were cultured on a clear-bottomed microplate (CellCarrier-96 Ultra, PerkinElmer), fixed with 4% paraformaldehyde at room temperature for 30 minutes, permeabilized with 70% ethanol at -30°C for at least 2 hours, rehydrated with 1×SSC solution prepared with diethyl dicarbonate (DEPC) water, and incubated in hybridization pretreatment buffer [2×SSC and 50% formamide prepared with DEPC water] at 55°C for 2 hours. The cells were then incubated overnight at 55°C in hybridization buffer [2×SSC, 50% formamide, 10% dextrin sulfate, and 0.1 ng / μL of Cy3-labeled (CAG)6-CA DNA / LNA probe (Gene Design Co., Ltd., 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 then washed twice at room temperature for 5 minutes with 1×SSC solution prepared in DEPC water. The cells were further incubated at 37°C for 60 minutes with 1×SSC prepared in DEPC water, and then washed twice with 1×SSC prepared in DEPC water. The cells were stained with 1 μg / mL of 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, Thermo Fisher Scientific) in PBS at room temperature for 10 minutes, and then washed with PBS. Images were captured and analyzed using the Opera Phenix® High Content Screening System (PerkinElmer) with a 40x water-immersion lens.

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

[0129] [Table 7]

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

[0131] As shown in Figure 1A, DM-1~3 and HC-1~3 all exhibited a morphology similar to human undifferentiated embryonic stem cells, with cells closely adhering to each other and individual cell boundaries indistinguishable under a phase-contrast microscope. Furthermore, DM-1~3 and HC-1~3 all expressed NANOG, OCT4, SSEA4, and TRA-1-60, which are markers for human undifferentiated iPS cells, thus possessing characteristics of human undifferentiated iPS cells.

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

[0133] As shown in Figure 1B, DM-1~3 and HC-1~3 all showed differentiation potential into mesodermal cells (α-smooth muscle actin), endodermal cells (SOX17), and ectoderm cells (βIII-tubulin). Therefore, it was proven that iPS1~3 and HC-1~3 are all pluripotent human iPS cell undifferentiated cells.

[0134] Figure 1C shows the capillary electrophoresis of the TPPCR reaction products of DM-1~3 and HC-1~3. As shown in Figure 1C, the number of repeats in the CTG repeat sequence of the DMPK gene of DM-1~3 and HC-1~3 did not contradict the number of repeats in the donor's genomic DNA analyzed by Southern blotting, as shown in Table 7.

[0135] Figure 2 is a panel of fluorescence micrographs of iPS(DM-3) cells derived from DM1 patients, obtained by fluorescence in situ hybridization (FISH) using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The upper left or lower left images show the results of FISH without RNase A treatment, while the upper right, A, and lower right images show the results of FISH after RNase A treatment. The upper left and upper right images are fluorescence micrographs of cells with counterstained nuclei with DAPI after FISH, while the lower left and lower right images are fluorescence micrographs of FISH alone without DAPI staining.

[0136] As shown in Figure 2, the bright spots observed when FISH was performed without RNase A treatment disappeared when FISH was performed after RNase A treatment. This experimental result indicates that the bright spots are signals from CUG-RNA aggregates (CUG-RNA foci) hybridized with the probe.

[0137] Figure 3A is a panel of fluorescence micrographs obtained by performing FISH using Cy3-labeled (CAG)6-CA DNA / LNA probe on undifferentiated cells of DM-1~3 and HC-1~3, respectively. The upper right corner of each image is a high-magnification fluorescence micrograph of a single nucleus. The scale bar in the lower 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 Phenix® High Content Screening System (PerkinElmer).

[0139] Figure 3B (left) is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from the FISH results in Figure 3A. Figure 3B (right) 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 in Figure 3A. "**" indicates a significant difference between the values ​​for DM-1 to DM-3 and HC-1 to DM-3 (p<0.01).

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

[0141] Based on these results, it was found that the iPS cells derived from DM1 patients created 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 ASO using iPS cells derived from DM1 patients (1) Undifferentiated cells (2.1) Materials and Methods Figure 4A is a schematic diagram showing the relative positional relationship of the complementary sequences of the ASOs in the embodiments of this application on DMPK-pre mRNA, as well as the structural type of the ASOs. ASO-1 to 3 are complementary to the non-coding region of exon 15 on DMPK-pre 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' end of the repeat sequence of the non-coding region of exon 15 on pre DMPK-mRNA. ASO-4 is a mixed mer consisting of ENA and a 2'-OMe-nucleotide analog, while the other ASO-1 to 3 and 5 are gapmers (hereinafter referred to as "ENA / DNA gapmers") consisting of ENA in the wing region and unmodified deoxyribonucleotides in the gap region. Note that ASO-C, used as a negative control in the embodiments of this specification, is an ENA / DNA gapmer that targets the transcript of a gene completely different from DMPK.

[0143] Figure 5A is a schematic diagram showing the relative positional relationship of the complementary sequences on DMPK-pre mRNA for ASO-2 and ASO-4 of the present invention and their corresponding control ASO-A and ASO-B, as well as the structural type of ASO. ASO-2 and ASO-A are complementary to the same sequence in the coding region of exon 15 on DMPK-mRNA, while ASO-4 and ASO-B are complementary to the same sequence in the repeat sequence of the CUG 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") whose wing region consists of MOE-nucleotide analogs and whose gap region consists of unmodified deoxyribonucleotides. ASO-4 is a mixed-mer consisting of ENA and 2'-O-Me-nucleotide analogs, while ASO-B consists entirely of 2'-O-Me-nucleotide analogs (RNase H inactive 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 in SEQ ID NOs. 11 to 14 and 3, respectively, in the sequence listings attached herein. The nucleotide sequence of ASO-A is listed in 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 Chemicals Co., Ltd. (Kobe) and oligonucleotides were synthesized. Administration of ASO to cultured iPS cells was performed by administering ASO at a final concentration of 5 or 10 nM with DharmaFECT1 (Horizon Discovery, Cambridge, UK) according to the manufacturer's instructions. FISH was performed 48 hours after administration of ASO to cultured iPS cells using a Cy3-labeled (CAG)6-CA DNA / LNA probe.

[0145] (2.2) Results Figure 4B shows fluorescence micrographs of undifferentiated iPS cells (DM-1) derived from DM1 patients, 48 ​​hours after administration of either ASO-C or ASO-1-5, followed by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The scale bar in the lower right corner of each fluorescence microscope image represents 10 μm.

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

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

[0148] As shown in Figures 4B-D, all of ASO-1 to ASO-5 significantly reduced RNA aggregates in undifferentiated iPS cells derived from DM1 patients. Of the ENA / DNA gapmers ASO-1 to ASO-3 and ASO-5, ASO-5 most strongly suppressed RNA aggregate formation (Figures 4C and D). When iPS cells were treated with ASO-4, a mixmer consisting of ENA targeting CUG repeat sequences and a 2'-OMe-nucleotide analog, RNA aggregates completely disappeared (Figures 4B-D).

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

[0150] As shown in Figure 4E, ASO-1, 4, and 5 did not affect the proliferation and survival of undifferentiated iPS cells at both 5 nM and 10 nM concentrations, but 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, 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 (Non-Patent Literature 5 (Wheeler, TM et al., Nature, 488, 111 (2012))). Similarly, ASO-4 and ASO-B target the same sequence on DMPK-mRNA. However, ASO-4 is a mixed nucleotide consisting of ENA and a 2'-OMe-nucleotide analog, while ASO-B is a mixed nucleotide consisting entirely of 2'-OMe-nucleotide analogs. ASO-B was reported under the code name PS58 (Non-Patent Literature 7 (Mulders, SA et al., Proc Natl Acad Sci USA, 106:13915 (2009))). Therefore, ASO-A and B were prepared and used as positive controls for ASO-2 and ASO-4, respectively.

[0152] Figure 5B shows fluorescence micrographs of undifferentiated iPS cells (DM-1) derived from DM1 patients after administration of ASO-2 or ASO-A, followed by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The scale bar in the lower right of each fluorescence micrograph represents 10 μm.

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

[0154] Figure 5D shows fluorescence micrographs of undifferentiated iPS cells (DM-1) derived from DM1 patients after administration of ASO-4 or ASO-B, followed by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe. The scale bar in the lower right of each fluorescence micrograph represents 10 μm.

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

[0156] As shown in Figure 5C, ASO-A reduced RNA aggregates without decreasing the number of undifferentiated iPS cells, regardless of whether it was at 5 nM or 10 nM, whereas ASO-2 reduced RNA aggregates but also reduced the number of undifferentiated iPS cells. Therefore, the effect of ASO-2 is thought to be due to cytotoxicity.

[0157] In contrast, as shown in Figure 5E, both ASO-4 and ASO-B reduced RNA aggregates without decreasing the number of undifferentiated iPS cells, regardless of whether they were at 5nM or 10nM concentrations. Therefore, the effects of ASO-4 and ASO-B cannot be attributed to cytotoxicity. Moreover, ASO-4 exhibits a significantly superior RNA aggregate suppression effect compared to ASO-B, which leaves more than half of the RNA aggregates intact, as it almost completely eliminates the RNA aggregates. The difference between ASO-B and ASO-4 is that ASO-4 is a micmer composed of ENA and 2'-OMe-nucleotide analogs, while ASO-B consists entirely of 2'-OMe-nucleotide analogs, with all internucleotide bonds being phosphorothioates. Therefore, it is thought that the micmer structure containing at least two types of RNase H-inactive nucleotide analogs and / or internucleotide bonds contributes to the significantly superior RNA aggregate suppression effect of ASO-4.

[0158] Example 3: Differentiation of muscle cells from iPS cells derived from patients with myotonic dystrophy. (3.1) Materials and Methods The method for differentiating muscle cells from undifferentiated iPS cells established in the embodiments of the present invention is as follows: The Tet-On-inducible MyoD expression system (Non-Patent Literature 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 created vector was cotransfected with the transpose-encoding pCyL43 vector, along with undifferentiated iPS cells from DM1 patients (DM-1~3) and healthy individuals (HC-1~3), using lipofectamine LTX (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Clones of the iPS cells were selected using puromycin (Nacalai Tesque Corporation). iPS cells were seeded in StemFit medium supplemented with 10 μM Rock inhibitor Y27632 (Nacalai Tesque Co., Ltd.) on cell culture vessels coated with Matrigel and cultured for 2 days. After that, the medium was switched to α-MEM (Nacalai Tesque Co., Ltd.) supplemented with 5% KnockOut® Serum Replacement (KSR, Thermo Fisher Scientific Co., Ltd.), 200 μM 2-mercaptoethanol (Thermo Fisher Scientific Co., Ltd.), 100 U / mL penicillin / streptomycin (Thermo Fisher Scientific Co., Ltd.), and 1 μg / mL doxycycline (Clontec, Takara Bio Inc., Shiga). Doxycycline was removed on day 8. The medium was changed every other day.

[0159] Figure 6A is a conceptual diagram showing the procedure for a differentiation experiment from undifferentiated cells to muscle cells using iPS cells derived from DM1 patients (DM-1~3) and healthy individuals (HC-1~3) with a tetracycline-inducible MyoD expression system. "StemFit" represents the period of culture in a medium for undifferentiated iPS cells, "KSR / α-MEM" represents the period of culture in a medium for differentiated muscle cells, and "KSR / α-MEM+Dox" represents the period of culture in a muscle cell medium supplemented with doxycycline as a differentiation inducer. On day 0, undifferentiated iPS cells were transfected with the tetracycline-inducible MyoD expression system. On day 2, the culture medium was changed to a muscle cell medium supplemented with a differentiation inducer to start differentiation induction. On day 8, the culture medium was changed to a muscle cell medium without a differentiation inducer to end differentiation induction, and analysis was performed on day 12.

[0160] Cells differentiated from undifferentiated DM-1~3 and HC-1~3 cells into muscle cells were analyzed using the immunocytochemical methods and nuclear RNA aggregate detection methods 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 differentiated from undifferentiated iPS cells (HC-1~3) derived from healthy individuals and iPS cells (DM-1~3) derived from DM1 patients, following 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 nucleus. The middle panel shows fluorescence micrographs stained with an antibody against α-actinin and counterstained with DAPI for the cell nucleus. The bottom panel shows fluorescence micrographs stained with an antibody against myogenin (MyoG) and counterstained with DAPI for the cell nucleus.

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

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

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

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

[0166] Figure 6F is a panel of fluorescence micrographs obtained by performing FISH using a Cy3-labeled (CAG)6-CA DNA / LNA probe on muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. The scale bar in the lower right of each fluorescence micrograph represents 10 μm.

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

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

[0170] (4.2) Results Figure 6F is a panel of fluorescence micrographs obtained by performing FISH using a Cy3-labeled (CAG)6-CA DNA / LNA probe on muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. The scale bar in the lower right of each fluorescence micrograph represents 10 μm.

[0171] Figure 6G (left) is a bar graph showing the average number of RNA aggregates per cell nucleus, calculated from FISH results for muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. Figure 6G (right) is a bar graph showing the percentage of RNA aggregate-positive cell nuclei detected by DAPI staining, calculated from FISH results for muscle cells differentiated from HC-1~3 and DM-1~3 cells according to the procedure shown in Figure 6A. "**" indicates a significant difference between the values ​​for DM-1~3 and HC-1~3 (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 DM-3) from DM1 patients. Comparing the graphs in Figure 3B and Figure 6G, there was no significant difference in the percentage of RNA aggregate-positive cell nuclei among all cell nuclei between undifferentiated DM-1 to DM-3 cells and muscle cells derived from DM-1 to DM-3. However, there was a very large difference in the average number of RNA aggregates per cell nucleus between undifferentiated DM-1 and DM-3 cells (2 to 3.5 aggregates) and muscle cells derived from DM-1 and DM-3 (11 to 16 aggregates). Notably, almost no RNA aggregates were detected in muscle cells derived from DM-2.

[0173] Figure 7A is a panel of fluorescence micrographs obtained by lipofection of DM-1~3 derived muscle cells with 10 nM ASO-C or ASO-4, followed by FISH and DAPI counterstaining using a Cy3-labeled (CAG)6-CA DNA / LNA probe.

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

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

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

[0177] As shown in Figures 7A-D, compared to ASO-C, administration of ASO-4 significantly reduced the average number of RNA aggregates per cell nucleus in DM-1 and DM-3 cell-derived muscle cells. Administration of ASO-4 showed almost no change in the average number of RNA aggregates per cell nucleus in DM-2 cell-derived muscle cells. This may be related to the fact that DM-2 cells originated from patients with a low number of CTG sequence repeats in the DMPK gene. As shown in Table 7 and Figure 1C, DM-2 cell donors had at most 150 CTG sequence repeats, which is significantly lower than the 1000-1150 and 2300-2850 CTG sequence repeats, respectively, of DM-1 and DM-3 cell donors. Therefore, it is thought that the effect of ASO-4 administration on reducing RNA aggregates is less pronounced in DM-2 cells compared to DM-1 and DM-3 cells.

[0178] Based on these results, we concluded that ASO-4 can reduce RNA aggregates in skeletal muscle cells derived from iPS cells of DM1 patients.

[0179] Example 5: Differentiation of neuromuscular organoids from iPS cells derived from patients with myotonic dystrophy. (5.1) Materials and Methods Neuromuscular organoids were created using a protocol published by Faustino Martins, JM et al. (Cell Stem Cell, 26: 172-186 e176. (2020)), with some modifications. A schematic diagram is shown in Figure 8A. 70% confluent iPS cells were dissociated into single cells and incubated at a rate of 75,000 cells / cm³ 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 in Matrigel-coated culture vessels. The nodal medium (NB) was a 1:1 mixture of Advanced Dulbecco's Modified Eagle Medium F12 (Gibco) supplemented with 1×N2 (Gibco) and Neurobasal medium (Gibco) supplemented with 1×B27 (Gibco), 2 mM L-glutamine (Gibco), 75 μg / mL BSA fraction V (Sigma), and 0.1 mM 2-mercaptoethanol (Gibco). The following day, Y27632 was removed, and the cells differentiated into neural mesodermal progenitor cells (NMPs) in two days. On day 0 of organoid formation, mNMPs were dissociated with Accumax (Innovative Cell Technologies), and 4,500 to 7,200 NMPs were seeded 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) per well into a PrimeSurfce 96-well plate. 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, the organoids were maintained in NB medium. On day 10, the organoids were transferred to 60 mm culture dishes, and on day 30, to 100 mm culture dishes. The organoids were cultured in a circular motion at 75 rpm and the medium was changed to fresh NB medium twice a week. For ASO treatment of organoids, the organoids were cultured in a swirling manner in NB medium containing 500 nM ASO.

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

[0181] (5.2) Results To verify whether the ASO according to the present invention reduces or eliminates RNA aggregates not only in muscle cells but also in progenitor cells and skeletal muscle fibers that differentiate into muscle, neuromuscular organoids (NMOs) were cultured in three dimensions for 50 days according to the method reported by Faustino Martins, JM et al. The three-dimensional aggregates were formed from iPS cell-derived neuromuscular progenitor cells (NMPs), and the three-dimensional aggregates were destined to differentiate into neuroectoderm and mesodermal cells. As shown in Figure 8B, the three-dimensional aggregates grew into organoids containing neural and mesodermal regions by day 5, and by day 50, the NMOs were observed to have a neural compartment with many neurons expressing TUJ1 and a skeletal muscle compartment with many MHC-expressing cells. As shown on the left side of the panel in Figure 8B, immunofluorescence analysis of the muscle region of the NMOs at day 50 revealed the expression of the sarcomere protein TITIN in the skeletal muscle fibers.

[0182] As shown in the upper panel of Figure 8C, muscle cells expressing desmin and muscle progenitor / satellite cells expressing PAX7 were observed along the myotubes in the skeletal muscle compartment of the organoid. 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 iPS cells of DM1 patients.

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

[0184] (6.2) Results As shown in the lower panel of Figure 8C and Figure 8D, treatment of neuromuscular organoids derived from iPS cells of DM1 patients 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 shows that, compared to neuromuscular organoids derived from iPS cells of healthy individuals, the number of PAX7-expressing muscle progenitor cells / satellite cells in DM1 patients' iPS cell-derived neuromuscular organoids at 50 days of culture was reduced. Therefore, in the skeletal muscle compartment of NMOs, the ratio of PAX7-expressing cells to the total number of cells (PAX7) was investigated. + (DAPI), and the ratio of the number of PAX7-expressing cells to the number of cells expressing MYOD that have the potential to differentiate into muscle (PAX7 + / MYOD + The following were analyzed (Figures 8E and 8F). The results showed that, compared to neuromuscular organoids from healthy individuals, the number of muscle progenitor cells / satellite cells expressing PAX7 was significantly reduced in neuromuscular organoids from DM1 patients. Conversely, the ratio of the number of cells expressing MYOD with muscle differentiation potential to the total number of cells, and the ratio of the number of cells expressing Ki67 to the number of muscle progenitor cells / satellite cells expressing PAX7, did not change between DM1 patients and healthy individuals. These results suggest that while satellite cells decrease in neuromuscular organoids from DM1 patients, they recovered to a level comparable to that of neuromuscular organoids from healthy individuals after ASO-4M treatment.

[0186] The results of this experiment showed that DM1-specific nuclear RNA aggregate formation could be 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. The effects of DMPK RNA with elongated CUG repeat sequences suggest that it may influence the entire process of skeletal muscle differentiation, from satellite cell behavior to muscle fiber formation. Therefore, the three-dimensional muscle organoid culture 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 myotonic dystrophy patients (7.1) Materials and Methods Lens epithelial cells and lens fiber cells were created from undifferentiated iPS cells based on Fu, Q. et al. (Invest Ophthalmol Vis Sci. 58: 517-527 (2017)), as listed in Table 6. First, approximately 80 iPS cell colonies, each containing about 20 cells, were seeded and cultured in mTesR medium (Stemcell, Vancouver, Canada) in Matrigel-coated culture dishes. Four hours after plating, the iPS cells were induced via 100 ng / ml of the BMP inhibitor noggin, and were induced to differentiate into ectoderm / neuroectoderm until epithelial-like cells first appeared around the periphery of the colonies on day 6 of culture. Next, approximately 50 differentiated iPS cells were mechanically separated along with the surrounding epithelial-like cells, and 30-50 differentiated iPS cell colonies were selected and reseeded in Matrigel-coated culture dishes. Subsequently, the BMP signaling pathway was reactivated via its agonists, BMP4 and BMP7 (20 ng / ml). Simultaneously, the fibroblast growth factor signaling pathway was activated with bFGF (100 ng / ml). Cell clusters containing lens epithelial cells that morphologically differed from surrounding cells and extended 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 signaling pathway, inducing differentiation of lens epithelial cells into lens fibrous cells.

[0188] The differentiation of lens epithelial cells and lens fiber cells was confirmed using αA-crystallin as a marker. Immunocytochemical detection of anti-αA-crystallin antibody was performed in the same manner as in Example 1. Furthermore, iPS cell cultures containing cells differentiated into lens epithelial cells and lens fiber cells were analyzed using the nuclear RNA aggregate detection method 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 healthy individuals and DM1 patients. 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 healthy individuals and DM1 patients, stained with anti-αA-crystallin antibody and counterstained with DAPI for the cell nuclei. The scale bar in Figure 9B represents 200 μm. From Figure 9B, it was confirmed 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 fluorescence micrographs of lens epithelial cells and lens fiber cells (HC and DM1, respectively) derived from iPS cells of healthy individuals and DM1 patients, after FISH and DAPI counterstaining. RNA aggregates were not detected in HC cells, but they were observed in DM1 cells.

[0191] Example 8 Evaluation of ASO using iPS cells derived from DM1 patients (4) Lens epithelial cells and lens fiber cells (8.1) Materials and Methods Forty-eight hours after administering 10 nM ASO to iPS cell-derived lens epithelial cells and lens fiber cells from DM1 patients described in Example 7 by lipofection, FISH was performed using a Cy3-labeled (CAG)6-CA DNA / LNA probe, 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 DM1 patients, but these RNA aggregates almost completely disappeared after treatment with ASO-4M.

[0193] The left graph in Figure 9D is a bar graph representing the mean number ± standard deviation of RNA aggregates per cell nucleus 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 epithelium and fibroblasts derived from DM1 patient cells. The right graph in Figure 9D is a bar graph representing the mean ± standard deviation of the percentage of RNA aggregates in all 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 cells in lens epithelium and fibroblasts derived from DM1 patient cells. From these results, it was concluded that ASO-4M treatment can reduce RNA aggregates in lens epithelium and fibroblasts derived from DM1 patient cells.

[0194] Example 9: Differentiation of skin organoids from iPS cells derived from patients with myotonic dystrophy. (9.1) Materials and Methods Skin organoids were prepared based on Lee, J. et al. (Nature, 582: 399-404. (2020)). iPS cells 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 viable cells, confirmed by trypan blue staining, were seeded per well in 100 μL of E8-20Y medium into a round-bottom 96-well plate, and the plate was centrifuged at 100 rcf to promote aggregate formation. After 24 hours, 100 μL of Essential 8 Flex (Gibco) medium (hereinafter referred to as E8Y medium) supplemented with 100 μg / ml Normocin was added, and the cells were further cultured. After 24 hours, the cell aggregates were individually harvested and transferred to a new round-bottom 96-well plate with 100 μL per well 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 to initiate differentiation into skin organoids. On day 3 of differentiation, 25 μL per well of E6 medium supplemented with 200 ng / mL LDN (BMP inhibitor, Stemgent) and 50 μg / mL FGF was added to induce neural crest cell formation, bringing the total volume to 125 μL. On day 6 of differentiation, 75 μL of fresh E6 medium was added, bringing 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-adhesion plate in 500 μl of organoid maturation medium (OMM) containing 1% Matrigel. To maintain constant medium circulation by suspension culture of the aggregates, the 24-well plate was placed in an orbital shaker shaken at a speed of 65 rpm in a 37°C incubator containing 5% CO2. The OMM was prepared by adding 1X GlutaMax to a medium mixed with Advanced DMEM / F12 (Gibco) and Neurobasal (Gibco) in a 1:1 ratio. TMThe culture medium was supplemented with Gibco vitamin A (0.5X B-27 Minus Vitamin A), Gibco vitamin A (0.5X N2), Gibco vitamin A (0.5X N2), Gibco vitamin A (0.1 mM), Gibco vitamin A (0.1 mM), and Normocin (100 μg / mL). On day 15 of differentiation, half of the used medium was replenished with OMM medium containing 1% Matrigel. From day 18 onward, 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), and from day 45 onward, the entire medium was replaced once a week. As the aggregates matured and grew larger, it was sometimes necessary to increase the total volume of medium per well to 1 mL after day 80.

[0195] The differentiation of skin organoids was confirmed by the formation of hair follicle structures. Furthermore, frozen sections of skin organoids were analyzed using the same nuclear RNA aggregate detection method 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 iPS cell-derived skin organoids from healthy individuals and DM1 patients (HC and DM1, respectively). As shown in Figures 10B and C, RNA aggregates were detected in sections of iPS cell-derived skin organoids from DM1 patients after 4 months of culture, but not in sections of iPS cell-derived skin organoids from 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 patients with myotonic dystrophy, and FISH using a Cy3-labeled (CAG)6-CA DNA / LNA probe to detect nuclear RNA aggregates, were performed in the same manner as in Example 9. For ASO treatment of the organoids, the organoids were cultured in a swirling manner in NB medium containing 500 nM ASO-4M.

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

[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 Imamura, K. et al. (Sci Rep., 6:34904. (2016)). Fig. 11A is a conceptual diagram showing the procedure of a differentiation experiment system from undifferentiated cells of DM1 patient-derived iPS cells (DM-1 to 3) and healthy individual-derived iPS cells (HC-1 to 3) using a tetracycline-inducible NGN2 expression system. As shown in Fig. 11A, "B27 / N2 / NB + DOX" and "B27 / NB + DOX" represent the periods cultured in a medium for inducing neurons with doxocycline added as a differentiation inducer, and "B27 / NB" represents the period cultured in a medium for differentiated neurons. Undifferentiated iPS cells transfected with the tetracycline-inducible NGN2 expression system were changed to a medium for inducing neurons on day 0 to start differentiation induction, changed to a medium for neurons without a differentiation inducer on day 8 to end differentiation induction, and analyzed on day 11.

[0200] (11.2) Results Figure 11B is a fluorescence micrograph of neurons differentiated from iPS cells derived from a DM1 patient, stained with an anti-tubulin βIII antibody and subjected to DAPI counterstaining. The scale bar represents 100 μm. As shown in Figure 11B, by the procedure of this example, neurons expressing tubulin βIII and having extended axons are homogeneously obtained. Figure 11C is a composite image of fluorescence micrographs of neurons differentiated from iPS cells derived from healthy individuals (HC-1 to 3) and iPS cells derived from DM1 patients (DM-1 to 3), stained with an anti-tubulin βIII antibody (upper row), an anti-MAP2 antibody (middle row), or an anti-tubulin βIII antibody and an anti-Tbr1 antibody, and subjected to DAPI counterstaining. As shown in Figure 11C, the differentiation of iPS cells derived from DM1 patients into neurons occurs without much difference from that of iPS cells derived from healthy individuals.

[0201] Figure 11D is a fluorescence micrograph of neurons differentiated from iPS cells derived from healthy individuals (HC-1 to 3) and iPS cells derived from DM1 patients (DM-1 to 3), subjected to FISH using a Cy3-labeled (CAG)6-CA DNA / LNA probe and DAPI counterstaining. From Figure 11D, similar to Examples 2 and 3, among the iPS cells derived from DM1 patients, in the neurons derived from iPS cell DM-2, both the average number of RNA aggregates per cell nucleus and the percentage of RNA aggregates in the whole cell are significantly less than those in the neurons derived from iPS cells DM-1 and DM-3. Different from Examples 2 and 3, there was almost no difference in both the average number of RNA aggregates per cell nucleus and the percentage of RNA aggregates in the whole cell in the 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 On the 8th day of induction of differentiation into neurons from the iPS cells of the DM1 patient described in Example 11, 10 nM of ASO was administered by lipofection, and on the 11th day, FISH was performed using a Cy3-labeled (CAG)6-CA DNA / LNA probe in the same manner as in Example 7.

[0203] (12.2) Results The upper left image of Figure 11F is a composite image (Control) of fluorescence microscopy images (FISH) performed using a Cy3-labeled (CAG)6-CA DNA / LNA probe, staining with anti-tubulin βIII antibody, and DAPI counterstaining on iPS cells (DM-1) derived from DM1 patients treated with Control ASO. The upper right image of Figure 11F is a composite image (ASO-4M) of fluorescence microscopy images (ASO-4M) performed using a Cy3-labeled (CAG)6-CA DNA / LNA probe, staining with anti-tubulin βIII antibody, and DAPI counterstaining on iPS cells (DM-1) derived from DM1 patients treated with ASO-4M (same as ASO-4). As shown in Figure 11F, the number of RNA aggregates in neurons was significantly reduced by ASO-4M treatment. [Industrial applicability]

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

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

Claims

1. An oligonucleotide containing a repeating sequence from the 5' end to the 3' end, in which the cytosine-adenine-guanine trinucleotide is repeated 5 to 13 times, Oligonucleotides having a mixed-mer structure in which all cytidines are β-D-ENA-cytidines, all adenosines are 2'-OMe-adenosine, and all guanosines are 2'-OMe-guanosine.

2. The aforementioned nucleic acid is (1) Oligonucleotides consisting of any of the base sequences of SEQ ID NOs: 1 to 9, (2) An oligonucleotide consisting of adenine-guanine, one of the base sequences of SEQ ID NOs: 1 to 9, and cytosine, from the 5' end to the 3' end, (3) Oligonucleotides having a base sequence consisting of guanine, 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 according to claim 1.

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

4. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier or diluent.

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

6. The pharmaceutical composition according to claim 4 or 5, delivered by parenteral administration selected from the group consisting of transdermal administration, intraocular administration, intralenical administration, intracrystalline lens administration, intra-gastrointestinal mucosal administration, submucosal administration, subcutaneous administration, intravenous administration, intra-arterial administration, intramuscular administration, intraperitoneal administration, intracranial administration, and intrathecal administration.

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

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

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