Pharmaceutical composition for muscle disease treatment

Abstract

To develop a nucleic acid complex that exhibits an excellent antisense effect in skeletal muscle and / or heart muscle, and a composition for treating or preventing muscle diseases that develop in skeletal muscle, heart muscle, and the like, comprising the nucleic acid complex as an active ingredient.SOLUTION: Provided is a double-stranded nucleic acid complex in which a first nucleic acid strand that hybridizes to the transcription product of a target gene and has an antisense effect on the transcription product is annealed with a second nucleic acid strand that comprises a base sequence complementary to the first nucleic acid strand and is bound to cholesterol or an analog thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a double-stranded nucleic acid complex capable of specifically suppressing the expression of a target gene expressed in skeletal muscle and cardiac muscle, and a pharmaceutical composition for treating or preventing a muscle disease, which contains the same as an active ingredient. [Background technology]

[0002] Muscular dystrophy, a type of muscle disease, is a progressive genetic muscle disorder in which degeneration or necrosis of skeletal muscle fibers causes muscle atrophy and muscle weakness. When the condition worsens, it can lead to motor impairments such as difficulty walking, and in many cases, death from respiratory or cardiac failure can occur. Muscular dystrophy is classified into various types, such as Duchamp, Becker, limb-girdle, and facioscapulohumeral types, depending on the inheritance pattern and clinical symptoms (Non-Patent Document 1).

[0003] To date, there is no cure for muscular dystrophies, and most are treated with symptomatic treatment. For example, Duchenne muscular dystrophy has traditionally been treated with steroids. In February 2017, the U.S. Food and Drug Administration (FDA) approved deflazacort (trade name Emflaza®) for the treatment of DMD in children aged 5 years and older and adults. This treatment is a corticosteroid that reduces immune system activity and suppresses inflammation. In September 2016, the FDA also approved eteplirsen (trade name EXONDYS 51) for the treatment of Duchenne muscular dystrophy. This treatment is a nucleic acid drug designed to induce exon skipping during pre-mRNA splicing during dystrophin gene expression, resulting in the synthesis of mRNA lacking the 51st exon.

[0004] The prevalence of muscular dystrophy is said to be 17 to 20 people per 100,000 population, but the market size for related therapeutic drugs is expanding year by year and is estimated to reach $78.5 billion by 2022.

[0005] In recent years, oligonucleotides have attracted attention in the development of pharmaceuticals known as nucleic acid drugs, and the development of nucleic acid drugs using the antisense method has been actively promoted, particularly because of their high selectivity for target genes and low toxicity. The antisense method is a method in which a partial sequence of mRNA or miRNA transcribed from a target gene is used as the target sense strand, and a complementary oligonucleotide (antisense oligonucleotide: often referred to as "ASO (AntiSense Oligonucleotide)" in this specification) is introduced into cells to selectively modify or inhibit the expression of a protein encoded by a target gene.

[0006] As a nucleic acid utilizing the antisense method, the present inventors have previously developed a double-stranded nucleic acid complex in which an antisense oligonucleotide is annealed to its complementary strand. For example, Patent Document 1 discloses that an antisense oligonucleotide annealed to a tocopherol-conjugated complementary strand is efficiently delivered to the liver and has a high antisense effect. Furthermore, Patent Document 2 discloses the development of a double-stranded antisense oligonucleotide with exon skipping effect, as well as a short gapmer antisense oligonucleotide in which additional nucleotides are added to the 5' end, 3' end, or both the 5' and 3' ends of the gapmer (antisense oligonucleotide). Furthermore, Patent Document 3 discloses the development of a double-stranded agent for delivering therapeutic oligonucleotides.

[0007] As mentioned above, many causes of death in muscular dystrophy are respiratory failure and heart failure caused by the expression of mutant genes. If the expression of these genes in skeletal muscles such as the diaphragm and cardiac muscle can be regulated using the aforementioned nucleic acid medicines, it may be possible to reduce the mortality rate from muscular dystrophy. Furthermore, similar methods may be possible to treat and prevent other muscle diseases such as myopathy and cardiomyopathy.

[0008] However, no nucleic acid complex has been developed to date that can be efficiently delivered to skeletal or cardiac muscle and exhibits an excellent antisense effect at the site. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2013 / 089283 [Patent Document 2] International Publication No. 2014 / 203518 [Patent Document 3] International Publication No. 2014 / 192310 [Non-patent literature]

[0010] [Non-Patent Document 1] Edited by Sugita, Hideo, Ozawa, Eijiro, and Nonaka, Masaya, 1995, New Muscle Diseases. Nankodo, Tokyo: pp. 469-550 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to develop a nucleic acid complex that can be efficiently delivered to skeletal muscle and / or cardiac muscle and exhibits excellent antisense effects at the relevant sites, and to develop a composition that contains such a nucleic acid complex as an active ingredient for treating or preventing muscle diseases that occur in skeletal muscle, cardiac muscle, etc. [Means for solving the problem]

[0012] In order to solve the above problems, the inventors have conducted extensive research and have found that a double-stranded nucleic acid complex consisting of an antisense oligonucleotide and a complementary strand bound to a lipid, particularly cholesterol, which was previously thought to be delivered mainly to the liver, can also be efficiently delivered to skeletal muscle and cardiac muscle, and exhibits extremely excellent antisense effects at these sites.

[0013] Therefore, by designing the target gene of the double-stranded nucleic acid complex (double-stranded nucleic acid agent) having the above configuration to be a causative gene of a muscle disease that is expressed in skeletal muscle and / or cardiac muscle, the antisense oligonucleotide can be efficiently delivered to skeletal muscle and / or cardiac muscle to regulate the expression of the target gene, thereby making it possible to use it as a therapeutic or preventive composition for treating a muscle disease. The present invention is based on the above findings and development results, and provides the following.

[0014] (1) A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, for suppressing or enhancing the expression level of a transcription product or translation product of a target gene in the skeletal or cardiac muscle of a subject, or for inhibiting the function of the transcription product or translation product of the target gene, wherein the first nucleic acid strand comprises a base sequence capable of hybridizing to all or part of the transcription product of the target gene and has an antisense effect on the transcription product; the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and is bound to cholesterol or an analog thereof; and the first nucleic acid strand is annealed to the second nucleic acid strand. (2) The double-stranded nucleic acid complex according to (1), wherein the first nucleic acid strand contains at least four consecutive deoxyribonucleosides. (3) The double-stranded nucleic acid complex according to (2), wherein the first nucleic acid strand is a gapmer. (4) The double-stranded nucleic acid complex according to (1) or (2), wherein the first nucleic acid strand is a mixmer. (5) The double-stranded nucleic acid complex according to any one of (1) to (4), wherein the second nucleic acid strand contains at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the first nucleic acid strand. (6) The double-stranded nucleic acid complex according to any one of (1) to (5), wherein the second nucleic acid strand does not contain natural ribonucleosides. (7) The double-stranded nucleic acid complex according to any one of (1) to (6), wherein the nucleic acid portion of the second nucleic acid strand is composed of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside bonds. (8) The double-stranded nucleic acid complex according to any one of (1) to (7), wherein the second nucleic acid strand is bound to cholesterol or an analog thereof. (9) The double-stranded nucleic acid complex according to any one of (1) to (8), wherein the cholesterol or an analog thereof is bound to the 5' end and / or the 3' end of the second nucleic acid strand. (10) The double-stranded nucleic acid complex according to any one of (1) to (9), wherein a ligand is bound to the second nucleic acid strand via a cleavable or uncleavable linker.

[0015] (11) The double-stranded nucleic acid complex according to any one of (1) to (10), wherein the first nucleic acid strand and the second nucleic acid strand are bound via the linker. (12) The double-stranded nucleic acid complex according to (10) or (11), wherein the linker is made of a nucleic acid. (13) A pharmaceutical composition comprising, as an active ingredient, the double-stranded nucleic acid complex according to any one of (1) to (12). (14) The pharmaceutical composition according to (13), which is for treating skeletal muscle dysfunction or cardiac dysfunction in a subject. (15) The pharmaceutical composition according to (13) or (14), wherein the skeletal muscle dysfunction or cardiac dysfunction is a disease selected from the group consisting of muscular dystrophy, myopathy, inflammatory myopathy, polymyositis, dermatomyositis, Danon disease, myasthenic syndrome, mitochondrial disease, myoglobinuria, glycogen storage disease, periodic paralysis, hereditary cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, hereditary arrhythmia, neurodegenerative disease, sarcopenia, and cachexia. (16) The pharmaceutical composition according to any one of (13) to (15), which is administered intravenously, intramuscularly, or subcutaneously. (17) The pharmaceutical composition according to any one of (13) to (16), wherein the amount of the double-stranded nucleic acid complex administered per dose is 0.1 mg / kg or more. (18) The pharmaceutical composition according to any one of (13) to (17), wherein the double-stranded nucleic acid complex is administered in a single dose of 0.01 mg / kg to 200 mg / kg. (19) The pharmaceutical composition according to any one of (13) to (18), wherein the transcription product of the target gene is any RNA selected from the group consisting of mRNA, microRNA, pre-mRNA, long non-coding RNA, and natural antisense RNA. (20) The pharmaceutical composition according to any one of (13) to (19), wherein the first nucleic acid strand is any RNA selected from the group consisting of steric blocking, splicing switch, exon skipping, and exon inclusion.

[0016] (21) The pharmaceutical composition according to any one of (13) to (20), wherein the base sequence of the first nucleic acid strand in the double-stranded nucleic acid complex is represented by SEQ ID NO: 24. (22) A double-stranded nucleic acid complex for inducing RNA editing, exon skipping, or exon inclusion of a target gene in skeletal or cardiac muscle of a subject, or for sterically blocking a target RNA, comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand comprises a base sequence capable of hybridizing to all or a part of a transcription product of the target gene and has an antisense effect on the transcription product, and the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and the first nucleic acid strand is annealed to the second nucleic acid strand. (23) The double-stranded nucleic acid complex according to (22), wherein the first nucleic acid strand comprises at least one morpholino nucleic acid or ribose 2'-modified nucleic acid. (24) The double-stranded nucleic acid complex according to (22) or (23), wherein 50% or more of the bases in the first nucleic acid strand are morpholino nucleic acids or ribose-2'-modified nucleic acids. (25) The double-stranded nucleic acid complex according to any one of (22) to (24), wherein the first nucleic acid strand is a mixmer. (26) The double-stranded nucleic acid complex according to any one of (22) to (25), wherein 100% of the bases in the first nucleic acid strand are morpholino nucleic acids or nucleic acids modified at the 2'-position of ribose. (27) The double-stranded nucleic acid complex according to any one of (22) to (26), wherein the second nucleic acid strand does not contain natural ribonucleosides. (28) The double-stranded nucleic acid complex according to any one of (22) to (27), wherein the nucleic acid portion of the second nucleic acid strand is composed of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside bonds. (29) The double-stranded nucleic acid complex according to any one of (22) to (28), wherein the second nucleic acid strand is bound to a functional moiety. (30) The double-stranded nucleic acid complex according to any one of (22) to (28), wherein the functional moiety is selected from the group consisting of cholesterol or an analog thereof, tocopherol or an analog thereof, phosphatidylethanolamine or an analog thereof, a substituted or unsubstituted C1-30 alkyl group, a substituted or unsubstituted C2-30 alkenyl group, and a substituted or unsubstituted C1-30 alkoxy group. (31) The double-stranded nucleic acid complex according to (30), wherein the functional moiety is cholesterol or an analog thereof. (32) The double-stranded nucleic acid complex according to any one of (22) to (31), wherein the cholesterol or an analog thereof is bound to the 5' end and / or the 3' end of the second nucleic acid strand.

[0017] (33) The double-stranded nucleic acid complex according to any one of (22) to (32), wherein a ligand is bound to the second nucleic acid strand via a cleavable or uncleavable linker. (34) A pharmaceutical composition comprising, as an active ingredient, the double-stranded nucleic acid complex according to any one of (22) to (33). (35) The pharmaceutical composition according to (34), which is for treating muscular dystrophy in a subject. (36) The pharmaceutical composition according to (35), wherein the muscular dystrophy is myotonic dystrophy or Duchenne muscular dystrophy. (37) The pharmaceutical composition according to any one of (34) to (36), which is administered intravenously or subcutaneously. (38) The pharmaceutical composition according to any one of (34) to (37), wherein the amount of the double-stranded nucleic acid complex administered per dose is 0.1 mg / kg or more. (39) The pharmaceutical composition according to any one of (34) to (38), wherein the double-stranded nucleic acid complex is administered in a single dose of 0.01 mg / kg to 200 mg / kg. (40) The pharmaceutical composition according to any one of (34) to (39), wherein the base sequence of the first nucleic acid strand in the double-stranded nucleic acid complex is represented by any one of SEQ ID NOs: 25 to 28. This specification includes the disclosure of Japanese Patent Application No. 2019-073832, from which this application claims priority. [Effects of the Invention]

[0018] The present invention provides a double-stranded nucleic acid complex that can efficiently deliver a double-stranded nucleic acid complex agent to skeletal muscle and cardiac muscle, thereby exerting an antisense effect at the site. The antisense effect enables the suppression or enhancement of target gene expression, functional inhibition, or induction of exon skipping. [Brief explanation of the drawings]

[0019] [Figure 1] Schematic diagrams of representative examples of double-stranded nucleic acid complexes of the present invention are shown. FIG. 1a shows a double-stranded nucleic acid complex in which tocopherol is bound to the 5' end of the second nucleic acid strand. FIG. 1b shows a double-stranded nucleic acid complex in which cholesterol is bound to the 5' end of the second nucleic acid strand. FIG. 1c shows a self-annealed single-stranded nucleic acid in which the double-stranded nucleic acid complex of FIG. 1b is linked via an RNA linker. Although not shown here, cholesterol or an analog thereof may be bound to the 3' end of the second nucleic acid strand. Cholesterol or an analog thereof may also be bound to both ends of the second nucleic acid strand. Furthermore, cholesterol or an analog thereof may be bound to an internal nucleotide of the second nucleic acid strand or to the RNA region of the single-stranded nucleic acid. [Figure 2] FIG. 1 shows the structures of various bridged nucleic acids. [Figure 3]This figure shows the inhibitory effect of the double-stranded nucleic acid complexes of the present invention, in which tocopherol or cholesterol is bound to the second nucleic acid strand (Toc#1HDO(mSR-B1) and Chol#1HDO(mSR-B1), respectively), on the expression of the target SR-B1 gene in the cardiac muscle (heart), quadriceps (quadriceps), diaphragm, and dorsal proper (back) muscles. In the figure, ASO is the positive control, and represents the single-stranded nucleic acid molecule ASO(mSR-B1), and PBS is the negative control, which uses PBS as a solvent. [Figure 4] This figure shows the inhibitory effect of the double-stranded nucleic acid complexes of the present invention, in which tocopherol or cholesterol is bound to the second nucleic acid strand (Toc#1HDO(mMalat1) and Chol#1HDO(mMalat1), respectively), on the expression of the target Malat1 gene in the cardiac muscle (Heart), quadriceps (Quadriceps), diaphragm (Diaphragm), and dorsal proper (Back) muscles. In the figure, ASO is the positive control, and represents the single-stranded nucleic acid molecule ASO(mMalat1), and PBS is the negative control, which uses PBS as a solvent. [Figure 5] This figure shows the inhibitory effect of administering 12.5 mg / kg of double-stranded nucleic acid complexes of the present invention, in which tocopherol or cholesterol is bound to the second nucleic acid strand (Toc#1HDO(mDMPK) and Chol#1HDO(mDMPK), respectively), on the expression of the target DMPK gene in the gastrocnemius (GC), tibialis anterior (TA), triceps brachii (TB), quadriceps femoris, diaphragm, dorsi propria (back), and cardiac muscle (heart). In the figure, ASO represents the positive control, which is a single-stranded nucleic acid molecule, ASO(mDMPK), and PBS represents the negative control, which is PBS used as a solvent. [Figure 6]This figure shows the inhibitory effect of administering 25 mg / kg of double-stranded nucleic acid complexes of the present invention, in which tocopherol or cholesterol is bound to the second nucleic acid strand (Toc#1HDO(mDMPK) and Chol#1HDO(mDMPK), respectively), on the expression of the target DMPK gene in the gastrocnemius (GC), tibialis anterior (TA), triceps brachii (TB), quadriceps femoris, diaphragm, dorsi propria (back), and cardiac muscle (heart). In the figure, ASO represents the positive control, which is a single-stranded nucleic acid molecule, ASO(mDMPK), and PBS represents the negative control, which is PBS used as a solvent. [Figure 7] This figure shows the inhibitory effect of administering 50 mg / kg of double-stranded nucleic acid complexes of the present invention, in which tocopherol or cholesterol is bound to the second nucleic acid strand (Toc#1HDO(mDMPK) and Chol#1HDO(mDMPK), respectively), on the expression of the target DMPK gene in the gastrocnemius (GC), tibialis anterior (TA), triceps (TB), quadriceps (quadriceps), diaphragm, dorsi propria (back), and cardiac muscle (heart). In the figure, ASO represents the positive control, which is a single-stranded nucleic acid molecule, ASO(mDMPK), and PBS represents the negative control, which is PBS used as a solvent. [Figure 8] This figure shows the inhibitory effect of double-stranded nucleic acid complexes of the present invention, in which tocopherol or cholesterol is bound to the second nucleic acid strand (Toc#1DNA / DNA(mMalat1) and Chol#1DNA / DNA(mMalat1), respectively), on the expression of the target malat1 gene in cardiac muscle (Heart), quadriceps (Quadriceps), and diaphragm (Diaphragm). The second nucleic acid strand constituting this double-stranded nucleic acid complex is also composed of DNA only. In the figure, ASO is the positive control, and represents the single-stranded nucleic acid molecule ASO(mMalat1), and PBS is the negative control, which uses PBS as a solvent. [Figure 9]This figure shows the inhibitory effect of the double-stranded nucleic acid complex of the present invention, which contains either cholesterol-conjugated Chol#1-cDNA(mMalat1)(PS) or Chol#1-cDNA(mMalat1)(PO), which is composed entirely of DNA and has phosphorothioate linkages between nucleosides as the second nucleic acid strand, on the expression of the target malat1 gene in cardiac muscle (heart), diaphragm, and dorsal muscle proper (back). In the figure, PBS represents the solvent used as a negative control. [Figure 10] This figure shows the inhibitory effect of the double-stranded nucleic acid complex of the present invention, which is composed of Chol#1HDO(mMalat1)(PO), Chol#1HDO(5'PS), and Chol#1HDO(3'PS), in which the second nucleic acid strand contains a phosphodiester or phosphorothioate bond between the nucleosides of the second nucleic acid strand, to which cholesterol is bound at the 5' end of the second nucleic acid strand, in cardiac muscle (heart), quadriceps (quadriceps), diaphragm, and dorsal muscle proper (back). In the figure, PBS represents the solvent used as a negative control. [Figure 11] This figure shows the inhibitory effect of multiple administrations of double-stranded nucleic acid complexes of the present invention, in which tocopherol or cholesterol is bound to the second nucleic acid strand (Toc#1HDO(mMalat1) and Chol#1HDO(mMalat1), respectively), on the expression of the target Malat1 gene in the myocardium (Heart), quadriceps (Quadriceps), diaphragm (Diaphragm), and dorsal proper (Back) muscles. In the figure, ASO represents the positive control, which is a single-stranded nucleic acid molecule, ASO(mMalat1), and PBS represents the negative control, which is PBS used as a solvent. [Figure 12]

[0023] Figure 1 shows the inhibitory effect of the double-stranded nucleic acid complex of the present invention (Chol#1HDO(mDMPK)) in which cholesterol is bound to the second nucleic acid strand in the expression of the target DMPK gene in cardiac muscle (Heart), quadriceps (Quadriceps), diaphragm, and back muscle (Back). In the figure, PBS represents the PBS used as a solvent, which serves as a negative control. [Figure 13]This figure shows the inhibitory effect of the double-stranded nucleic acid complex Chol#1HDO (mMalat1) of the present invention, in which cholesterol is bound to the second nucleic acid strand, on the expression of a target gene (malat1) in (a) cardiac muscle (Heart), (b) dorsal muscle (Back), (c) quadriceps femoris (QF), and (d) diaphragm (Dia). The vertical axis indicates the relative expression level of malat1 non-coding RNA, and the horizontal axis indicates the time (days) elapsed since administration. In the figure, PBS indicates the PBS used as a solvent, which serves as a negative control. [Figure 14] The relative expression level of malat1 non-coding RNA in each tissue 8 weeks (56 days) after administration is shown. In the figure, PBS indicates the PBS used as a solvent, which serves as a negative control. [Figure 15] FIG. 1 shows the suppressive effect of single administration of the double-stranded nucleic acid complex Chol#1HDO(mMalat1) of the present invention at various doses on the expression of the target malat1 gene in (a) quadriceps, (b) cardiac muscle, (c) back muscle, and (d) diaphragm. [Figure 16] 1 shows the inhibitory effect of the double-stranded nucleic acid complex of the present invention, in which a linker consisting of a hexyl group having six carbon atoms is attached between the cholesterol attached to the second nucleic acid strand and the nucleic acid terminus, on the expression of the target malat1 gene in cardiac muscle (heart), quadriceps (quadriceps), diaphragm (diaphragm), and dorsal muscle proper (back). In the figure, PBS represents the PBS used as a solvent as a negative control. [Figure 17] 1 shows the inhibitory effect of double-stranded nucleic acid complexes containing first nucleic acid strands with different lengths of complementary strands to the target gene on the expression of a target gene (malat1) in cardiac muscle (heart), quadriceps (quadriceps), diaphragm, and dorsal muscle proper (back). In the figure, PBS represents the PBS used as a solvent, which serves as a negative control. [Figure 18] FIG. 1 shows the effect of subcutaneous administration of the double-stranded nucleic acid complex agent of the present invention on the suppression of target gene (malat1) expression in (a) myocardium (Heart), (b) quadriceps (Quadriceps), and (c) diaphragm. [Figure 19] FIG. 1 shows the inhibitory effects of a single-stranded nucleic acid complex agent having cholesterol bound to its terminus, the double-stranded nucleic acid complex agent Chol#1HDO (mMalat1) of the present invention, and negative control PBS on the expression of the malat1 gene in cardiac muscle (Heart), quadriceps (Quadriceps), diaphragm, and dorsal muscle proper (Back). [Figure 20] FIG. 1 shows the platelet counts in mouse blood 72 hours after administration of single-stranded nucleic acid complex agents having cholesterol attached to their termini (5′Chol-ASO-DNA, 3′Chol-ASO-DNA), the double-stranded nucleic acid complex agent Chol#1HDO (mMalat1) of the present invention, and negative control PBS. [Figure 21] Representative electropherograms of PCR results obtained using a Bioanalyzer 2100 (Agilent) are shown. (a) shows the PCR products from the heart, and (b) shows the PCR products from the quadriceps. In the figure, the arrowhead indicates the unskipped band, and the arrow indicates the skipped band. [Figure 22] Figure 1 shows the exon skipping rates of the dystrophin gene in mdx mice (a model mouse of Duchenne muscular dystrophy) treated with a single-stranded nucleic acid complex (PMO), the double-stranded nucleic acid complex Toc#1HDO (PMO) of the present invention with a tocopherol-terminated terminal, and negative control PBS. (a) Cardiac muscle (Heart), (b) Diaphragm, (c) Back, (d) Quadriceps, (e) Tibialis anterior, and (f) Triceps. [Figure 23]This is a Western blot diagram showing the expression of dystrophin protein. The dystrophin levels are shown after administration of a single-stranded nucleic acid complex (PMO), a double-stranded nucleic acid complex (Toc#1HDO) with a tocopherol terminally attached (Toc-HDO) of the present invention, and negative control PBS to (a) the cardiac muscle (heart) and (b) the dorsal muscle proper (back) of mdx mice. B10 (normal mouse) shows dystrophin as a positive control. [Figure 24] FIG. 1 shows immunostaining results showing the expression of dystrophin protein in the cardiac muscle and dorsal muscle proper after administration of a single-stranded nucleic acid complex (PMO) and the double-stranded nucleic acid complex Toc#1HDO (Toc-HDO) of the present invention, which has a tocopherol attached to its end, in mdx mice. [Figure 25] FIG. 1 shows exon skipping of the dystrophin gene in the myocardium, diaphragm, quadriceps, tibialis anterior, and triceps brachii muscles of mdx mice using a single-stranded nucleic acid complex agent (PMO), the double-stranded nucleic acid complex agents Toc#1HDO (Toc-HDO) and Chol#1HDO (Chol-HDO) of the present invention having a tocopherol attached to the terminus, and negative control PBS. [Figure 26] FIG. 1 shows exon skipping of the dystrophin gene in the cardiac muscle, quadriceps, tibialis anterior, triceps brachii, and dorsi proper muscles of mdx mice using a single-stranded nucleic acid complex agent (Mixmer), a double-stranded nucleic acid complex agent Toc#1HDO (Toc-Mixmer) of the present invention having a tocopherol attached to its terminus, and negative control PBS. [Figure 27] This figure shows the inhibitory effect of the double-stranded nucleic acid complex of the present invention (Chol#1HDO(mMalat1)) and the nucleic acid molecule Chol-sHDO, which is a self-annealed nucleic acid molecule formed by linking Chol-HDO with an RNA linker (Fig. 1c), in the cardiac muscle (heart), quadriceps (quadriceps), diaphragm, and dorsal muscle proper (back). PBS indicates the solvent used as a negative control. [Figure 28]This figure shows the inhibitory effect of the double-stranded nucleic acid complex of the present invention, in which cholesterol is bound to the second nucleic acid strand (Chol#1HDO(mMalat1)), and the nucleic acid (3'Chol(TEG)HDO(mMalat1)), in which the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is bound to cholesterol at its 3' end, with a tetraethylene glycol (TEG) linker between the cholesterol and the end of the second nucleic acid strand, on the expression of the target Malat1 gene in cardiac muscle (heart), quadriceps (quadriceps), diaphragm, and dorsal muscles proper (back). PBS indicates the PBS used as a solvent, which serves as a negative control. [Figure 29] FIG. 1 shows the running duration in an exercise stress test for normal mice (B10), and mdx mice administered PBS alone (mdx), a single-stranded nucleic acid complex (PMO), a double-stranded nucleic acid complex with tocopherol attached to the end, Toc#1HDO (Toc-HDO), or a double-stranded nucleic acid complex with cholesterol attached to the end, Chol#1HDO (Chol-HDO). [Figure 30] FIG. 1 shows the results of measuring (a) grip power and (b) holding impulse in normal mice (B10) and mdx mice administered PBS alone (mdx), a single-stranded nucleic acid complexing agent (PMO), a double-stranded nucleic acid complexing agent Toc#1HDO (Toc-HDO) with a tocopherol terminally attached, or a double-stranded nucleic acid complexing agent Chol#1HDO (Chol-HDO) with a cholesterol terminally attached. [Figure 31] FIG. 1 shows the measured values of (a) creatine kinase (CK), (b) aspartate aminotransferase (AST), and (c) alanine aminotransferase (ALT) in the serum of normal mice (B10) and mdx mice administered PBS alone (mdx), a single-stranded nucleic acid complexing agent (PMO), a tocopherol-terminated double-stranded nucleic acid complexing agent Toc#1HDO (Toc-HDO), or a cholesterol-terminated double-stranded nucleic acid complexing agent Chol#1HDO (Chol-HDO). [Figure 32]FIG. 1 shows the corrected QT interval (QTc) measured by electrocardiogram in normal mice (B10), and mdx mice administered PBS alone (mdx), a single-stranded nucleic acid complexing agent (PMO), a double-stranded nucleic acid complexing agent Toc#1HDO (Toc-HDO) having a tocopherol attached to its end, or a double-stranded nucleic acid complexing agent Chol#1HDO (Chol-HDO) having a cholesterol attached to its end. [Figure 33] 1 is a Western blot diagram showing the expression of dystrophin protein in cardiac muscle, (a) dystrophin protein and (b) vinculin protein in cardiac muscle of a normal mouse (B10) and mdx mice administered with PBS alone (mdx), a single-stranded nucleic acid complex (PMO), a tocopherol-terminated double-stranded nucleic acid complex Toc#1HDO (Toc-HDO), or a cholesterol-terminated double-stranded nucleic acid complex Chol#1HDO (Chol-HDO). [Figure 34] 1 is a Western blot diagram showing the expression of dystrophin protein in the quadriceps muscle of a normal mouse (B10), and mdx mice administered with PBS alone (mdx), a single-stranded nucleic acid complex (PMO), a tocopherol-terminated double-stranded nucleic acid complex Toc#1HDO (Toc-HDO), or a cholesterol-terminated double-stranded nucleic acid complex Chol#1HDO (Chol-HDO). [Figure 35] Immunostaining images showing dystrophin protein expression in cardiac muscle are shown. These images show dystrophin protein expression in cardiac muscle of normal mice (B10) and mdx mice administered PBS alone (mdx), single-stranded nucleic acid complex (PMO), tocopherol-terminated double-stranded nucleic acid complex Toc#1HDO (Toc-HDO), or cholesterol-terminated double-stranded nucleic acid complex Chol#1HDO (Chol-HDO). The scale bar indicates 200 μm. [Figure 36]This is an immunostaining image showing dystrophin protein expression in the quadriceps muscle of a normal mouse (B10) and an mdx mouse administered PBS alone (mdx), a single-stranded nucleic acid complex (PMO), a double-stranded nucleic acid complex with tocopherol attached at its termini (Toc#1HDO (Toc-HDO)), or a double-stranded nucleic acid complex with cholesterol attached at its termini (Chol#1HDO (Chol-HDO)). The scale bar indicates 200 μm. DETAILED DESCRIPTION OF THE INVENTION

[0020] 1. Double-stranded nucleic acid complex 1-1. Overview A first aspect of the present invention relates to a double-stranded nucleic acid complex, which can suppress or enhance the expression level of a transcription product or translation product of a target gene in skeletal or cardiac muscle of a subject, inhibit the function of the transcription product or translation product of the target gene, or induce steric blocking, splicing switching, RNA editing, exon skipping, or exon inclusion by virtue of its antisense effect.

[0021] The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand annealed to each other. The first nucleic acid strand comprises a base sequence capable of hybridizing to all or part of a transcription product of a target gene and has an antisense effect on the transcription product. The second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and has a functional moiety bound to its 5'-end and / or 3'-end.

[0022] 1-2.Definition of Terms As used herein, a "target gene" refers to a gene whose transcription or translation product expression level can be suppressed or enhanced, whose transcription or translation product function can be inhibited, or whose steric blocking, splicing switch, RNA editing, exon skipping, or exon inclusion can be induced by the antisense effect of the double-stranded nucleic acid complex of the present invention. The type of target gene is not particularly limited as long as it is expressed in vivo. Examples of such a gene include genes derived from organisms into which the double-stranded nucleic acid complex of the present invention is introduced, such as genes whose expression increases in various diseases. Examples of such genes include the scavenger receptor B1 (often referred to herein as "SR-B1") gene, the metastasis associated lung adenocarcinoma transcript 1 (often referred to herein as "Malat1") gene, the DMPK (dystrophia myotonica-protein kinase) gene, and the dystrophin gene.

[0023] Here, scavenger receptors are receptor membrane proteins for denatured lipoproteins and are known to be involved in cholesterol and lipoprotein metabolism. SR-B1 is a two-transmembrane protein belonging to the evolutionarily conserved CD36 family, and has a long extracellular domain and two short intracellular domains, one at the amino terminus and one at the carboxyl terminus.

[0024] Malat1 is a long non-coding RNA (lncRNA) that is highly expressed in malignant tumors, including lung cancer, and is known to reside in the nucleus of muscle cells.

[0025] The DMPK gene encodes myotonin protein kinase and is known to be the causative gene for myotonic dystrophy, the most common muscular dystrophy in adults. Abnormal expansion of the CTG repeat sequence in the 3' untranslated region of the DMPK gene is believed to be the cause of the disease.

[0026] As used herein, the term "target transcript" refers to any RNA that is a direct target of the nucleic acid complex of the present invention and is synthesized by RNA polymerase. Generally, this term refers to a "transcription product of a target gene." Specifically, this term may include mRNA (including mature mRNA, pre-mRNA, and mRNA without base modifications) transcribed from a target gene, non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA. Examples of transcription products of a target gene include SR-B1 mRNA, which is a transcription product of the SR-B1 gene; Malat1 non-coding RNA, which is a transcription product of the Malat1 gene; and DMPK mRNA, which is a transcription product of the DMPK gene.

[0027] As a specific example, SEQ ID NO: 1 shows the nucleotide sequence of mouse SR-B1 mRNA, and SEQ ID NO: 2 shows the nucleotide sequence of human SR-B1 mRNA. SEQ ID NO: 3 shows the nucleotide sequence of mouse malat1 non-coding RNA, and SEQ ID NO: 4 shows the nucleotide sequence of human Malat1 non-coding RNA. SEQ ID NO: 5 shows the nucleotide sequence of mouse DMPK mRNA, and SEQ ID NO: 6 shows the nucleotide sequence of human DMPK mRNA. Note that in all of SEQ ID NOs: 1 to 6, the mRNA nucleotide sequence has been replaced with the DNA nucleotide sequence. Information on the nucleotide sequences of these genes and transcription products can be obtained from known databases, such as the NCBI (National Center for Biotechnology Information) database.

[0028] In addition, nucleotide sequences of known antisense drugs can also be used. For example, the nucleotide sequence shown in SEQ ID NO: 24 constituting ISIS 598769 (IONIS), a therapeutic drug for myotonic dystrophy that targets the DMPK gene, the causative gene of myotonic dystrophy; the nucleotide sequence shown in SEQ ID NO: 25 constituting Eteplirsen (Sarepta; Exondys 51), a known therapeutic drug for Dusenne muscular dystrophy that induces exon skipping of the pre-mRNA of the dystrophin gene; the nucleotide sequence shown in SEQ ID NO: 26 constituting Golodirsen (Sarepta); the nucleotide sequence shown in SEQ ID NO: 27 constituting NS-065 / NCNP-01 (Nippon Shinyaku); and the nucleotide sequence shown in SEQ ID NO: 28 constituting Casimersen (Sarepta) may be used.

[0029] As used herein, the term "antisense oligonucleotide (ASO)" refers to a single-stranded oligonucleotide that contains a complementary base sequence capable of hybridizing to all or a portion of a target transcript, e.g., any target region, and that can inhibit and control the expression of the transcript of the target gene or the level of the target transcript through its antisense effect. In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand functions as an ASO, and its target region may include a 3' UTR, a 5' UTR, an exon, an intron, a coding region, a translation initiation region, a translation termination region, or any other nucleic acid region. The target region of the target transcript can be at least 8 bases long, e.g., 10 to 35 bases long, 12 to 25 bases long, 13 to 20 bases long, 14 to 19 bases long, or 15 to 18 bases long.

[0030] The term "antisense effect" refers to the effect of ASOs hybridizing to a target transcript (e.g., the RNA sense strand) to modulate the expression or editing of that target transcript. "Modulating the expression or editing of a target transcript" refers to suppression or reduction of the expression of a target gene or the expression level of the target transcript (herein, "expression level of a target transcript" is often referred to as "target transcript level"), translation inhibition, RNA editing, splicing function modification effects (e.g., splicing switch, exon inclusion, exon skipping, etc.), or transcript degradation. For example, in post-transcriptional inhibition of a target gene, when an RNA oligonucleotide is introduced into a cell as an ASO, the ASO forms a partial duplex with the mRNA, the transcript of the target gene. This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of the target protein encoded by the target gene at the translational level (steric blocking). On the other hand, when an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in degradation of the target gene's mRNA and inhibition of the expression of the protein encoded by the target gene. Furthermore, the antisense effect can also be achieved by targeting an intron in a pre-mRNA. Furthermore, the antisense effect can also be achieved by targeting an miRNA. In this case, inhibition of the function of the miRNA can increase the expression of the gene whose expression the miRNA normally controls. In one embodiment, the modulation of the expression of the target transcript can be a reduction in the amount of the target transcript.

[0031] As used herein, the term "translation product of a target gene" refers to any polypeptide or protein that is a direct target of the nucleic acid complex of the present invention and is synthesized by translation of the target transcription product or the transcription product of the target gene. Examples of translation products of target genes include the SR-B1 protein, which is a translation product of the SR-B1 gene, the Malat1 protein, which is a translation product of the Malat1 gene, and the DMPK protein, which is a translation product of the DMPK gene.

[0032] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a monomeric nucleoside or nucleotide, an oligomeric oligonucleotide, or a polymeric polynucleotide.

[0033] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, specific examples of which include ribose and deoxyribose. The base portion (nucleobase) of a nucleoside is typically a heterocyclic base moiety. Examples include, but are not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).

[0034] A "nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides containing a pentofuranosyl sugar, the phosphate group is typically linked to the 2', 3', or 5' hydroxyl group of the sugar.

[0035] An "oligonucleotide" refers to a linear oligomer formed by covalently linking several to several tens of hydroxyl groups in the sugar moieties and phosphate groups between adjacent nucleotides. A "polynucleotide" refers to a linear polymer formed by linking several tens or more, preferably several hundred or more, of nucleotides, more numerous than an oligonucleotide, by such covalent bonds. Within an oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.

[0036] As used herein, the term "nucleic acid strand" or simply "strand" refers to an oligonucleotide or polynucleotide. A nucleic acid strand can be produced in full length or in partial lengths by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions. A nucleic acid strand can contain natural and / or non-natural nucleotides.

[0037] As used herein, "natural nucleosides" refer to nucleosides that exist in nature. Examples include ribonucleosides consisting of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and a base such as adenine, cytosine, guanine, or thymine. Ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to herein as "DNA nucleosides" and "RNA nucleosides," respectively.

[0038] As used herein, the term "natural nucleotide" refers to a naturally occurring nucleotide molecule in which a phosphate group is covalently bound to the sugar moiety of the natural nucleoside. Examples include ribonucleotides, which are known as building blocks of RNA and in which a phosphate group is bound to a ribonucleoside, and deoxyribonucleotides, which are known as building blocks of DNA and in which a phosphate group is bound to a deoxyribonucleoside.

[0039] As used herein, the term "unnatural nucleoside" refers to any nucleoside other than a natural nucleoside. For example, this term includes modified nucleosides and nucleoside mimetics. As used herein, the term "modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase. Nucleic acid chains, including unnatural oligonucleotides, are often preferred over natural forms due to desirable properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0040] As used herein, the term "mimetic" refers to functional groups that replace sugars, nucleobases, and / or internucleoside linkages. Generally, mimetics are used in place of sugars or sugar-internucleoside linkage combinations, while maintaining the nucleobases for hybridization to a selected target. As used herein, "nucleoside mimetics" includes structures used to replace sugars, or sugars and bases, or linkages between monomeric subunits that comprise an oligomeric compound at one or more positions. An "oligomeric compound" refers to a polymer of linked monomeric subunits that is capable of hybridizing at least to a region of a nucleic acid molecule. Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units.

[0041] As used herein, the term "bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). As used herein, nucleosides containing a bicyclic sugar moiety may also be referred to as "bridged nucleosides." Some examples of bridged nucleic acids are shown in Figure 2.

[0042] A bicyclic sugar may be a sugar in which the 2' and 4' carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. One subgroup of nucleic acids (BNAs) containing bicyclic sugars is the 4'-(CH2) p -O-2',4'-(CH2) p -CH2-2',4'-(CH2) p -S-2',4'-(CH2) p -OCH2O-2',4'-(CH2) n -N(R3)-O-(CH2) m-2' [wherein p, m, and n represent an integer of 1 to 4, an integer of 0 to 2, and an integer of 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a unit substituent (a fluorescent or chemiluminescent labeled molecule, a functional group having nucleic acid cleavage activity, an intracellular or intranuclear localization signal peptide, etc.)]. Furthermore, with respect to BNAs according to certain embodiments, in the OR2 substituent on the 3' carbon atom and the OR1 substituent on the 5' carbon atom, R1 and R2 are typically hydrogen atoms, but may be the same or different from each other and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R4)R5 (wherein R4 and R5 may be the same or different from each other and represent, respectively, a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms). Non-limiting examples of such BNAs include methyleneoxy (4'-CH2-O-2') BNA (also known as LNA (Locked Nucleic Acid®, 2',4'-BNA), e.g., α-L-methyleneoxy (4'-CH2-O-2') BNA or β-D-methyleneoxy (4'-CH2-O-2') BNA, ethyleneoxy (4'-(CH2)2-O-2') BNA (also known as ENA), β-D-thio (4'-CH2-S-2') BNA, aminooxy (4'-CH2-ON(R3)-2') BNA, oxyamino (4'-CH2-N(R3)-O-2') BNA (2',4'-BNA NC (also known as 2',4'-BNA) cocBicyclic nucleosides containing methyleneoxy (4'-CH-O-2') bridges are often referred to herein as "LNA nucleosides." Examples of such nucleosides include 3'-amino-2',4'-BNAs, 5'-methyl BNAs, (4'-CH(CH)-O-2') BNAs (also known as cEt BNAs), (4'-CH(CHOCH)-O-2') BNAs (also known as cMOE BNAs), amide BNAs (4'-C(O)-N(R)-2') BNAs (R = H, Me) (also known as AmNAs), 2'-O,4'-C-spirocyclopropylene-bridged nucleic acids (also known as scpBNAs), and other BNAs known to those skilled in the art. Bicyclic nucleosides containing methyleneoxy (4'-CH-O-2') bridges are often referred to herein as "LNA nucleosides."

[0043] As used herein, the term "non-natural nucleotide" refers to any nucleotide other than a natural nucleotide, including modified nucleotides and nucleotide mimetics. As used herein, "modified nucleotide" refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase. The term "nucleotide mimetics" includes structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound. Examples of nucleotide mimetics include peptide nucleic acids and morpholino nucleic acids (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Peptide nucleic acids (PNAs) are nucleotide mimetics with a backbone in which N-(2-aminoethyl)glycine is linked via an amide bond instead of a sugar. Nucleic acid chains, including non-natural oligonucleotides, often have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Therefore, they are preferred over natural nucleotides.

[0044] As used herein, the term "modified internucleoside linkage" refers to an internucleoside linkage that has a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage). Modified internucleoside linkages include phosphorus-containing internucleoside linkages that contain a phosphorus atom and non-phosphorus-containing internucleoside linkages that do not contain a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages, alkylphosphonate linkages, alkylthiophosphonate linkages, boranophosphate linkages, and phosphoramidate linkages. A phosphorothioate linkage is an internucleoside linkage in which the non-bridging oxygen atom of a phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known. Modified internucleoside linkages are preferably linkages that are more nuclease-resistant than naturally occurring internucleoside linkages.

[0045] As used herein, "modified nucleobase" or "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified nucleobases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, or 8-bromoguanine. A preferred modified nucleobase is 5-methylcytosine.

[0046] "Unmodified nucleobase" or "unmodified base" is synonymous with natural nucleobases and refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0047] As used herein, the term "modified sugar" refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)). Nucleic acid strands herein may contain one or more modified nucleosides, optionally containing modified sugars. Sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the nucleic acid strand. Nucleosides may also contain chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNAs), and the substitution of S, N(R), or C(R1)(R2) of a ribosyl ring oxygen atom (where R, R1, and R2 are each independently H, C1-C). 12 Examples of nucleosides having modified sugar moieties, as used herein, include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F (2'-fluoro), 2'-OCH (2'-OMe or 2'-O-methyl), and 2'-O(CH)OCH substituents. The 2'-position substituent may also be an allyl, amino, azido, thio, -O-allyl, -O-C-C 10 alkyl, —OCF, —O(CH)SCH, —O(CH)—ON(R)(R), and O—CH—C(═O)—N(R)(R), where each R and R is independently H or a substituted or unsubstituted C—C 10 As used herein, the term "2'-modified sugar" refers to a furanosyl sugar modified at the 2' position.

[0048] Generally, modifications can be made so that nucleotides in the same chain can be independently modified. The same nucleotide can also have a modified internucleoside linkage (e.g., a phosphorothioate linkage) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar) to confer resistance to enzymatic cleavage. The same nucleotide can also have a modified nucleobase (e.g., a 5-methylcytosine) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar).

[0049] The number, type, and position of non-natural nucleotides in a nucleic acid strand can affect the antisense effect, etc., provided by the nucleic acid complex of the present invention. The choice of modification can vary depending on the sequence of the target gene, etc., but those skilled in the art can determine a suitable embodiment by referring to the descriptions in literature related to antisense methods (e.g., WO 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085). Furthermore, when the antisense effect of a modified nucleic acid complex is measured, if the measured value thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of that of the nucleic acid complex before modification), the relevant modification can be evaluated.

[0050] As used herein, the term "complementary" refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds. In the present invention, the first nucleic acid strand does not necessarily have to be completely complementary to all or a portion of the target transcript (e.g., the transcript of the target gene), but it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. Similarly, the complementary region in the second nucleic acid strand does not necessarily have to be completely complementary to all or a portion of the first nucleic acid strand, but it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.

[0051] As used herein, the term "muscular disease" refers to a disease that causes muscle atrophy and muscle weakness. Examples include muscular dystrophy, myopathy, inflammatory myopathy (including polymyositis and dermatomyositis), Danon disease, myasthenic syndrome, mitochondrial disease, myoglobinuria, glycogen storage disease, and periodic paralysis. In the present invention, a preferred muscular disease is muscular dystrophy. Various types of muscular dystrophy are known, including Duchenne, Becker, Emery-Dreyfus, limb-girdle, facioscapulohumeral, and oculopharyngeal myopathy. The muscular dystrophy referred to herein may be any of these types. Similarly, myopathy is known to include congenital, distal, hypothyroid, and steroid-induced myopathy, and the myopathy referred to herein may be any of these types.

[0052] As used herein, the term "functional moiety" refers to a moiety that binds to a double-stranded nucleic acid complex, thereby enabling efficient delivery of the double-stranded nucleic acid complex to skeletal muscle, cardiac muscle, or the like. Examples of functional moieties include, but are not limited to, lipid ligands, lipid derivative ligands, peptide ligands, antibody ligands, aptamers, small molecule ligands, and ligand molecules that are taken up by cardiac or skeletal muscle. Examples of functional moieties include cholesterol or analogs thereof, tocopherol or analogs thereof, phosphatidylethanolamine or analogs thereof, substituted or unsubstituted C1-30 alkyl groups, substituted or unsubstituted C2-30 alkenyl groups, and substituted or unsubstituted C1-30 alkoxy groups.

[0053] As used herein, "tocopherol" refers to a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) with a ring structure called chroman. Tocorol has a strong antioxidant effect and therefore functions in vivo as an antioxidant to eliminate free radicals generated by metabolism and protect cells from damage.

[0054] Several different types of tocopherol are known, consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, based on the position of the methyl group bound to the chroman. The tocopherol referred to herein may be any tocopherol. Examples of tocopherol analogs include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Preferably, the tocopherol is α-tocopherol.

[0055] As used herein, "cholesterol" refers to a type of sterol, also known as steroid alcohol, which is particularly abundant in animals. Cholesterol plays an important role in metabolic processes in vivo, and is also a major component of the cell membrane system in animal cells, along with phospholipids. Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols having a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0056] As used herein, the term "analog" refers to a compound having a similar structure and properties, which has the same or a similar basic skeleton. Analogs include, for example, biosynthetic intermediates, metabolic products, compounds with substituents, etc. Whether a compound is an analog of another compound can be determined by one skilled in the art based on common general technical knowledge.

[0057] As used herein, the term "subject" refers to a target to which the double-stranded nucleic acid complex or pharmaceutical composition of the present invention is applied. Subjects include individuals as well as organs, tissues, and cells. When the subject is an individual, it can be any animal, including humans. Examples of non-human subjects include various livestock, poultry, pets, and laboratory animals. The subject may be, but is not limited to, an individual in need of reducing the expression level of a target transcript in skeletal or cardiac muscle, or an individual in need of treatment or prevention of a muscle disease.

[0058] 1-3.Configuration The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand. The specific structure of each nucleic acid strand is shown below.

[0059] The first nucleic acid strand is a single-stranded oligonucleotide strand that contains a base sequence capable of hybridizing to all or part of a transcription product of a target gene and exerts an antisense effect on the target transcription product.

[0060] The second nucleic acid strand is a single-stranded oligonucleotide strand containing a base sequence complementary to that of the first nucleic acid strand. The second nucleic acid strand is bound to cholesterol or its analog. In the double-stranded nucleic acid complex, the second nucleic acid strand is annealed to the first nucleic acid strand through hydrogen bonds of the complementary base pairs.

[0061] The base lengths of the first and second nucleic acid strands are not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base lengths of the first and second nucleic acid strands may be 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The first and second nucleic acid strands may be the same length or different lengths (e.g., one of them may be 1 to 3 bases shorter or longer). The double-stranded structure formed by the first and second nucleic acid strands may include a bulge. The length selected can be determined by balancing the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost, synthesis yield, etc.

[0062] The internucleoside linkages in the first and second nucleic acid strands may be naturally occurring internucleoside linkages and / or modified internucleoside linkages. Preferably, at least one, at least two, or at least three internucleoside linkages from the termini (5'-terminus, 3'-terminus, or both) of the first and / or second nucleic acid strands are modified internucleoside linkages. Here, for example, the two internucleoside linkages from the terminus of the nucleic acid strand refer to the internucleoside linkage closest to the terminus of the nucleic acid strand and the adjacent internucleoside linkage located opposite the terminus. Modified internucleoside linkages in the terminal region of the nucleic acid strand are preferred because they can suppress or inhibit undesired degradation of the nucleic acid strand. In one embodiment, all internucleoside linkages in the first and / or second nucleic acid strands may be modified internucleoside linkages. The modified internucleoside linkages may be phosphorothioate linkages.

[0063] At least one (e.g., three) internucleoside linkages from the 3'-end of the second nucleic acid strand may be a modified internucleoside linkage such as a phosphorothioate linkage, which has high RNase resistance. Inclusion of a modified internucleoside linkage such as a phosphorothioate modification at the 3'-end of the second nucleic acid strand is preferred because it improves the gene silencing activity of the double-stranded nucleic acid complex.

[0064] At the 5'-end and 3'-end of the second nucleic acid strand, the internucleoside bond of 2 to 6 bases from the end where cholesterol or an analogue thereof is not bound may be a modified internucleoside bond (for example, a phosphorothioate bond).

[0065] At least one (e.g., three) nucleosides from the 3'-end of the second nucleic acid strand may be, for example, a modified nucleoside with high RNase resistance, such as 2'F-RNA or 2'-OMe. Inclusion of a modified nucleoside, such as 2'F-RNA or 2'-OMe, at the 3'-end of the second nucleic acid strand is preferred because it enhances the gene suppression activity of the double-stranded nucleic acid complex.

[0066] At the 5'-end and 3'-end of the second nucleic acid strand where cholesterol or an analogue thereof is not bound, 1 to 5 nucleosides from the end may be modified nucleosides such as 2'F-RNA, which has high RNase resistance.

[0067] The nucleosides in the first and second nucleic acid strands can be natural nucleosides (deoxyribonucleosides, ribonucleosides, or both) and / or non-natural nucleosides.

[0068] As used herein, the base sequence of the first nucleic acid strand is complementary to all or part of the base sequence of the target transcript, and therefore can hybridize (or anneal) to the target transcript. The complementarity of the base sequences can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to the target transcript, for example, based on information on the base sequence of the target gene.

[0069] Hybridization conditions may be of various stringent conditions, such as low stringency conditions and high stringency conditions. Low stringency conditions may be conditions of relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS. High stringency conditions may be conditions of relatively high temperature and low salt concentration, for example, 65°C, 0.1xSSC, 0.1% SDS. Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0070] The first nucleic acid strand, when hybridized to a target transcript, can contain at least 4, at least 5, at least 6, or at least 7 consecutive nucleosides recognized by RNase H. Typically, the region may contain 4 to 20, 5 to 16, or 6 to 12 consecutive nucleosides. Examples of nucleosides recognized by RNase H include natural deoxyribonucleosides. Modified deoxyribonucleosides and suitable nucleosides containing other bases are well known in the art. It is also known that nucleosides containing a hydroxy group at the 2' position, such as ribonucleosides, are unsuitable as such nucleosides. The suitability of nucleosides for use in this region containing "at least 4 consecutive nucleosides" can be easily determined. In one embodiment, the first nucleic acid strand can contain at least 4 consecutive deoxyribonucleosides.

[0071] In one embodiment, the entire length of the first nucleic acid strand is not composed solely of natural ribonucleosides. Preferably, the first nucleic acid strand does not contain natural ribonucleosides, or the first nucleic acid strand comprises less than half of the total length of natural ribonucleosides.

[0072] In one embodiment, the second nucleic acid strand may contain at least four consecutive ribonucleosides complementary to the at least four consecutive nucleosides (e.g., deoxyribonucleosides) in the first nucleic acid strand, such that the second nucleic acid strand forms a partial DNA-RNA heteroduplex with the first nucleic acid strand and is recognized and cleaved by RNase H. The at least four consecutive ribonucleosides in the second nucleic acid strand are preferably linked by naturally occurring internucleoside linkages, i.e., phosphodiester bonds.

[0073] The second nucleic acid strand may be composed of all nucleosides that are ribonucleosides and / or modified nucleosides. The second nucleic acid strand may be composed of all nucleosides that are deoxyribonucleosides and / or modified nucleosides, or may not contain ribonucleosides. In one embodiment, the second nucleic acid strand may be composed of all nucleosides that are deoxyribonucleosides and / or modified nucleosides.

[0074] The first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex of the present invention may be a gapmer. As used herein, the term "gapmer" refers, in principle, to a single-stranded nucleic acid comprising a central region (DNA gap region) and wing regions (referred to as the 5' wing region and the 3' wing region, respectively) located directly at the 5' and 3' ends of the central region. In a gapmer, the central region contains at least four consecutive deoxyribonucleosides, and the wing regions contain at least one unnatural nucleoside. Although not limited thereto, the unnatural nucleosides contained in the wing regions typically have stronger RNA-binding strength and higher resistance to nucleases (such as nucleases) than natural nucleosides. When the unnatural nucleosides constituting the wing regions contain or consist of bridged nucleosides, the gapmer is specifically referred to as a "BNA / DNA gapmer." The number of bridged nucleosides contained in the 5' wing region and the 3' wing region is at least one, and may be, for example, two or three. The bridged nucleosides contained in the 5' wing region and the 3' wing region may be contiguous or non-contiguous within the 5' wing region and the 3' wing region. The bridged nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). When the bridged nucleoside is an LNA nucleoside, the gapmer is referred to as an "LNA / DNA gapmer." When the unnatural nucleosides comprising the 5' wing region and the 3' wing region comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer." When the unnatural nucleosides comprising the 5' wing region and the 3' wing region comprise or consist of morpholino nucleic acids, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer." The base length of the 5' wing region and the 3' wing region may each independently be at least 2 bases, for example, 2 to 10 bases, 2 to 7 bases, or 3 to 5 bases. The 5' wing region and the 3' wing region may contain at least one unnatural nucleoside, and may further contain a natural nucleoside.

[0075] The first nucleic acid strand and / or the second nucleic acid strand constituting the gapmer may be composed of, in order from the 5' end, a bridged nucleoside having a length of 2 to 7 bases or 3 to 5 bases, a ribonucleoside or deoxyribonucleoside having a length of 4 to 15 bases or 8 to 12 bases, and a bridged nucleoside having a length of 2 to 7 bases or 3 to 5 bases.

[0076] In addition, a nucleic acid strand having a wing region only on either the 5'-end or the 3'-end is called a "hemigapmer" in the art, and in this specification, hemigapmers are also included in the term "gapmer."

[0077] The first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex of the present invention may be a mixmer. As used herein, a "mixmer" refers to a nucleic acid strand that contains alternating natural and unnatural nucleosides of periodic or random segment lengths, but does not contain four or more consecutive deoxyribonucleosides and ribonucleosides. A mixmer in which the unnatural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "BNA / DNA mixmer." A mixmer in which the unnatural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "peptide nucleic acid / DNA mixmer." In a mixmer, a mixmer in which the non-natural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid / DNA mixmer." A mixmer is not limited to containing only two types of nucleosides. A mixmer can contain any number of types of nucleosides, regardless of whether they are natural or modified nucleosides or nucleoside mimics. For example, a mixmer may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside). The bridged nucleoside may further contain a modified nucleobase (e.g., 5-methylcytosine).

[0078] At least one, at least two, at least three, or at least four nucleosides from the end (5'-end, 3'-end, or both ends) of the second nucleic acid strand may be modified nucleosides. The modified nucleosides may contain a modified sugar and / or a modified nucleobase. The modified sugar may be a 2'-modified sugar (e.g., a sugar containing a 2'-O-methyl group). The modified nucleobase may also be 5-methylcytosine.

[0079] The second nucleic acid strand may be composed of, from the 5'-terminus, modified nucleosides (e.g., modified nucleosides containing 2'-modified sugars) each having a length of 2 to 7 bases or 3 to 5 bases, ribonucleosides or deoxyribonucleosides (optionally linked via modified internucleoside linkages) each having a length of 4 to 15 bases or 8 to 12 bases, and modified nucleosides (e.g., modified nucleosides containing 2'-modified sugars). In this case, the first nucleic acid strand may be a gapmer.

[0080] The first and second nucleic acid strands may comprise, in whole or in part, a nucleoside mimic or a nucleotide mimic. The nucleotide mimic may be a peptide nucleic acid and / or a morpholino nucleic acid. The first nucleic acid strand may comprise at least one modified nucleoside. The modified nucleoside may comprise a 2'-modified sugar. The 2'-modified sugar may comprise a sugar containing a 2'-O-methyl group. Thus, one embodiment of the present invention relates to a double-stranded nucleic acid complex, in which a first nucleic acid strand comprises a base sequence capable of hybridizing to all or part of a transcription product of a target gene and has an antisense effect on the transcription product, a second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, the first nucleic acid strand is annealed to the second nucleic acid strand, and the first nucleic acid strand comprises, in whole (100%) or in part (e.g., 80% or more of the total) a morpholino nucleic acid.

[0081] The first and second nucleic acid strands may contain any combination of the modified internucleoside linkages and modified nucleosides described above.

[0082] The first and second nucleic acid strands may be linked via a linker. In this case, the first and second nucleic acid strands may be linked via the linker to form a single strand. However, even in this case, the functional region has the same structure as in the double-stranded nucleic acid complex, and therefore, in this specification, such single-stranded nucleic acids are also included as an embodiment of the double-stranded nucleic acid complex of the present invention. The linker may be any polymer. Examples include polynucleotides, polypeptides, and alkylenes. Specifically, it may be composed of natural nucleotides such as DNA and RNA, or unnatural nucleotides such as peptide nucleic acids and morpholino nucleic acids. When the linker is composed of a nucleic acid, the chain length of the linker may be at least one base, for example, 3 to 10 bases or 4 to 6 bases. A chain length of 4 bases is preferred. The linker can be located on either the 5'- or 3'-side of the first nucleic acid strand, but for example, in a configuration in which cholesterol or an analog thereof is bound to the 5'-side of the second nucleic acid strand, the 5'-end of the first nucleic acid strand and the 3'-end of the second nucleic acid strand are linked via a linker. In one embodiment, the first nucleic acid strand is a hemi-gapmer having a wing region only on the 3'-end side, and the second nucleic acid strand is a nucleic acid strand that does not contain sugar-modified nucleosides.

[0083] The second nucleic acid strand is bound to cholesterol or an analog thereof.

[0084] The second nucleic acid strand bound to cholesterol or an analog thereof may have a group represented by the following general formula (I):

[0085] [ka] [In the formula, R c represents an alkylene group having 4 to 18 carbon atoms, preferably 5 to 16 carbon atoms, which may have a substituent (wherein the substituent is a halogen atom or an alkyl group having 1 to 3 carbon atoms which may be substituted with a hydroxy group, such as a hydroxymethyl group, and non-adjacent carbon atoms in the alkylene group may be substituted with an oxygen atom).

[0086] Rc may be, but is not limited to, -(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-, -(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-CH2-CH(CH2OH)-, or -(CH2)6-.

[0087] The group represented by the above general formula (I) or (II) can be bound to the 5'-end or 3'-end of the second nucleic acid strand via a phosphate ester bond.

[0088] Cholesterol or an analog thereof may be bound to the 5'-end, 3'-end, or both ends of the second nucleic acid strand. Cholesterol or an analog thereof may also be bound to an internal nucleotide of the second nucleic acid strand. Although not limited thereto, cholesterol or an analog thereof bound to the 5'-end of the second nucleic acid strand is particularly preferred.

[0089] When the second nucleic acid strand contains multiple cholesterols or their analogs, they may be the same or different. For example, one cholesterol may be bound to the 5'-end of the second nucleic acid strand and one other cholesterol analog may be bound to the 3'-end. Regarding the binding positions, cholesterol or its analogs may be bound to multiple positions on the second nucleic acid strand and / or may be bound as a group to one position. One cholesterol or its analog may be linked to each of the 5'-end and 3'-end of the second nucleic acid strand.

[0090] The bond between the second nucleic acid strand and cholesterol or an analog thereof may be a direct bond or an indirect bond mediated by another substance.

[0091] When the second nucleic acid strand and cholesterol or an analog thereof are directly bound to each other, they may be bound to the second nucleic acid strand via, for example, a covalent bond, an ionic bond, a hydrogen bond, etc. In view of the fact that a more stable bond can be obtained, a covalent bond is preferred.

[0092] When the second nucleic acid strand and cholesterol or an analog thereof are indirectly bound to each other, they may be bound via a linking group (often referred to as a "linker" in this specification). The linker may be either a cleavable linker or an uncleavable linker.

[0093] "Cleavable linker" refers to a linker that can be cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within a human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases. Cleavable linkers include, but are not limited to, amides, esters, one or both phosphodiesters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers. As an example, cholesterol or an analog thereof may be linked via a disulfide bond.

[0094] The term "non-cleavable linker" refers to a linker that is not cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within the human body). Examples of non-cleavable linkers include, but are not limited to, linkers consisting of a phosphorothioate bond, and a modified or unmodified deoxyribonucleoside or modified or unmodified ribonucleoside linked by a phosphorothioate bond. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but may typically be 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.

[0095] A specific example of the linker is a linker represented by the following general formula II.

[0096] [ka] [In the formula, n represents 0 or 1.]

[0097] The second nucleic acid strand may further include at least one functional moiety bound to the polynucleotide constituting the nucleic acid strand. The term "functional moiety" refers to a moiety that confers a desired function to the double-stranded nucleic acid complex and / or the nucleic acid strand to which the functional moiety is bound. Examples of the desired function include labeling or purification. Examples of moieties that confer labeling function include compounds such as fluorescent proteins and luciferase. Examples of moieties that confer purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide. The binding position and type of the functional moiety on the second nucleic acid strand are as described above for the binding between cholesterol or its analog and the second nucleic acid strand.

[0098] The antisense effect of the double-stranded nucleic acid complex of the present invention on the target transcript of the first nucleic acid strand in skeletal muscle or cardiac muscle can be measured by methods known in the art. For example, after introducing the double-stranded nucleic acid complex into cells, measurement can be performed using known techniques such as Northern blotting, quantitative PCR, or Western blotting. By measuring the expression level of the target gene or the level of the target transcript (e.g., mRNA level, RNA level such as microRNA, cDNA level, protein level, etc.) in skeletal muscle cells or cardiac muscle cells, it can be determined whether the double-stranded nucleic acid complex suppresses target gene expression at those sites. The criteria for this determination are not limited, but may include a reduction of the target gene expression level or target transcript level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 40% compared to the measurement value of a negative control (e.g., vehicle administration).

[0099] As described above, exemplary embodiments of the double-stranded nucleic acid complex of the present invention have been described, but the double-stranded nucleic acid complex of the present invention is not limited to the above exemplary embodiments.

[0100] 1-4. Method for producing double-stranded nucleic acid complex Those skilled in the art can prepare the double-stranded nucleic acid complex of the present invention by appropriately selecting a known method. While not limited thereto, the process typically begins with designing and preparing each of the first and second nucleic acid strands that make up the double-stranded nucleic acid complex. For example, the first nucleic acid strand is designed based on the base sequence information of the target transcription product (e.g., the base sequence of the target gene), and the second nucleic acid strand is designed as its complementary strand. Next, based on the designed base sequence information, each nucleic acid strand can be synthesized using a commercially available automated nucleic acid synthesizer, for example, from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, or the like. The resulting oligonucleotides can then be purified using a reverse-phase column or the like.

[0101] Furthermore, in the case of a double-stranded nucleic acid complex to which a functional moiety is bound, the first nucleic acid strand may be produced according to the above-described method. Meanwhile, the second nucleic acid strand to which a functional moiety is bound can be produced by carrying out the above-described synthesis and purification using a nucleic acid species to which a functional moiety has already been bound. For example, the second nucleic acid strand may be produced by carrying out the above-described synthesis and purification using a nucleic acid species to which cholesterol or an analog thereof has already been bound. Alternatively, cholesterol or an analog thereof may be bound by a known method to the second nucleic acid strand produced by carrying out the above-described synthesis and purification. After producing each nucleic acid strand, the first nucleic acid strand and the second nucleic acid strand are annealed as described below to produce a double-stranded nucleic acid complex to which the desired functional moiety is bound.

[0102] Methods for linking functional moieties to nucleic acids are well known in the art. Nucleic acids prepared by this method are mixed in an appropriate buffer solution and denatured at about 90°C to 98°C for several minutes (e.g., 5 minutes). The nucleic acids are then annealed at about 30°C to 70°C for about 1 to 8 hours to produce one of the double-stranded nucleic acid complexes of the present invention. Alternatively, nucleic acid strands can be ordered from various manufacturers (e.g., Gene Design, Inc.) by specifying the base sequence and the modification site and type. The annealing step can be carried out by allowing the solution to stand at room temperature (about 10°C to about 35°C) for about 5 to 60 minutes. The first and second nucleic acid strands may be dissolved in a buffer solution (e.g., phosphate-buffered saline) or water at about 70°C to 98°C, respectively, and the resulting two solutions may be mixed. The mixture may be maintained at about 70°C to 98°C for several minutes (e.g., 5 minutes), followed by maintaining the mixture at about 30°C to 70°C (or 30°C to 50°C) for about 1 to 8 hours to prepare a double-stranded nucleic acid complex according to some embodiments of the present invention. The first nucleic acid strand and the second nucleic acid strand can also be dissolved in a buffer solution (e.g., phosphate-buffered saline) or water at room temperature (about 10°C to about 35°C). The annealing conditions (time and temperature) for preparing the double-stranded nucleic acid complex are not limited to those described above. Conditions suitable for promoting annealing of nucleic acid strands are well known in the art.

[0103] 1-5.Effects The double-stranded nucleic acid complex of the present invention can be efficiently delivered to the skeletal or cardiac muscle of a subject, where it exerts an antisense effect on the target gene at that site, thereby suppressing its expression. Therefore, by using this double-stranded nucleic acid complex as an active ingredient, it is possible to treat or prevent diseases such as muscle diseases that can be developed or aggravated by the expression of the target gene in the skeletal or cardiac muscle of a subject.

[0104] 2. Pharmaceutical Compositions 2-1. Overview A second aspect of the present invention is a pharmaceutical composition. The pharmaceutical composition of the present invention comprises the double-stranded nucleic acid complex of the first aspect as an active ingredient and / or as a drug delivery molecule for skeletal muscle or cardiac muscle. The double-stranded nucleic acid complex of the first aspect can regulate the expression level of a target transcript in skeletal muscle or cardiac muscle through its antisense effect. Therefore, by administering the pharmaceutical composition of the present invention to a subject, the double-stranded nucleic acid complex can be delivered to the subject's skeletal muscle or cardiac muscle, allowing for the treatment of diseases that may occur in those areas, such as muscle diseases. The pharmaceutical composition of the present invention may essentially consist of the double-stranded nucleic acid complex of the first aspect. That is, the pharmaceutical composition of the present invention may further contain auxiliary components such as a carrier in addition to the double-stranded nucleic acid complex of the first aspect. Alternatively, the pharmaceutical composition of the present invention may consist solely of the double-stranded nucleic acid complex of the first aspect.

[0105] 2-2.Configuration The pharmaceutical composition of the present invention may contain an active ingredient and a carrier as essential components. Each component will be specifically described below.

[0106] 2-2-1. Active ingredients The active ingredient is an essential component of the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention contains at least the double-stranded nucleic acid complex described in the first aspect as an active ingredient. The pharmaceutical composition of the present invention can contain two or more types of the double-stranded nucleic acid complex.

[0107] The amount (content) of the double-stranded nucleic acid complex contained in the pharmaceutical composition varies depending on the type of double-stranded nucleic acid complex, the site of delivery (skeletal muscle or cardiac muscle), the dosage form of the pharmaceutical composition, the dosage of the pharmaceutical composition, and the type of carrier (described below). Therefore, it can be determined appropriately taking into account each condition. Typically, a single dose of the pharmaceutical composition is adjusted to contain an effective amount of the double-stranded nucleic acid complex. The term "effective amount" refers to the amount of the double-stranded nucleic acid complex necessary for the complex to function as an active ingredient, while causing little or no harmful side effects in the living body to which it is administered. This effective amount may vary depending on various conditions, such as information about the subject, the route of administration, and the number of administrations. Ultimately, it is determined by the judgment of a physician, veterinarian, or pharmacist. "Subject information" refers to various individual information about the living body to which the pharmaceutical composition is administered. For example, if the subject is a human, this information includes age, weight, sex, diet, health condition, disease progression and severity, drug sensitivity, and the presence or absence of concomitant medications.

[0108] 2-2-2. Carrier The pharmaceutical composition of the present invention can contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonicity adjusters, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizers, and other additives.

[0109] The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0110] The above-mentioned carriers are used to avoid or inhibit the decomposition of the double-stranded nucleic acid complex, which is the active ingredient, by enzymes and the like in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and medicinal efficacy, and may be used appropriately as needed.

[0111] 2-2-3. Dosage form The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that can deliver the double-stranded nucleic acid complex described in the first embodiment, which is the active ingredient, to the target site, skeletal muscle or cardiac muscle, without inactivating it by degradation or the like, and can exert the pharmacological effect of the active ingredient in vivo (antisense effect on the expression of the target gene).

[0112] The specific dosage form varies depending on the administration method and / or formulation conditions. The administration method can be roughly divided into parenteral administration and oral administration, so a dosage form suitable for each administration method may be used.

[0113] If the administration method is parenteral administration, the preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. Examples of liquids include injections. Injections can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. Other forms include ointments, plasters, cataplasms, transdermal agents, lotions, inhalants, aerosols, eye drops, and suppositories.

[0114] When the administration method is oral administration, preferred dosage forms include solids (including tablets, capsules, drops, and lozenges), granules, powders, powders, and liquids (including oral solutions, emulsions, and syrups). If a solid is used, it can be made into a dosage form coated with a coating known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets, as needed.

[0115] The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The pharmaceutical composition of the present invention may be formulated according to a conventional method in the art.

[0116] 2-3. Dosage form and dosage Herein, there is no particular limitation on the preferred administration form of the pharmaceutical composition. For example, oral administration or parenteral administration may be used. Specific examples of parenteral administration include intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration (including implantable continuous subcutaneous administration), tracheal / bronchial administration, rectal administration, and administration by blood transfusion. Considering that the target application site of the present invention is skeletal muscle or cardiac muscle, intramuscular injection administration and intravenous drip administration, which are the target sites, are preferred.

[0117] When the pharmaceutical composition is administered or ingested, the dosage or intake may be, for example, such that the amount of the double-stranded nucleic acid complex contained therein is 0.00001 mg / kg / day to 10,000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg / day. The pharmaceutical composition may be administered in a single dose or multiple doses. In the case of multiple doses, the composition may be administered daily or at appropriate intervals (e.g., every 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times. The single dose of the double-stranded nucleic acid complex may be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.25 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 80 mg / kg or more, 90 mg / kg or more, 100 mg / kg or more, 120 mg / kg or more, 140 mg / kg or more, 150 mg / kg or more, 160 mg / kg or more, 170 mg / kg or more, 180 mg / kg or more, 190 mg / kg or more, 210 mg / kg or more, 220 mg / kg or more, 230 mg / kg or more, 240 mg / kg or more, 250 mg / kg or more, 260 mg / kg or more, 270 mg / kg or more, 280 mg / kg or more, 290 mg / kg or more, 300 mg / kg or more, 310 mg / kg or more, 320 mg / kg or more, 330 mg / kg or more, 340 mg / kg or more, 350 mg / kg or more, 360 mg / kg or more, 370 mg / kg or more, 380 mg / kg or more, 390 mg / kg or more, 400 mg / kg or more, 410 mg / kg or The dose can be 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, and can be selected appropriately from any amount within the range of, for example, 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg).

[0118] The double-stranded nucleic acid complex of the present invention may be administered at a dose of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg) twice a week for four doses. Alternatively, the double-stranded nucleic acid complex may be administered at a dose of 0.05 to 30 mg / kg (e.g., about 25 mg / kg) once or twice a week for two to four doses, for example, twice a week for two doses. The adoption of such a dosing regimen (divided administration) can reduce toxicity (e.g., avoid platelet reduction) and the burden on the subject compared to a single administration of a higher dose.

[0119] Repeated administration of the pharmaceutical composition results in additive intracellular inhibitory effects. Furthermore, when administering repeatedly, efficacy can be improved by leaving a certain interval between administrations (e.g., half a day or more).

[0120] 2-4.Applicable diseases The diseases to which the pharmaceutical composition is applied are diseases that may develop or become more severe as a result of expression of the target gene in skeletal or cardiac muscles, including, but not limited to, muscle diseases.

[0121] In the present invention, "muscle disease" is a general term for diseases that cause muscle weakness due to muscle cells (including skeletal muscle cells or cardiac muscle cells). Examples include muscular dystrophy, myopathy, inflammatory muscle diseases (including polymyositis and dermatomyositis), Danon disease, myasthenic syndrome, mitochondrial disease, myoglobinuria, glycogen storage disease, periodic paralysis, hereditary cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmias, including hereditary ones. Also included are diseases that have a primary cause in another organ and may secondary cause dysfunction of skeletal muscle or cardiac muscle cells. Examples include neurodegenerative diseases, sarcopenia, cachexia, etc.

[0122] 2-5.Drug delivery The pharmaceutical composition of the present invention utilizes the fact that the double-stranded nucleic acid complex of the first embodiment, which contains the active ingredient, can be efficiently delivered to skeletal or cardiac muscle. By conjugating a specific drug to the first and / or second nucleic acid strand, the drug can be delivered to skeletal or cardiac muscle. Drugs delivered to skeletal or cardiac muscle include, but are not limited to, peptides, proteins, or nucleic acid drugs, as well as other organic compounds, such as antitumor drugs, hormone drugs, antibiotics, antiviral agents, and anti-inflammatory drugs. Preferred drugs are small molecule drugs. Small molecule drugs are well understood by those skilled in the art. Typically, small molecule drugs refer to drugs with a molecular weight of less than 1,000 daltons. The drug may be a lipophilic drug. Examples of nucleic acid drugs include, but are not limited to, ASOs, antagomir (miR), splice-switching oligonucleotides, aptamers, single-stranded siRNAs, microRNAs, and pre-microRNAs. The binding site and type of the drug on the second nucleic acid strand are the same as those described above for the binding of cholesterol or its analogs to the second nucleic acid strand.

[0123] Effects It is possible to treat or prevent muscle diseases and the like that can develop due to the expression of specific genes in skeletal or cardiac muscles.

[0124] As disclosed in the Examples below, the pharmaceutical composition of the present invention can be efficiently delivered to skeletal muscle or cardiac muscle and effectively suppress the expression of a target gene or the level of a target transcript at the site. Therefore, a method for reducing the expression level of a target transcript in the skeletal muscle or cardiac muscle of a subject is provided, comprising administering to the subject a pharmaceutical composition containing the double-stranded nucleic acid complex described above. The method may also be a method for treating a muscle disease in a subject. Also provided is a drug delivery method for delivering a drug to the skeletal muscle or cardiac muscle of a subject, comprising administering to the subject a pharmaceutical composition containing the double-stranded nucleic acid complex described above. [Example]

[0125] Example 1 (the purpose) We will verify the in vivo inhibitory effect of double-stranded nucleic acid complexes consisting of antisense oligonucleotides targeting the SR-B1 gene and tocopherol- or cholesterol-bound complementary strands on mRNA expression in tissues.

[0126] (method) (1) Preparation of nucleic acids The target gene was scavenger receptor B1 (SR-B1). The names and base sequences of the first and second nucleic acid strands constituting the double-stranded nucleic acid complex agent used in this example are shown in Table 1.

[0127] [Table 1]

[0128] The first nucleic acid strand targets the mouse SR-B1 gene and is composed of a 14-mer single-stranded LNA / DNA gapmer having a base sequence complementary to positions 2479 to 2492 of its transcription product, SR-B1 mRNA (GenBank accession number NM_016741, SEQ ID NO: 1). More specifically, this LNA / DNA gapmer is composed of two LNA nucleosides at each of the 5' and 3' ends, and ten DNA nucleosides between them.

[0129] The second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is composed of tocopherol-bound complementary RNA (Toc#1-cRNA(mSR-B1)) having tocopherol bound to its 5' end, or cholesterol-bound complementary RNA (Chol#1-cRNA(mSR-B1)) having cholesterol bound to its 5' end.

[0130] The double-stranded nucleic acid complex agent of the present invention, a tocopherol-conjugated heteroduplex oligonucleotide (hereinafter referred to as "Toc-HDO") or a cholesterol-conjugated heteroduplex oligonucleotide (hereinafter referred to as "Chol-HDO"), was prepared by annealing the first nucleic acid strand with one of two types of second nucleic acid strand. The first nucleic acid strand and the second nucleic acid strand were mixed in equimolar amounts, and the solution was heated to 95°C for 5 minutes, then cooled to 37°C and maintained for 1 hour. This annealed the nucleic acid strands to prepare the double-stranded nucleic acid complex agent. The annealed nucleic acid was stored at 4°C or on ice. The prepared double-stranded nucleic acid complex agent is referred to as "Toc#1HDO(mR-B1)" or "Chol#1HDO(mSR-B1)."

[0131] A conventional single-stranded antisense oligonucleotide (ASO) (control ASO) was used as a comparison for the double-stranded nucleic acid complex. This control ASO has the same structure as the first nucleic acid strand of the double-stranded nucleic acid complex. The prepared single-stranded ASO was designated "ASO (mSR-B1)."

[0132] (2) In vivo experiments Male C57BL / 6 mice weighing 20 g and aged 6 to 7 weeks were used as mice to be administered with the double-stranded nucleic acid complex agent, etc. In this example, all experiments using mice were performed with n=4. The double-stranded nucleic acid complex and the control ASO (mSR-B1) were each intravenously injected into mice via the tail vein at a dose of 50 mg / kg. A negative control group of mice was also injected with a single dose of PBS alone.

[0133] (3) Expression analysis At 72 hours after administration, mice were perfused with PBS and then dissected to remove the myocardium, quadriceps, diaphragm, and dorsi proper muscles. Subsequently, mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, the MagNA Pure 96 (Roche Life Sciences), according to the protocol. cDNA was synthesized according to the Transcriptor Universal cDNA Master (Roche Life Sciences) protocol. Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific, based on various gene numbers. PCR conditions (temperature and time) consisted of 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second. The resulting amplified products were quantified by quantitative RT-PCR. Based on the results, the expression level of SR-B1 mRNA was divided by the expression level of ACTB mRNA (an internal control gene) to obtain relative expression levels. The mean and standard error of the relative expression levels were calculated. Results were compared between groups and further evaluated by t-test.

[0134] (result) The results are shown in Figure 3. Figure 3 shows the inhibitory effect of the double-stranded nucleic acid complexes of the present invention, which target the SR-B1 gene bound to tocopherol or cholesterol (Toc#1HDO(mSR-B1) and Chol#1HDO(mSR-B1), respectively), on the expression of the target SR-B1 gene in the myocardium (Heart), quadriceps (Quadriceps), diaphragm, and back muscles (Back). Error bars indicate the standard error of each value.

[0135] Toc#1HDO (mSR-B1) and Chol#1HDO (mSR-B1) showed significant suppressive effects on target gene expression in various skeletal and cardiac muscles compared to the single-stranded control ASO (mSR-B1).

[0136] <Example 2> (the purpose) We investigated the in vivo inhibitory effect of a single administration of a double-stranded nucleic acid complex targeting the malat1 gene, in which the second nucleic acid strand is a tocopherol- or cholesterol-bound complementary strand, on mRNA expression in tissues.

[0137] (method) (1) Preparation of nucleic acids The target gene was the metastasis-associated lung adenocarcinoma transcript (malat1). The names and base sequences of the first and second nucleic acid strands constituting the double-stranded nucleic acid complex agent used in this example are shown in Table 2.

[0138] [Table 2]

[0139] The first nucleic acid strand targets the mouse malat1 gene and is composed of a 16-mer single-stranded LNA / DNA gapmer having a base sequence complementary to positions 1316 to 1331 of its transcription product, the malat1 non-coding RNA (GenBank accession number NR_002847, SEQ ID NO: 3). More specifically, this LNA / DNA gapmer is composed of three LNA nucleosides at each of the 5' and 3' ends, and ten DNA nucleosides between them.

[0140] On the other hand, the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is composed of tocopherol-bound complementary RNA (Toc#1-cRNA(mMalat1)) with tocopherol bound to its 5' end, or cholesterol-bound complementary RNA (Chol#1-cRNA(mMalat1)) with cholesterol bound to its 5' end.

[0141] The first nucleic acid strand was mixed with one of two second nucleic acid strands in equimolar amounts, and the solution was heated to 95°C for 5 minutes. The solution was then cooled to 37°C and maintained for 1 hour, annealing the two nucleic acid strands to prepare the double-stranded nucleic acid complex. The annealed nucleic acid was stored at 4°C or on ice. The prepared double-stranded nucleic acid complex was designated "Toc#1HDO(mMalat1)" or "Chol#1HDO(mMalat1)."

[0142] A conventional single-stranded antisense oligonucleotide (ASO) (control ASO) was used as a comparison for the double-stranded nucleic acid complex agent. This control ASO has the same structure as the first nucleic acid strand of the double-stranded nucleic acid complex agent. The prepared single-stranded ASO was designated "ASO (mMalat1)."

[0143] (2) In vivo experiments The mice to be administered with the double-stranded nucleic acid complex agent and the like were male C57BL / 6 mice weighing 20 g and aged 6 to 7 weeks. The double-stranded nucleic acid complex and control ASO were each intravenously injected into mice via the tail vein at a dose of 50 mg / kg. A negative control group of mice was also injected with a single dose of PBS alone.

[0144] (3) Expression analysis At 72 hours after administration, mice were perfused with PBS and then dissected to remove the myocardium, quadriceps, diaphragm, and dorsi proper muscles. Subsequently, mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, the MagNA Pure 96 (Roche Life Sciences), according to the protocol. cDNA was synthesized according to the Transcriptor Universal cDNA Master (Roche Life Sciences) protocol. Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific, based on various gene numbers. PCR conditions (temperature and time) consisted of 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second. The resulting amplified products were quantified by quantitative RT-PCR. Based on the results, the expression level of mRNA (malat1) was calculated relative to the expression level of mRNA (ACTB; an internal control gene). The mean and standard error of the relative expression levels were calculated. The results were compared between groups and further evaluated by t-test.

[0145] (result) The results are shown in Figure 4. Figure 4 shows the suppressive effect of tocopherol- or cholesterol-bound double-stranded nucleic acid complexes (Toc#1HDO(mMalat1) and Chol#1HDO(mMalat1) respectively) on the expression of the target Malat1 gene in the myocardium (Heart), quadriceps (Quadriceps), diaphragm, and back muscles (Back). Error bars indicate the standard error of each value.

[0146] Toc#1HDO (mMalat1) and Chol#1HDO (mMalat1) showed significant suppressive effects on target gene expression in various skeletal and cardiac muscles compared to the single-stranded control ASO (mMalat1).

[0147] Example 3 (the purpose) The in vivo inhibitory effect of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide targeting the DMPK gene and a tocopherol- or cholesterol-bound complementary strand on mRNA expression in tissues was examined in the same manner as in Examples 1 and 2.

[0148] (method) (1) Preparation of nucleic acids The target gene was the DMPK (dystrophia myotonica-protein kinase) gene. The DMPK gene encodes myotonin protein kinase, which is known to be the causative gene for myotonic dystrophy, the most common muscular dystrophy in adults. The disease is thought to be caused by an abnormal expansion of a CTG repeat sequence in the 3' untranslated region of the DMPK gene.

[0149] Table 3 shows the names and base sequences of the first and second nucleic acid strands constituting the double-stranded nucleic acid complex agent used in this example.

[0150] [Table 3]

[0151] The first nucleic acid strand targets the mouse DMPK gene and is composed of a 16-mer single-stranded LNA / DNA gapmer having a base sequence complementary to positions 2682 to 2697 of its transcription product, DMPK mRNA (GenBank accession number NM_032418, SEQ ID NO: 5). More specifically, this LNA / DNA gapmer is composed of three LNA nucleosides at each of the 5' and 3' ends, and ten DNA nucleosides between them.

[0152] The second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is composed of tocopherol-bound complementary RNA (Toc#1-cRNA(mDMPK)) with tocopherol bound to its 5' end, or cholesterol-bound complementary RNA (Chol#1-cRNA(mDMPK)) with cholesterol bound to its 5' end.

[0153] The double-stranded nucleic acid complex agent of the present invention, Toc-HDO or cholesterol-linked heteroduplex oligonucleotide Chol-HDO, was prepared by annealing the first nucleic acid strand with either the second nucleic acid strand. Specifically, the first nucleic acid strand and the second nucleic acid strand were mixed in equimolar amounts, the solution was heated to 95°C for 5 minutes, then cooled to 37°C and maintained for 1 hour, thereby annealing the nucleic acid strands to prepare the double-stranded nucleic acid complex agent. The annealed nucleic acid was stored at room temperature, 4°C, or on ice. The prepared double-stranded nucleic acid complex agent is referred to as "Toc#1HDO(mDMPK)" or "Chol#1HDO(mDMPK)."

[0154] A conventional single-stranded antisense oligonucleotide (ASO) (control ASO) was used as a comparison for the double-stranded nucleic acid complex agent. This control ASO has the same structure as the first nucleic acid strand of the double-stranded nucleic acid complex agent. The prepared single-stranded ASO was designated "ASO (mDMPK)."

[0155] (2) In vivo experiments The mice to be administered with the double-stranded nucleic acid complex agent and the like were male C57BL / 6 mice weighing 20 g and aged 6 to 7 weeks. The double-stranded nucleic acid complex and control ASO were intravenously injected into mice via the tail vein at a single dose of 12.5 mg / kg, 25 mg / kg, or 50 mg / kg, respectively. A negative control group of mice was also injected with a single dose of PBS alone.

[0156] (3) Expression analysis At 72 hours after administration, mice were perfused with PBS and then dissected to remove the heart muscle (heart), quadriceps (quadriceps), diaphragm (diaphragm), dorsi proper (back), tibialis anterior (TA), gastrocnemius (GC), and triceps brachii (TB). Subsequently, mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, MagNA Pure 96 (Roche Life Sciences), according to the protocol. cDNA was synthesized according to the Transcriptor Universal cDNA Master (Roche Life Sciences) protocol. Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific, based on various gene numbers. The PCR conditions (temperature and time) were 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second, repeated 40 times. The resulting amplified products were quantified by quantitative RT-PCR, and the expression levels of mRNA (DMPK) and mRNA (ACTB; internal control gene) were calculated based on the results to obtain relative expression levels. The mean and standard error of the relative expression levels were calculated. The results of each group were compared and further evaluated by t-test.

[0157] (result) The results are shown in Figures 5 to 7. Figures 5, 6, and 7 show the inhibitory effects of tocopherol- or cholesterol-bound double-stranded nucleic acid complexes (Toc#1HDO(mDMPK) and Chol#1HDO(mDMPK) respectively) at 12.5 mg / kg, 25 mg / kg, and 50 mg / kg on the expression of the target DMPK gene in the gastrocnemius (GC), tibialis anterior (TA), triceps (TB), quadriceps (quadriceps), diaphragm, back, and heart muscle (heart). Error bars indicate the standard error of each value.

[0158] Both Toc#1HDO(mDMPK) and Chol#1HDO(mDMPK) showed significant suppressive effects on target genes in various skeletal and cardiac muscles compared with the single-chain control ASO(mDMPK). In particular, the suppressive effect of Chol#1HDO(mDMPK) was remarkable in a dose-dependent manner.

[0159] Example 4: DNA only (the purpose) In double-stranded nucleic acid complexes in which the second nucleic acid strand is composed of DNA, the in vivo inhibitory effect of a single administration of a double-stranded nucleic acid complex bound to tocopherol and cholesterol on target gene expression in cardiac and skeletal muscles was examined.

[0160] (method) (1) Preparation of nucleic acids The basic structure of the double-stranded nucleic acid complex agent of this example is that α-tocopherol and cholesterol are bound to the second nucleic acid strand. This differs from Example 2 in that the second nucleic acid strand is composed entirely of DNA. The names and base sequences of the first and second nucleic acid strands that make up the double-stranded nucleic acid complex agent used in this example are shown in Table 4.

[0161] [Table 4]

[0162] The first nucleic acid strand used was the first nucleic acid strand prepared in Example 2. The second nucleic acid strand has a sequence complementary to the first nucleic acid strand and has cholesterol bound to its 5' end, similar to the cholesterol-conjugated complementary strand RNA of the second nucleic acid strand (Chol#1-cRNA(mMalat1)) prepared in Example 2, but unlike the second nucleic acid strand in Example 2, it is composed entirely of DNA.

[0163] The double-stranded nucleic acid complex agents were prepared according to the method described in Example 2. The double-stranded nucleic acid complex agent prepared using Toc#1-cDNA(mMalat1) as the second nucleic acid strand is referred to as "Toc#1DNA / DNA," and the double-stranded nucleic acid complex agent prepared using Chol#1-cDNA(mMalat1) is referred to as "Chol#1DNA / DNA." In addition, a single-stranded antisense oligonucleotide (ASO) (ASO(mMalat1)) was used as a comparison control for the double-stranded nucleic acid complex agents.

[0164] (2) In vivo experiments The basic procedure was the same as that described in Example 2. Mice were administered a single dose of 50 mg / kg of the double-stranded nucleic acid complex agent.

[0165] (3) Expression analysis Seventy-two hours after the final administration, the mice were perfused with PBS, and then dissected to remove the myocardium, quadriceps, and diaphragm. RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 mRNA expression levels from the tissues were performed as described in Example 2.

[0166] (result) The results are shown in Figure 8. Figure 8 shows the inhibitory effect of cholesterol-bound double-stranded nucleic acid complex Chol#1DNA / DNA on the expression of the target malat1 gene in the cardiac muscle (heart), quadriceps (quadriceps), and diaphragm (diaphragm). Error bars indicate the standard error of each.

[0167] Even when cholesterol was bound to the second nucleic acid strand, which was composed only of DNA, a significant inhibitory effect on target gene expression was confirmed in various skeletal and cardiac muscles compared to the single-stranded control ASO (mMalat1) and tocopherol.

[0168] <Example 5> (the purpose) As in Example 4, the in vivo inhibitory effect of a single administration of a double-stranded nucleic acid complex in which the second nucleic acid strand was composed only of DNA and a cholesterol-linked complementary strand having various internucleoside bond modification patterns on target gene expression in cardiac and skeletal muscles was examined.

[0169] (method) (1) Preparation of nucleic acids The target gene was malat 1. Table 5 shows the names and base sequences of the first and second nucleic acid strands constituting the double-stranded nucleic acid complex agent used in this example.

[0170] [Table 5]

[0171] In Chol#1-cDNA(mMalat1)(PS), all internucleosides are phosphorothioate bonds, and in Chol#1-cDNA(mMalat1)(PO), all internucleosides are phosphodiester bonds.

[0172] The double-stranded nucleic acid complex agents were prepared according to the method described in Example 2. The double-stranded nucleic acid complex agent prepared using Chol#1-cDNA(mMalat1) as the second nucleic acid strand is referred to as "Chol#1DNA / DNA," the double-stranded nucleic acid complex agent prepared using Chol#1-cDNA(mMalat1)(PS) is referred to as "Chol#1DNA / DNA-PS," and the double-stranded nucleic acid complex agent prepared using Chol#1-cDNA(mMalat1)(PO) is referred to as "Chol#1DNA / DNA-PO."

[0173] (2) In vivo experiments The basic procedure was the same as that described in Example 2. Mice were administered a single dose of 50 mg / kg of the double-stranded nucleic acid complex agent.

[0174] (3) Expression analysis At 72 hours after administration, the mice were perfused with PBS, and then dissected to remove the myocardium, diaphragm, and dorsal muscle. RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 mRNA expression levels from the tissues were performed as described in Example 2.

[0175] (result) The results are shown in Figure 9. As can be seen from Figure 9, when the second nucleic acid strand was composed of DNA only, a significant effect of suppressing the expression of the target gene was confirmed in various skeletal muscles and cardiac muscles, similar to Example 4, regardless of whether all internucleoside bonds were phosphorothioate bonds or all internucleoside bonds were phosphodiester bonds.

[0176] Example 6 (the purpose) The in vivo inhibitory effects in cardiac and skeletal muscles were examined when a single dose of a double-stranded nucleic acid complex in which the second nucleic acid strand was composed of a cholesterol-linked complementary strand with various internucleoside bond modification patterns was administered.

[0177] (method) (1) Preparation of nucleic acids The target gene was malat 1. Table 6 shows the names and base sequences of the first and second nucleic acid strands constituting the double-stranded nucleic acid complex agent used in this example.

[0178] [Table 6]

[0179] Chol#1-cRNA(mMalat1)(PO) is composed solely of unmodified phosphorothioate internucleoside bonds, while Chol#1-cRNA(mMalat1)(5'PS) and Chol#1-cRNA(mMalat1)(3'PS) are composed of phosphorothioate (PS) linkages between the three nucleosides from the 5' and 3' ends, respectively, where cholesterol is bound.

[0180] The double-stranded nucleic acid complex agents were prepared according to the method described in Example 2. The double-stranded nucleic acid complex agent prepared using ASO(mMalat1) as the first nucleic acid strand and Chol#1-cRNA(mMalat1)(PO) as the second nucleic acid strand is referred to as "Chol#1HDO(PO)," the double-stranded nucleic acid complex agent prepared using Chol#1-cRNA(mMalat1)(5'PS) is referred to as "Chol#1HDO(5'PS)," and the double-stranded nucleic acid complex agent prepared using Chol#1-cRNA(mMalat1)(3'PS) is referred to as "Chol#1HDO(3'PS)."

[0181] (2) In vivo experiments The basic procedure was the same as that described in Example 2. Mice were administered a single dose of 50 mg / kg of the double-stranded nucleic acid complex agent. A negative control group of mice was also prepared, receiving only PBS.

[0182] (3) Expression analysis At 72 hours after administration, the mice were perfused with PBS, and then dissected to remove the myocardium, quadriceps femoris, diaphragm, and dorsal proper muscles. RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 mRNA expression levels from the tissues were performed as described in Example 2.

[0183] (result) The results are shown in Figure 10. Figure 10 shows the suppressive effect of cholesterol-bound double-stranded nucleic acid complexes Chol#1HDO(PO), Chol#1HDO(5'PS), and Chol#1HDO(3'PS) on target Malat1 gene expression in cardiac muscle (Heart), quadriceps (Quadriceps), diaphragm, and back muscle (Back). Error bars indicate standard error of each value.

[0184] All double-stranded nucleic acid complexes significantly suppressed the expression of malat1 non-coding RNA. In particular, the inhibitory activity tended to increase when the complex contained a modified internucleoside linkage at the 3' end, such as Chol#1HDO(3'PS).

[0185] Example 7 (the purpose) We investigated the in vivo inhibitory effect of multiple administration of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide targeting the malat1 gene and a tocopherol- or cholesterol-conjugated complementary strand on mRNA expression in tissues.

[0186] (method) (1) Preparation of nucleic acids The double-stranded nucleic acid complex agents used were Toc#1HDO(mMalat1) and Chol#1HDO(mMalat1) prepared in Example 2, and the single-stranded control ASO used was ASO(mMalat1).

[0187] (2) In vivo experiments The mice to be administered with the double-stranded nucleic acid complex agent and the like were male C57BL / 6 mice weighing 20 g and aged 6 to 7 weeks. The double-stranded nucleic acid complex and control ASO were intravenously injected into mice via the tail vein at a dose of 50 mg / kg per injection. The injections were administered weekly for a total of four times over a four-week period. A negative control group of mice was also injected with a single dose of PBS alone.

[0188] (3) Expression analysis At 72 hours after the final administration, mice were perfused with PBS and then dissected to remove the myocardium, quadriceps, diaphragm, and dorsi proper muscles. Subsequently, mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, the MagNA Pure 96 (Roche Life Sciences), according to the protocol. cDNA was synthesized according to the Transcriptor Universal cDNA Master (Roche Life Sciences) protocol. Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific, based on various gene numbers. PCR conditions (temperature and time) consisted of 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second. The resulting amplified products were quantified by quantitative RT-PCR. Based on the results, the expression level of mRNA (malat1) was calculated relative to the expression level of mRNA (ACTB; an internal control gene). The mean and standard error of the relative expression levels were calculated. The results were compared between groups and further evaluated by t-test.

[0189] (result) The results are shown in Figure 11. Figure 11 shows the suppressive effect of tocopherol- or cholesterol-bound double-stranded nucleic acid complexes (Toc#1HDO(mMalat1) and Chol#1HDO(mMalat1) respectively) on the expression of the target Malat1 gene in the cardiac muscle (Heart), quadriceps (Quadriceps), diaphragm, and back proper muscles (Back). Error bars indicate the standard error of each value.

[0190] It was revealed that the inhibitory effect of Toc#1HDO(mMalat1) and Chol#1HDO(mMalat1) on target gene (malat1) expression was further enhanced by multiple administrations in various skeletal and cardiac muscles.

[0191] Example 8 (the purpose) We investigated the in vivo inhibitory effect of multiple administrations of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide targeting the DMPK gene and a cholesterol-conjugated complementary strand on mRNA expression in tissues.

[0192] (method) (1) Preparation of nucleic acids The double-stranded nucleic acid complex agent used was Chol#1HDO(mDMPK) prepared in Example 3.

[0193] (2) In vivo experiments The mice to be administered with the double-stranded nucleic acid complex agent and the like were male C57BL / 6 mice weighing 20 g and aged 6 to 7 weeks. The double-stranded nucleic acid complex was intravenously injected into mice via the tail vein at a dose of 50 mg / kg per administration, twice a week for a total of four doses. In addition, a negative control group of mice was also created that received a single injection of PBS alone.

[0194] (3) Expression analysis At 72 hours after the final administration, the mice were perfused with PBS, and then dissected to remove the myocardium, quadriceps femoris, diaphragm, and dorsal proper muscles. RNA extraction from each tissue, cDNA synthesis, quantitative RT-PCR, and evaluation of DMPK mRNA expression level were performed as described in Example 8.

[0195] (result) The results are shown in Figure 12. Figure 12 shows the inhibitory effect of cholesterol-bound double-stranded nucleic acid complex (Chol#1HDO(mDMPK)) on the expression of the target DMPK gene in the myocardium (Heart), quadriceps (Quadriceps), diaphragm, and back muscles. The error bars indicate the standard error of each value.

[0196] It was revealed that the inhibitory effect of Chol#1HDO(mDMPK) on target gene (DMPK) expression was further enhanced by multiple administrations in various skeletal muscles and cardiac muscles, compared to the negative control (PBS only).

[0197] Example 9 (the purpose) Experiments were conducted to evaluate the long-term in vivo inhibitory effects of double-stranded nucleic acid complexes on mRNA expression in cardiac and skeletal muscle tissues by a single administration.

[0198] (method) (1) Preparation of nucleic acids The double-stranded nucleic acid complex agent used was Chol#1HDO (mMalat1) prepared in Example 2.

[0199] (2) In vivo experiments The double-stranded nucleic acid complex agent was administered once to mice in the same manner as in Example 2.

[0200] (3) Expression analysis Mice were perfused with PBS on days 3, 7, 14, 28, 56, and 168 after administration, and then dissected to remove the myocardium, quadriceps, diaphragm, dorsi propria, liver, kidney, colon, and lung. RNA extraction, cDNA synthesis, and quantitative RT-PCR from each tissue were performed, and the expression level of malat1 non-coding RNA was evaluated according to the methods described in Example 2.

[0201] (result) The results are shown in Figures 13 and 14. Figure 13 is a graph showing the inhibitory effect of cholesterol-bound double-stranded nucleic acid complexes on the expression of a target gene (malat1) in cardiac and skeletal muscles. The vertical axis shows the relative expression level of malat1 non-coding RNA, and the horizontal axis shows the time (days) elapsed since administration. The error bars show the standard error of each. Figure 13a shows cardiac muscle (Heart), Figure 13b shows back muscle (Back), Figure 13c shows quadriceps muscle (Quadriceps), and Figure 13d shows diaphragm (Diaphragm).

[0202] FIG. 14 shows the relative expression level of malat1 non-coding RNA in each tissue 8 weeks (56 days) after administration, compared with the negative control (PBS only).

[0203] 13 and 14, it was revealed that Chol#1HDO(mMalat1) significantly suppressed the expression of malat1 non-coding RNA in cardiac muscle, quadriceps femoris, diaphragm, and dorsal proper muscle for a long period of time compared with the negative control. Furthermore, it was also found that the effect persisted in skeletal muscle even 8 weeks after administration.

[0204] Example 10 (the purpose) The in vivo inhibitory effect of a single administration of the double-stranded nucleic acid complex on mRNA expression in cardiac and skeletal muscle tissues was examined when administered at various doses.

[0205] (method) (1) Preparation of nucleic acids The double-stranded nucleic acid complex agent Chol#1HDO (mMalat1) prepared in Example 2 was used.

[0206] (2) In vivo experiments The double-stranded nucleic acid complex agent Chol#1HDO (mMalat1) was intravenously injected into mice via the tail vein in a single dose at 12.5 mg / kg, 25.0 mg / kg, 50 mg / kg, or 75 mg / kg.

[0207] (3) Expression analysis Seventy-two hours after administration, the mice were perfused with PBS, and then dissected to remove the myocardium, quadriceps femoris, diaphragm, and dorsal proper muscles. RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 mRNA expression levels from each tissue were performed as described in Example 2.

[0208] (result) The results are shown in Figure 15. It was revealed that Chol#1HDO(mMalat1) can dose-dependently suppress the expression of malat1 non-coding RNA in both cardiac and skeletal muscles.

[0209] Example 11 (the purpose) The in vivo inhibitory effect of a double-stranded nucleic acid complex, in which cholesterol and a saturated fatty acid group are bound to the second nucleic acid strand, on mRNA expression in cardiac and skeletal muscle tissues was examined by single administration.

[0210] (method) (1) Preparation of nucleic acids The target gene was malat1, as in Example 2. The first nucleic acid strand constituting the double-stranded nucleic acid complex agent used in this example was the first nucleic acid strand described in Example 2, i.e., a 16-mer single-stranded LNA / DNA gapmer ASO (mMalat1) targeting malat1 non-coding RNA, a transcription product of the mouse malat1 gene. Meanwhile, the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is a strand with cholesterol bound to its 5' or 3' end, with a linker (C6) consisting of a saturated fatty acid group with six carbon atoms (hexyl group) interposed between the cholesterol and the end of the second nucleic acid strand. The names of the second nucleic acid strands are as shown in Table 7.

[0211] [Table 7]

[0212] The double-stranded nucleic acid complex agents of the present invention were prepared by annealing the first nucleic acid strand with either 5'Chol(C6)-cRNA(mMalat1) or 3'Chol(C6)-cRNA(mMalat1) of the second nucleic acid strand. Specific preparation methods were similar to those described in Example 2. The prepared double-stranded nucleic acid complex agents are referred to as 5'Chol(C6)HDO and 3'Chol(C6)HDO.

[0213] (2) In vivo experiments The basic procedure was the same as that described in Example 2. Mice were administered a single dose of 50 mg / kg of the double-stranded nucleic acid complex agent.

[0214] (3) Expression analysis At 72 hours after administration, the mice were perfused with PBS and then dissected to remove the myocardium (heart), quadriceps (quadriceps), diaphragm, and dorsal proper muscles (back). RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 expression levels from each tissue were performed according to the methods described in Example 2.

[0215] (result) The results are shown in Figure 16. Even when a C6 linker was inserted between the end of the second nucleic acid strand and the cholesterol, the in vivo inhibitory effect of a single administration of the double-stranded nucleic acid complex on mRNA expression in cardiac and skeletal muscle tissue was confirmed. The effect was stronger when the second nucleic acid strand was bound to the 5' end.

[0216] Example 12 (the purpose) The in vivo inhibitory effect of double-stranded nucleic acid complexes of different lengths on mRNA expression in tissues targeting target genes was examined.

[0217] (method) (1) Preparation of nucleic acids The target gene was malat1, as in Example 2. The first nucleic acid strand constituting the double-stranded nucleic acid complex agent used in this example was the first nucleic acid strand described in Example 2, i.e., a 13-mer and 16-mer single-stranded LNA / DNA gapmer ASO (mMalat1) targeting malat1 non-coding RNA, a transcription product of the mouse malat1 gene. The second nucleic acid strand is a complementary strand to each first nucleic acid strand, and is configured with cholesterol bound to the 5' end. The names and sequences of the first and second nucleic acid strands used in this example are shown in Table 8.

[0218] [Table 8]

[0219] The prepared 16mer and 13mer double-stranded nucleic acid complex agents are referred to as "16mer Chol-HDO" and "13mer Chol-HDO," respectively.

[0220] (2) In vivo experiments The basic procedure was the same as that described in Example 2. Mice were administered a single dose of 50 mg / kg of the double-stranded nucleic acid complex agent.

[0221] (3) Expression analysis At 72 hours after administration, the mice were perfused with PBS and then dissected to remove the myocardium (heart), quadriceps (quadriceps), diaphragm, and dorsal proper muscles (back). RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 expression levels from each tissue were performed according to the methods described in Example 2.

[0222] (result) The results are shown in Figure 17. Figure 17 is a graph showing the inhibitory effect of double-stranded nucleic acid complexes of different lengths on the target gene on the expression of a target gene (malat1) in cardiac and skeletal muscle throughout the body. The results are shown for cardiac muscle, quadriceps femoris, diaphragm, and dorsal proper muscle. Error bars indicate standard error.

[0223] Both 13mer Chol-HDO and 16mer Chol-HDO showed significant inhibitory effects on expression compared to the negative control PBS, with 13mer Chol-HDO showing a particularly pronounced effect.

[0224] Example 13 (the purpose) The long-term in vivo expression inhibitory effect of a single subcutaneous administration of the double-stranded nucleic acid complex was examined.

[0225] (method) (1) Preparation of nucleic acids The double-stranded nucleic acid complex agent Chol#1 HDO (malat1) prepared in Example 2 was used.

[0226] (2) In vivo experiments The basic procedure was similar to that described in Example 7, but in this example, a double-stranded nucleic acid complex agent was administered subcutaneously to mice in a single dose of 50 mg / kg.

[0227] (3) Expression analysis On days 7, 14, and 28 after subcutaneous administration, mice were perfused with PBS and then dissected to remove the myocardium, quadriceps, and diaphragm. RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 expression levels from each tissue were performed as described in Example 2.

[0228] (result) The results are shown in Figure 18. Figure 18 is a graph showing the effect of subcutaneously administering a double-stranded nucleic acid complex on the expression of a target gene (malat1) in the cardiac muscle, quadriceps, and diaphragm. Error bars indicate standard error.

[0229] It has been demonstrated that the double-stranded nucleic acid complex agent of the present invention can maintain the effect of suppressing target gene expression over a long period of time not only when administered intravenously but also when administered subcutaneously.

[0230] Example 14 (the purpose) The toxicity of the cholesterol-conjugated double-stranded nucleic acid complex of the present invention and cholesterol-conjugated ASO administered to living organisms was examined.

[0231] (method) (1) Preparation of nucleic acids Table 9 shows the names and base sequences of the first nucleic acid strands constituting the single-stranded nucleic acid complexes used in this example.

[0232] [Table 9]

[0233] The first nucleic acid strand is an ASO (mMalat1) described in the above examples that targets the mouse malat1 gene, and has a structure in which cholesterol is bound to its 5' or 3' end with or without a DNA linker (ccttc).

[0234] (2) In vivo experiments The basic procedure was the same as that described in Example 2. A single dose of 50 mg / kg of the double-stranded nucleic acid complex agent was administered subcutaneously and intravenously to mice.

[0235] (3) Expression analysis Blood was collected from each mouse 72 hours after administration, and blood counts were measured by outsourcing to LSI Medience Corporation.

[0236] (result) The results are shown in Figures 19 and 20. Figure 19 is a graph showing the inhibitory effects of the single-stranded nucleic acid complex preparation described above, the positive control double-stranded nucleic acid complex preparation Chol#1HDO(mMalat1), and the negative control PBS on the expression of the malat1 gene in the myocardium (heart), quadriceps (quadriceps), diaphragm (diaphragm), and back muscle (back). "sc" indicates subcutaneous administration. Error bars indicate standard error. Compared to the negative control PBS, both Chol-HDO and Chol-HDO s.c. demonstrated a significant inhibitory effect on the expression of the target gene (malat1 gene) in all of the myocardium, quadriceps, diaphragm, and back muscle. Furthermore, the double-stranded nucleic acid complex of the present invention (Chol-HDO(mMalat1)) significantly inhibited the expression of the target gene, even compared to the single-stranded nucleic acid complex (3'-Chol-DNA-ASO(mMalat1)). In particular, when the single-stranded nucleic acid complex was administered subcutaneously (3'-Chol-DNA-ASO(mMalat1)sc), no inhibition of target gene expression was observed, whereas when the double-stranded nucleic acid complex of the present invention was administered subcutaneously (Chol-HDO(mMalat1)sc), a strong inhibitory effect was observed.

[0237] Figure 20 shows the platelet count in the blood after administration of each nucleic acid complex. "sc" indicates subcutaneous administration, and "iv" indicates intravenous administration. Compared to single-stranded nucleic acid complexes with cholesterol attached to the termini (5'-Chol-DNA-ASO(mMalat1) and 3'-Chol-DNA-ASO(mMalat1)), the double-stranded nucleic acid complexes of the present invention (5'-Chol-HDO(mMalat1)iv and 5'-Chol-HDO(mMalat1)sc) did not show a decrease in platelet count. This result suggests that the double-stranded nucleic acid complexes of the present invention are less toxic to living organisms than single-stranded nucleic acid complexes.

[0238] Example 15 (the purpose) The purpose of this study is to evaluate the exon skipping effect and dystrophin expression in muscles throughout the body by multiple administration of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide (morpholino oligomer) that targets the exon 23 / intron 23 boundary region of mdx mice (Duchenne muscular dystrophy model mice) for exon skipping and a tocopherol-conjugated complementary strand.

[0239] We conducted experiments to evaluate the in vivo effects of inducing exon skipping expression in tissues and dystrophin protein expression using a double-stranded nucleic acid agent consisting of an antisense oligonucleotide targeting exon 23 / intron 23 of the mouse dystrophin gene and a tocopherol-conjugated complementary strand.

[0240] (method) (1) Preparation of nucleic acids The double-stranded nucleic acid complex agent was compared with a conventional single-stranded antisense oligonucleotide (ASO) control. The control (ASO) was a 25-mer single-stranded morpholino targeting exon 23 / intron 23 of the mouse dystrophin pre-mRNA. This ASO consisted entirely of morpholino. This morpholino had a sequence complementary to positions 83803536–83803512 of mouse dystrophin pre-mRNA (GenBank accession number: NC_000086.7). The morpholino (first strand) was annealed with tocopherol-conjugated Toc#1-cRNA (mDystrophin) to prepare a double-stranded nucleic acid agent, a tocopherol-conjugated heteroduplex oligonucleotide (Toc-HDO). The first and second strands were mixed in equimolar amounts, and the solution was heated to 95°C for 5 minutes, then cooled to 37°C and held for 1 hour, thereby annealing the nucleic acid strands to prepare the double-stranded nucleic acid agent described above. The annealed nucleic acid was stored at 4°C or on ice. The prepared double-stranded nucleic acid agent is designated Toc#1HDO.

[0241] The names and base sequences of the first and second strands used in this example are shown in Table 10.

[0242] [Table 10]

[0243] (2) In vivo experiments The mice used were male mdx mice, 6-7 weeks old and weighing 20 g. All experiments using mice were performed with a n=4. Toc#1HDO was intravenously injected into the mice at a dose of 100 mg / kg. The administration was once a week for a total of five doses. Additionally, as negative controls, mice were injected with PBS alone or PMO instead of Toc#1HDO.

[0244] (3) Expression analysis Two weeks after the final administration, mdx mice were perfused with PBS. The mice were then dissected to remove the myocardium, quadriceps, diaphragm, and dorsi proper muscles. mRNA was then extracted from each tissue using Isogen. One-Step RT-PCR was performed using 200 ng of the extracted total RNA using the Qiagen One Step RT-PCR Kit (Qiagen). The reaction mixture was prepared according to the protocol provided with the kit. A LifeECO thermal cycler (Bioer Technology) was used. The RT-PCR program consisted of reverse transcription at 42°C for 30 minutes, followed by heat denaturation at 95°C for 15 minutes, followed by 35 cycles of PCR amplification (94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds), followed by a final extension reaction at 72°C for 7 minutes.

[0245] The nucleotide sequences of the forward (Fw) primer and reverse (Rv) primer used in RT-PCR are as follows: Fw primer: 5'-ATCCAGCAGTCAGAAAGCAAA-3' (SEQ ID NO: 20) Rv primer: 5'-CAGCCATCCATTTCTGTAAGG-3' (SEQ ID NO: 21)

[0246] 1 μL of the RT-PCR reaction product was analyzed using a Bioanalyzer 2100 (Agilent) with the Agilent DNA1000 kit. The electrophoresis image of the Bioanalyzer 2100 is shown in Figure 21. The polynucleotide amount of the band in which exon 23 was skipped (arrow) ("A") and the polynucleotide amount of the band in which exon 23 was not skipped (arrowhead) ("B") were measured.

[0247] Mdx mice have an abnormal stop codon in exon 23. Therefore, in mRNA (B) in which exon 23 was not skipped, subsequent exons are not translated, and normal dystrophin is not expressed. On the other hand, mRNA (A) in which exon 23 was skipped is shorter because it does not contain exon 23, but normal dystrophin is expressed. Based on the measured values of "A" and "B," the skipping efficiency was calculated according to the following formula.

[0248] Skipping efficiency (%) = A / (A+B) x 100 The muscle specimens were sliced into 40 25 μM slices using a cryostat (Leica CM3050 S) and then lysed in 150 μL of buffer (125 mM Tris-HCl pH 6.4, 10% glycerol, 4% SDS, 4 M urea, 10% mercaptoethanol, 0.005% BPB, HO).

[0249] Subsequently, the mixture was subjected to ultrasonic disruption, heated at 100°C for 3 minutes, and centrifuged at 10,000 g for 5 minutes, after which the supernatant was collected.

[0250] Proteins were electrophoresed using 4-15% gradient 10-well plates (Mini-PROTEAN® TGX Precast Gels). After transfer to a membrane, Western blotting was performed using rabbit anti-dystrophin antibody (Abcam ab15277-rabbit) as the primary antibody and HRP-conjugated goat anti-rabbit IgG (Jackson Lab) as the secondary antibody. Luminescence was assessed using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) ChemiDoc Imaging System (Biorad).

[0251] Next, 10 μM-thick thin sections of the muscle specimens were prepared using a cryostat (Leica CM3050 S) and mounted on coverslips (MAS-02, Matsunami Kogyo). After immersing the slides in -30°C acetone for 10 minutes, the specimens were immersed in TBS and allowed to settle. The sections were then blocked with 5% goat serum and treated with the primary antibody solution: rabbit anti-dystrophin antibody (Abcam ab15277-rabbit) (1 / 400) / 5% goat serum / 0.25% Tween 20 / TBS, followed by overnight incubation. After washing the primary antibody solution three times with 0.25% Tween 20 / TBS, the sections were incubated for 1 hour with a secondary antibody solution of goat anti-rabbit IgG antibody (Invitrogen; Alexa Fluor 568) (1 / 1000) / 5% goat serum / 0.25% Tween 20 / TBS. The secondary antibody solution was washed three times with 0.25% Tween 20 / TBS, mounted with VECTASHIELD, and then observed with a Keyence (BZ-X700) microscope. Immunostained images were photographed.

[0252] (result) The results of the skipping rate are shown in Figure 22, the Western blot in Figure 23, and the immunostaining in Figure 24. Figure 22 shows that exon skipping was hardly observed with the single-stranded nucleic acid complex (PMO) in (a) the heart, whereas exon skipping of 27% or more was observed with the double-stranded nucleic acid complex (Toc-HDO) of the present invention. Furthermore, in other skeletal muscles shown in Figures 22(b) to (f), exon skipping was observed with the double-stranded nucleic acid complex (Toc-HDO) of the present invention, which was two to four times higher than that with the single-stranded nucleic acid complex (PMO). The Western blot in Figure 23 also showed higher dystrophin expression with the double-stranded nucleic acid complex (Toc-HDO) than with the single-stranded nucleic acid complex (PMO) in (a) the heart and (b) the quadriceps. Furthermore, in the immunostaining images of Figure 24, in the heart (a), (b) and quadriceps (c), (d), dystrophin expression was greater in the double-stranded nucleic acid complex (Toc-HDO) (b), (d) than in the single-stranded nucleic acid complex (PMO) (a), (c), as in Figure 23.

[0253] Example 16 (the purpose) The purpose of this study was to evaluate the exon skipping effect and dystrophin expression in whole-body muscles following a single administration of a double-stranded nucleic acid complex (Chol-HDO) consisting of an antisense oligonucleotide (morpholino oligomer) targeting the exon 23 / intron 23 boundary region of mdx mice and a cholesterol-conjugated complementary strand. The basic evaluation method was similar to that described in Example 15.

[0254] (method) (1) Preparation of nucleic acids The first strand of the double-stranded nucleic acid complex agent used was that prepared in Example 15. This first strand was annealed with cholesterol-conjugated Chol#1-cRNA (mDystrophin) to prepare a cholesterol-conjugated heteroduplex oligonucleotide (Chol-HDO), a double-stranded nucleic acid agent. The first and second strands were mixed in equimolar amounts, and the solution was heated to 95°C for 5 minutes, then cooled to 37°C and maintained for 1 hour. This annealed the nucleic acid strands to prepare the double-stranded nucleic acid agent. The annealed nucleic acid was stored at 4°C or on ice. The prepared double-stranded nucleic acid agent is designated Chol#1HDO.

[0255] Table 11 shows the names and base sequences of the first and second nucleic acid strands used in this example.

[0256] [Table 11]

[0257] (2) In vivo experiments Male mdx mice weighing 20 g and aged 6–7 weeks were used. All experiments were performed with an n=1. Chol#1HDO was intravenously injected into the mice via the retroorbital vein at a dose of 100 mg / kg. A single dose was administered. Additionally, negative control groups were prepared in which mice were injected with PBS, PMO alone, or Toc-HDO instead of Chol#1HDO.

[0258] (3) Expression analysis Expression analysis was carried out according to the method described in Example 15.

[0259] (result) The results are shown in Figure 25. There was no difference in the effects of Chol-HDO and Toc-HDO in the heart (a), but in skeletal muscle (b) to (e), Chol-HDO had a greater exon skipping effect than Toc-PMO.

[0260] Example 17 (the purpose) The purpose of this study was to evaluate the exon skipping effect and dystrophin expression in the muscles of the whole body following a single subcutaneous administration of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide (mixmer oligomer) targeting the exon 23 / intron 23 boundary region of mdx mice and a tocopherol-conjugated complementary strand. The basic evaluation method was similar to that described in Example 15.

[0261] (method) (1) Preparation of nucleic acids The double-stranded nucleic acid complex agent was compared with a conventional single-stranded antisense oligonucleotide (ASO) control. The control (ASO) was a 13-mer single-stranded mixmer targeting exon 23 / intron 23 of the mouse dystrophin gene pre-mRNA. This ASO was composed of LNA and DNA and had a sequence complementary to mouse dystrophin pre-mRNA (GenBank accession number: NC_000086.7). The mixmer was used as the first strand and annealed with tocopherol-conjugated Toc#1-cRNA (mDystrophin) to prepare a double-stranded nucleic acid agent, a tocopherol-conjugated heteroduplex oligonucleotide (Toc-HDO). The first and second strands were mixed in equimolar amounts, and the solution was heated to 95°C for 5 minutes, then cooled to 37°C and maintained for 1 hour, thereby annealing the nucleic acid strands to prepare the double-stranded nucleic acid agent. The annealed nucleic acid was stored at 4°C or on ice. The prepared double-stranded nucleic acid agent was designated Toc#2HDO. Table 12 shows the names and base sequences of the first and second nucleic acid strands used in this example.

[0262] [Table 12]

[0263] (2) In vivo experiments Male mdx mice, 6-7 weeks old and weighing 20 g, were used. All experiments were performed in duplicate. Mice were subcutaneously injected with 100 mg / kg of Toc#2HDO. The administration was single. Additionally, negative control groups were prepared by injecting mice with PBS alone or single-stranded mixmer instead of Toc#2HDO.

[0264] (3) Expression analysis Expression analysis was carried out according to the method described in Example 17.

[0265] (result) The results are shown in Figure 26. It was revealed that the double-stranded mixmer Toc#2HDO (Toc-Mixmer) had a higher exon skipping effect than the single-stranded mixmer (Mixmer) in the heart (a) and skeletal muscles (b) to (e).

[0266] Example 18 (the purpose) Targeting the malat1 gene, we examined the in vivo inhibitory effect of mRNA expression in tissues by single-dose administration of a double-stranded nucleic acid complex (Chol#1HDO(mMalat1)), consisting of a double-stranded nucleic acid complex in which cholesterol is bound to the second nucleic acid strand, and a nucleic acid molecule formed by self-annealing a single-stranded nucleic acid in which the first nucleic acid strand and a cholesterol-bound complementary strand are linked by an RNA linker.

[0267] (method) (1) Preparation of nucleic acids The target gene was malat1. The double-stranded nucleic acid complex agent used was Chol#1HDO(mMalat1) prepared in Example 2. The name and base sequence of the single-stranded nucleic acid, in which the first nucleic acid strand (ASO) and its complementary strand (cholesterol-conjugated cRNA) Chol#1-cRNA(mMalat1) were linked via an RNA linker, are shown in Table 13. Chol#1sHDO(mMalat1) (or CholsHDO) was prepared by self-annealing this single-stranded nucleic acid as shown in Figure 1c. Specifically, the Chol#1-sHDO(mMalat1) solution was heated to 95°C for 5 minutes, then cooled to 37°C and maintained for 1 hour, thereby allowing the nucleic acid strand to self-anneal. The annealed nucleic acid was stored at room temperature, 4°C, or on ice. The prepared nucleic acid is referred to as "Chol#1sHDO(mMalat1)."

[0268] [Table 13]

[0269] (2) In vivo experiments The basic procedure was the same as that described in Example 2. A single dose of 50 mg / kg in terms of ASO was administered to mice.

[0270] (3) Expression analysis At 72 hours after administration, the mice were perfused with PBS, and then dissected to remove the myocardium, quadriceps femoris, diaphragm, and dorsal proper muscles. RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 mRNA expression levels from the tissues were performed as described in Example 2.

[0271] (result) The results are shown in Figure 27. Figure 27 confirms that even single-stranded nucleic acids in which a first nucleic acid strand and a complementary strand (cholesterol-bound) are linked via an RNA linker have a significant effect of suppressing the expression of target genes in various skeletal muscles and cardiac muscles, comparable to double-stranded nucleic acid complex agents consisting of cholesterol-bound complementary strands.

[0272] Example 19 (the purpose) The in vivo inhibitory effect of mRNA expression in tissues was examined by administering a single dose of a double-stranded nucleic acid complex (Chol#1HDO(mMalat1)) targeting the malat1 gene and consisting of a double-stranded nucleic acid complex in which cholesterol is bound to the second nucleic acid strand, and a nucleic acid (3'Chol(TEG)HDO(mMalat1)) in which the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is bound to cholesterol at its 3' end, with a linker consisting of tetraethylene glycol (TEG) interposed between the cholesterol and the end of the second nucleic acid strand.

[0273] (1) Preparation of nucleic acids The target gene was malat1, as in Example 2. The double-stranded nucleic acid complex agent (Chol#1HDO(mMalat1)) and first nucleic acid strand used in Example 2 were the first nucleic acid strand described in Example 2, i.e., a 16-mer single-stranded LNA / DNA gapmer ASO (mMalat1) targeting malat1 non-coding RNA, a transcription product of the mouse malat1 gene. Meanwhile, the double-stranded nucleic acid complex agent of the present invention was prepared by annealing the first nucleic acid strand with 3'Chol(TEG)-cRNA (mMalat1) of the second nucleic acid strand. The specific preparation method was the same as in Example 2. The prepared double-stranded nucleic acid complex agent is referred to as "3'Chol(TEG)HDO." The names and base sequences of the first and second nucleic acid strands are as shown in Table 14.

[0274] [Table 14]

[0275] (2) In vivo experiments The basic procedure was the same as that described in Example 2. Mice were administered a single dose of 50 mg / kg of the double-stranded nucleic acid complex agent.

[0276] (3) Expression analysis At 72 hours after administration, the mice were perfused with PBS and then dissected to remove the myocardium (heart), quadriceps (quadriceps), diaphragm, and dorsal proper muscles (back). RNA extraction, cDNA synthesis, quantitative RT-PCR, and evaluation of malat1 expression levels from each tissue were performed according to the methods described in Example 2.

[0277] (result) The results are shown in Figure 28. The effect was weakened when the antibody was attached to the 3' end, so it is important to attach it to the 5' end.

[0278] Example 20 (the purpose) The purpose of this study is to evaluate the effect on the athletic performance of multiple administrations of double-stranded nucleic acid complexes (Chol-HDO or Toc-HDO) consisting of an antisense oligonucleotide (morpholino oligomer) that targets the exon 23 / intron 23 boundary region of mdx mice for exon skipping and a cholesterol- or tocopherol-conjugated complementary strand.

[0279] (method) (1) Preparation of nucleic acids The first strand of the double-stranded nucleic acid complex agent used was that prepared in Example 15. The first strand was annealed with cholesterol-conjugated Chol#1-cRNA (mDystrophin) or tocopherol-conjugated Toc#1-cRNA (mDystrophin) to prepare the double-stranded nucleic acid agents, cholesterol-conjugated heteroduplex oligonucleotide (Chol-HDO) and tocopherol-conjugated heteroduplex oligonucleotide (Toc-HDO). The first and second strands were mixed in equimolar amounts, heated to 95°C for 5 minutes, then cooled to 37°C and maintained for 1 hour. The nucleic acid strands were annealed to prepare the double-stranded nucleic acid agent. The annealed nucleic acids were stored at 4°C or on ice. The prepared double-stranded nucleic acid agents are referred to as Chol#1HDO and Toc#1HDO.

[0280] Table 15 shows the names and base sequences of the first and second nucleic acid strands used in this example.

[0281] [Table 15]

[0282] (2) In vivo experiments Mice were intravenously injected with Chol#1HDO or Toc#1HDO at a dose of 100 mg / kg. The injection was administered once a week for a total of five times. Mice were injected with PBS alone or PMO instead of Chol#1HDO or Toc#1HDO as negative controls, and B10 (normal mice) were used as a positive control.

[0283] (3) Exercise stress test An exercise stress test was conducted more than one week after the fifth and final administration. The exercise stress test was performed using a rat / mouse compatible treadmill (belt-type forced running device) (Muromachi Kikai, MK-680S) with electrical stimulation and no incline. The running speed was 5 m / min for the first 5 minutes, and then the speed was increased by 1 m / min every minute, and the running time was measured.

[0284] (result) The results are shown in Figure 29. Compared to negative control mdx mice (mdx, n = 6) administered only PBS, mdx mice (n = 3) administered single-stranded nucleic acid complex (PMO) showed a slight increase in running duration. In contrast, mdx mice administered double-stranded nucleic acid complex (Toc-HDO, n = 4) or double-stranded nucleic acid complex (Chol-HDO, n = 6) showed a significant increase in running duration, and in particular, the double-stranded nucleic acid complex (Chol-HDO) restored motor ability to a level equivalent to that of the positive control B10 (n = 7).

[0285] Example 21 (the purpose) The purpose of this study was to evaluate the effects on grip strength and motor ability of multiple administrations of double-stranded nucleic acid complexes (Chol-HDO or Toc-HDO) consisting of an antisense oligonucleotide (morpholino oligomer) that targets the exon 23 / intron 23 boundary region of mdx mice for exon skipping and a cholesterol- or tocopherol-conjugated complementary strand.

[0286] (method) (1) Preparation of nucleic acids The nucleic acid complex agent used in this example was prepared according to the method described in Example 20. The names and sequences of the first and second nucleic acid strands used in this example are also shown in Table 15.

[0287] (2) In vivo experiments The mice used in this example and the method of administering the nucleic acid complex agent were similar to those in Example 20.

[0288] (3) Grip strength measurement test A grip strength measurement test was conducted at least one week after the fifth and final administration. A mouse grip strength measurement device (Muromachi Kikai, MK-380CM), a stainless steel mesh (Muromachi Kikai, MK-380CM-F / MM), and a digital force gauge (IMADA, DS2-50N) were used for the grip strength measurement test. The mouse's tail was pulled while grasping the wire mesh with its forelimbs, and the tension was measured until the mouse released the mesh. Three measurements were taken and the average was calculated.

[0289] (4) Wire hang test A wire hang test was performed at least one week after the fifth and final administration. The wire hang test was performed by having the mouse cling to a wire mesh, then flipping the mesh over and measuring the time it took for the mouse to fall off (holding time). The product of the mouse's weight (g) and the holding time (s) was calculated as the holding impulse (s*g) (Holding Impulse (s*g) = Body mass (g) × Hang Time (s)). The average of two measurements was calculated.

[0290] (result) The results are shown in Figure 30. Compared to negative control mdx mice (mdx, n = 7) administered only PBS, mdx mice (n = 9) administered single-stranded nucleic acid complexes (PMO) showed slight increases in grip power and holding impulse. In contrast, mdx mice administered double-stranded nucleic acid complexes (Toc-HDO, n = 9) or double-stranded nucleic acid complexes (Chol-HDO, n = 6) showed significant increases in grip power and holding impulse. In Figure 30, B10 was used as a positive control (n = 6 for grip strength and n = 5 for holding impulse).

[0291] Example 22 (the purpose) The blood concentrations of muscle-derived creatine kinase (CK), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) are elevated in mdx mice. Therefore, we aimed to evaluate serum CK, AST, and ALT levels after multiple administration of double-stranded nucleic acid complexes (Chol-HDO or Toc-HDO) consisting of an antisense oligonucleotide (morpholino oligomer) targeting the exon 23 / intron 23 boundary region of mdx mice for exon skipping, and a cholesterol- or tocopherol-conjugated complementary strand.

[0292] (method) (1) Preparation of nucleic acids The nucleic acid complex agent used in this example was prepared according to the method described in Example 20. The names and sequences of the first and second nucleic acid strands used in this example are also shown in Table 15.

[0293] (2) In vivo experiments The mice used in this example and the method of administering the nucleic acid complex agent were similar to those in Example 20.

[0294] (3) Serum analysis At least one week after the fifth and final administration, blood was collected from the mice and serum was separated. The serum was sent to SRL, Inc., and the results of CK, AST, and ALT measurements were obtained.

[0295] (result) The results are shown in Figure 31. Compared to negative control mdx mice (mdx, n = 11) administered only PBS, mdx mice (n = 7) administered single-stranded nucleic acid complexes (PMO) showed slight decreases in CK, AST, and ALT levels. In contrast, mdx mice administered double-stranded nucleic acid complexes (Toc-HDO, n = 7) or double-stranded nucleic acid complexes (Chol-HDO, n = 5 or 6) showed significant decreases in CK, AST, and ALT levels, particularly in Chol-HDO, which showed decreases to levels comparable to those of the positive control B10 (n = 8).

[0296] Example 23 (the purpose) The purpose of this study is to evaluate the effects on myocardial function of multiple administrations of double-stranded nucleic acid complexes (Chol-HDO or Toc-HDO) consisting of an antisense oligonucleotide (morpholino oligomer) that targets the exon 23 / intron 23 boundary region of mdx mice for exon skipping and a cholesterol- or tocopherol-conjugated complementary strand.

[0297] (method) (1) Preparation of nucleic acids The nucleic acid complex agent used in this example was prepared according to the method described in Example 20. The names and sequences of the first and second nucleic acid strands used in this example are also shown in Table 15.

[0298] (2) In vivo experiments The mice used in this example and the method of administering the nucleic acid complex agent were similar to those in Example 20.

[0299] (3) Electrocardiogram measurement Electrocardiograms were performed at least one week after the fifth and final administration. Electrocardiograms were performed under isoflurane anesthesia using a PowerLab 2 / 26 PL2602 with two analog input channels and a High Performance Differential Bio Amplifier ML132. Corrected QT interval (QTc) was calculated by correcting the QT interval by the RR interval.

[0300] (result) The results are shown in Figure 32. The QTc interval was prolonged in negative control mdx mice (mdx, n = 5) administered with PBS alone compared with normal mice (B10, n = 5). The QTc interval was barely improved in mdx mice administered with a single-stranded nucleic acid complex (PMO, n = 5), whereas the QTc interval was significantly improved in mdx mice administered with a double-stranded nucleic acid complex (Toc-HDO, n = 5) or a double-stranded nucleic acid complex (Chol-HDO, n = 5).

[0301] Example 24 (the purpose) The purpose of this study was to evaluate the expression of dystrophin protein in the myocardium and quadriceps muscle of mdx mice after multiple administration of a double-stranded nucleic acid complex (Chol-HDO or Toc-HDO) consisting of an antisense oligonucleotide (morpholino oligomer) that targets the exon 23 / intron 23 boundary region of the mouse for exon skipping, and a cholesterol- or tocopherol-conjugated complementary strand.

[0302] (method) (1) Preparation of nucleic acids The nucleic acid complex agent used in this example was prepared according to the method described in Example 20. The names and sequences of the first and second nucleic acid strands used in this example are also shown in Table 15.

[0303] (2) In vivo experiments The mice used in this example and the method of administering the nucleic acid complex agent were similar to those in Example 20.

[0304] (3) Expression analysis At least one week after the fifth and final administration, mice were dissected and subjected to expression analysis by Western blot and immunostaining. Expression analysis was performed according to the method described in Example 15. However, in this example, Western blot for vinculin was also performed simultaneously as a control. The vinculin antibody (anti-Vinculin (hVIN-1) antibody; Novus Biologicals, NB600-1293) was used at a dilution of 1 / 1000.

[0305] (result) The results are shown in Figures 33 to 37. Compared to mdx mice administered with single-stranded nucleic acid complex (PMO), mdx mice administered with double-stranded nucleic acid complex (Toc-HDO) or double-stranded nucleic acid complex (Chol-HDO) showed higher dystrophin expression in the myocardium (Figure 33) and quadriceps (Figure 34). Furthermore, the immunostaining images in Figures 35 and 36 also showed higher dystrophin expression in the myocardium (Figure 35) and quadriceps (Figure 36) in mdx mice administered with double-stranded nucleic acid complex (Toc-HDO) or double-stranded nucleic acid complex (Chol-HDO) than in mdx mice administered with single-stranded nucleic acid complex (PMO).

[0306] SEQUENCE LISTING <120> Pharmaceutical Composition for Treating Muscle Disease <150> JP 2019-073832 <151> 2019-04-08 <160> 30 <210> 1 <211> 2534 <212> DNA <213> Mus musculus <220> <223> SR-B1 mRNA <400> 1 ggaatcccgc gccgaactcg ggggcgggct gcccgggcca tggcgcataa agcctctggc 60 cacctgcagg gctactgctg ctccggccac cgccaggcac acaccttgct gctgagggag 120 tctcggcttc tgtcatctct gtggcctccg tcacctctgt ctccgtctcc ttcaggtcct 180 gagccccgag agccccttcc gcgcacgcgg acatgggcgg cagctccagg gcgcgctggg 240 tggccttggg gttgggcgcc ctggggctgc tgtttgctgc gctcggcgtt gtcatgatcc 300 tcatggtgcc ctccctcatc aagcagcagg tgctcaagaa tgtccgcata gacccgagca 360 gcctgtcctt cgggatgtgg areagatcc ccgtcccttt ctacttgtct gtctacttct 420 tcgaagtggt caacccaaac gaggtcctca acggccagaa gccagtagtc cgggagcgtg 480 gaccctatgt ctacagggag ttcagacaaa aggtcaacat caccttcaat gaacgaca 540 ccgtgtcctt cgtggagaac cgcagcctcc atttccagcc tgacaagtcg catggctcag 600 agagtgacta cattgtactg cctaacatct tggtcctggg gggctcgata ttgatggaga 660 gcaagcctgt gagcctgaag ctgatgatga ccttggcgct ggtcaccatg ggccagcgtg 720 cttttatgaa ccgcacagtt ggtgagatcc tgtggggcta tgacgatccc ttcgtgcatt 780 ttctcaacac gtacctccca gacatgcttc ccataaaggg caaatttggc ctgtttgttg 840 ggatgaacaa ctcgaattct ggggtcttca ctgtcttcac gggcgtccag aatttcagca 900 ggatccatct ggtggacaaa tggaacggac tcagcaagat cgattattgg cattcagagc 960 agtgtaacat gatcaatggg acttccgggc agatgtgggc acccttcatg acacccgaat 1020 cctcgctgga attcttcagc ccggaggcat gcaggtccat gaagctgacc tacaacgaat 1080 caagggtgtt tgaaggcatt cccacgtatc gcttcacggc ccccgatact ctgtttgcca 1140 acgggtccgt ctacccaccc aacgaaggct tctgcccatg ccgagagtct ggcattcaga 1200 atgtcagcac ctgcaggttt ggtgcgcctc tgtttctctc ccacccccac ttttacaacg 1260 ccgaccctgt gttgtcagaa gctgttcttg gtctgaaccc taacccaaag gagcattcct 1320 tgttcctaga catccatccg gtcactggga tccccatgaa ctgttctgtg aagatgcagc 1380 tgagcctcta catcaaatct gtcaagggca tcgggcaaac agggaagatc gagccagtag 1440 ttctgccgtt gctgtggttc gaacagagcg gagcaatggg tggcaagccc ctgagcacgt 1500 tctacacgca gctggtgctg atgccccagg ttcttcacta cgcgcagtat gtgctgctgg 1560 ggcttggagg cctcctgttg ctggtgccca tcatctgcca actgcgcagc caggagaat 1620 gctttttgtt ttggagtggt agtaaaaagg gctcccagga taaggaggcc attcaggcct 1680 actctgagtc cctgatgtca ccagctgcca agggcacggt gctccaagaa gccaagctat 1740 agggtcctga agacactata agccccccaa acctgatagc ttggtcagac cagccaccca 1800 gtccctacac cccgcttctt gaggactctc tcagcggaca gcccaccagt gccatggcct 1860 gagcccccag atgtcacacc tgtccgcacg cacggcacat ggatgcccac gcatgtgcaa 1920 aaacaactca gggaccaggg acagacctgc tgccaagtga gcctgatggg ccacaggtgt 1980 gctcttctaa atggcctgtg agccaggctg tgggaactct agctgctgtc agcccctcct 2040 gtaggagctg gccctgccca ggctcctgac ttccctcagg aagtctttct gtctttctcc 2100 atcagtctga aagccttagt tcccacagag gacggatctg tcactcctag gggctgggca 2160 tatgtcggcc tcttgtgcca aggccaggca agcagctcca ggtcctgacc agtttgcaca 2220 cacactctgg agctgtatct ggcgcttttt ctatcgtctc tgctatgtca ctgaattaac 2280 cactgtacgt ggcagaggtg gcaggcccct cagggtcctt atttttcagg catggggtca 2340 aagctagagg tatgggccgt ctacaccccc ccgccccccg gcatctagtg tacctcacca 2400 gagggtattc ggaggcccag catcctgcaa ccgacccctt ttttctactg gaagagaaat 2460 tttatcatct ttgaaagga agtcatgact gaagcaataa accttttcac tgattcaaca 2520 aaaaaaaaaaaaaa 2534 <210> 2 <211> 2759 <212> DNA <213> Homo sapiens <220> <223> SR-B1 mRNA <400> 2 gctcaggccc cgcccctgcc gccggaatcc tgaagcccaa ggctgcccgg gggcggtccg 60 gcggcgccgg cgatggggca taaaaccact ggccacctgc cgggctgctc ctgcgtgcgc 120 tgccgtcccg gatccaccgt gcctctgcgg cctgcgtgcc cggagtcccc gctgtgtcg 180 tctctgtcgc cgtccccgtc tcctgccagg cgcggagccc tgcgagccgc gggtgggccc 240 caggcgcgca gacatgggct gctccgccaa agcgcgctgg gctgccggg cgctgggcgt 300 cgcggggcta ctgtgcgctg tgctgggcgc tgtcatgatc gtgatgggc cgtcgctcat 360 caagcagcag gtccttaaga acgtgcgcat cgaccccagt agcctgtcct tcaacatgtg 420 gaaggagatc cctatcccct tctatctctc cgtctacttc tttgacgtca tgaaccccag 480 cgagatcctg aagggcgaga agccgcaggt gcgggagcgc gggccctacg tgtacaggga 540 gttcaggcac aaaagcaaca tcaccttcaa caacaacgac accgtgtcct tcctcgagta 600 ccgcaccttc cagttccagc cctccaagtc ccacggctcg gagagcgact acatcgtcat 660 gcccaacatc ctggtcttgg gtgcggcggt gatgatggag aataagccca tgaccctgaa 720 gctcatcatg accttggcat tcaccaccct cggcgaacgt gccttcatga accgcactgt 780 gggtgagatc atgtggggct acaaggaccc ccttgtgaat ctcatcaaca agtactttcc 840 aggcatgttc cccttcaagg acaagttcgg attatttgct gagctcaaca actccgactc 900 tgggctcttc acggtgttca cgggggtcca gaacatcagc aggatccacc tcgtggacaa 960 gtggaacggg ctgagcaagg ttgacttctg gcattccgat cagtgcaaca tgatcaatgg 1020 aacttctggg caaatgtggc cgcccttcat gactcctgag tcctcgctgg agttctacag 1080 cccggaggcc tgccgatcca tgaagctaat gtacaaggag tcaggggtgt ttgaaggcat 1140 ccccacctat cgcttcgtgg ctcccaaaac cctgtttgcc aacgggtcca tctacccacc 1200 caacgaaggc ttctgcccgt gcctggagtc tggaattcag aacgtcagca cctgcaggtt 1260 cagtgccccc ttgtttctct cccatcctca cttcctcaac gctgacccgg ttctggcaga 1320 agcggtgact ggcctgcacc ctaaccagga ggcacactcc ttgttcctgg acatccaccc 1380 ggtcacggga atccccatga actgctctgt gaaactgcag ctgagcctct acatgaaatc 1440 tgtcgcaggc attggacaaa ctgggaagat tgagcctgtg gtcctgccgc tgctctggtt 1500 tgcagagagc ggggccatgg agggggagac tcttcacaca ttctacactc agctggtgtt 1560 gatgcccaag gtgatgcact atgcccagta cgtcctcctg gcgctgggct gcgtcctgct 1620 gctggtccct gtcatctgcc aaatccggag ccaagaagaa tgctatttat tttggagtag 1680 tagtaaaaag ggctcaaagg ataaggaggc cattcaggcc tattctgaat ccctgatgac 1740 atcagctccc aagggctctg tgctgcagga agcaaaactg tagggtcctg aggacaccgt 1800 gagccagcca ggcctggccg ctgggcctga ccggcccccc agcccctaca ccccgcttct 1860 cccggactct cccagcggac agccccccag ccccacagcc tgagcctccc agctgccatg 1920 tgcctgttgc acacctgcac acacgccctg gcacacatac acacatgcgt gcaggcttgt 1980 gcagacactc agggatggag ctgctgctga agggacttgt agggagaggc tcgtcaacaa 2040 gcactgttct ggaaccttct ctccacgtgg cccacaggcc tgaccacagg ggctgtgggt 2100 cctgcgtccc cttctcgggg tgagcctggc ctgtcccgtt cagccgttgg gcccaggctt 2160 cctcccctcc aaggtgaaac actgcagtcc cggtgtggtg gctccccatg caggacgggc 2220 caggctggga gtgccgcctt cctgtgccaa attcagtggg gactcagtgc ccaggccctg 2280 gccacgagct ttggccttgg tctacctgcc aggccaggca aagcgccttt acacaggcct 2340 cggaaaacaa tggagtgagc acaagatgcc ctgtgcagct gccgagggt ctccgcccac 2400 cccggccgga ctttgatccc cccgaagtct tcacaggcac tgcatcgggt tgtctggcgc 2460 ccttttcctc cagcctaaac tgacatcatc ctatggactg agccggccac tctctggccg 2520 aagtggccgc aggctgtgcc cccgagctgc ccccaccccc tcacagggtc cctcagatta 2580 taggtgccca ggctgaggtg aagaggcctg ggggccctgc cttccgggcg ctcctggacc 2640 ctggggcaaa cctgtgaccc ttttctactg gaatagaaat gagttttatc atctttgaaa 2700 aataattcac tcttgaagta ataaacgttt aaaaaaatgg gaaaaaaaaa aaaaaaaa 2759 <210> 3 <211> 6982 <212> DNA <213> Mus musculus <220> <223> Malat1 mRNA <400> 3 aggcattcag gcagcgagag cagagcagcg tagagcagca cagctgagct cgtgaggcag 60 gagactcagc ccgaggaaat cgcagataag ttttaatta aaaagattga gcagtaaaaa 120 gaattagaac tctaaactta agctaataga gtagcttatc gaaatattac ttagtcttaa 180 taatctaaga agatcttaag agataacatg aaggcttatt taaacagttt gaaaaaggaa 240 atgaggagaa aagtatttgt actgtataat ggaggctgac cagagcagtt taggagattg 300 taaagggagg ttttgtgaag ttctaaaagg ttctagtttg aaggtcggcc ttgtagatta 360 aaacgaaggt tacctaaata gaatctaagt ggcatttaaa acagtaaagt tgtagagaat 420 agtttgaaaa tgaggtgtag ttttaaaaga ttgagaaaag taggttaagt tgacggccgt 480 tataaaaatc cttcgactgg cgcatgtacg tttgaaggca tgagttggaa acaggggaaga 540 tggaagtgtt aggctagccg ggcgatggtg gcgcacgcct ttaatcctag cacttgggag 600 gcagaggcag gcggatttct gagttcgagg ccagcctggt ctacagagtg agttccagga 660 cagccagggc tacacagaga aaccctgtct tgaaaaaaca aaaaggttag gctagtattt 720 gggaaagaa gattagaaaa tggaagtgaa agacgaagaa gacatacagg aaggtgaagaa 780 aaaagctgtt agaagagata ggaaaataga agacaaagca tctttagaag acagaaaagg 840 tacttaagg cacaggtagt aggaagccga agaatagaag atagaaaagaa ccaagataga 900 aaaacaaaat ggaagttaag acaactttgg atgccagcat tcaagatagg caaagagat 960 aagattgagg ccaaaaggtt ggataagata taagtcaga aggaaattat ctttaaagcc 1020 ataagttcaa atttctgatg gagcgagcag tttagaagag tctttagaca gccacataca 1080 1140 ttgaagagtt agaagaatat taaaagcctt aacttgtagc ttaatttgc ttgatgacaa 1200 aaggacttt gataacagtt tcaagattgt cagcattttg cattggactt gagctgaggt 1260 gctttaaaa tcctaacgac tagcattggc agctgaccca ggtctacaca gaagtgcatt 1320 cagtgaacta ggaagacagg agcggcagac aggagtcccg aagccagttt ggtgaagcta 1380 ggaaggactg aggagccagc agcagcagtg catggtgaag atagcccagg aaagagtgcg 1440 gttcggtgga ggaagctagg aagaaggagc catacggatg tggtggtgaa gctgggaaag 1500 ggttccagga tggtggagcg agagcgagtt ggtgatgaag ctagctggcg gcttggcttg 1560 tcaactgcgc ggaggaggcg agcaggcatt gtggagagga tagatagcgg ctcctagacc 1620 agcatgccag tgtgcaagaa aggctgcagg gagagcatgc ggtgcggtaa cattccttga 1680 ggtcggcaac atggtggtgg ttttctgtaa cttggatggt aacttgttta ctttgtctta 1740 atagttatgg gggagttgta ggcttctgtg taagagata tatctggggc tgtatgtagg 1800 ccttgcggg tgttgtaggt tttttttt cagggttg tcctcttgca tcttgtcaga 1860 agcttttgag ggctgactgc aaggcccag aaagaat ggtagatggc aagttgtctt 1920 taaccgctca gagggaatg aatggtagag ccagcacac ctcccagtttt tgtaagacgt 1980 tgtagtttga acgatgacc taccacaagc ctcactcctg tgtagggg gtaattgggc 2040 aaagtgcttt tggggatg ggggcaaat atatttgag ttctttccc cttaggtctg 2100 tctgaatcc taaggcaga tgactcagg gaaccagaaaaggaatc cactctcagg 2160 atagcagag ctcgccaggt ttacagtttg taggagtag aggatg ctagctttca 2220 cactgagtgt ggaggagctg gccatggcgg aattgctgt agtttactct ttcccctcc 2280 cttaatgaga tttgtaaaat cctaacact tttacttgaa atatttggga gtggtcttaa 2340 cagggaggag tgggtgggg aaacgtttt tttctaagat tttccacaga tgctagagtt 2400 gtgttgacac actgggttag agaaggcgtg tactgctatg ctgttggcac gacaccttca 2460 gggactggag ctgccttttg tccttggaag agttttccca gttgccgctg aagtcagcac 2520 agtgcggctt tggttcacag tcacctcagg agaacctcag gagcttggct aggccagagg 2580 ttgaagttaa gttttacagc accgtgattt aaaatatttc attaaagggg aggggtaaaa 2640 cttagttggc tgtggccttg tgtttgggtg ggtgggggtg ttaggtaatt gtttagttta 2700 tgatttcaga taatcatacc agagaactta aatatttgga aaaacaggaa atctcagctt 2760 tcaagttggc aagtaactcc caatccagtt tttgcttctt tttcctttt tcttttttg 2820 aggcgggcag ctaaggaagg ttggttcctc tgccggtccc tcgaaagcgt agggcttggg 2880 ggttggtctg gtccactggg atgatgtgat gctacagtgg ggactcttct gaagctgttg 2940 gatgaatata gattgtagtg tgtggttctc ttttgaaatt tttttcaggt gacttaatgt 3000 atcttaataa ctactatagg aacaaaggaa gtggctttaa tgaccctgaa ggaatttctt 3060 ctggtgatag cttttatatt atcaagtaag agatactatc tcagttttgt ataagcaagt 3120 ctttttccta gtgtaggaga aatgattttc cttgtgacta aacaagatgt aaaggtatgc 3180 tttttttctt cttgtgcatt gtatacttgt gtttatttgt aacttataat ttaagaatta 3240 tgataattca gcctgaatgt cttttagagg gtgggctttt gttgatgagg gaggggaaac 3300 cttttttttt ctgtagacct ttttcagata acaccatctg agtcataacc agcctggcag 3360 tgtgatgacg tagatgcaga gggagcagct ccttggtgaa tgagtgataa gtaaaggcag 3420 aaaaaataat gtcatgtctc catggggaat gagcatgagc cagagattgt tcctactgat 3480 gaaaagctgc atatgcaaaa atttaagcaa atgaaagcaa ccagtataaa gttatggcaa 3540 tacctttaaa agttatggct tatctaccaa gctttatcca caaaagtaaa gaattgatga 3600 aaaacagtga agatcaaatg ttcatctcaa aactgctttt acaaaagcag aatagaaatg 3660 aagtgaaaat gctgcattaa gcctggagta aaaagaagct gagcttgttg agatgagtgg 3720 gatcgagcgg ctgcgaggcg gtgcagtgtg ccaatgtttc gtttgcctca gacaggtttc 3780 tcttcataag cagaagagtt gcttcattcc atctcggagc aggaaacagc agactgctgt 3840 tgacagataa gtgtaacttg gatctgcagt attgcatgtt agggatagat aagtgccttt 3900 tttctctttt tccaaaaaga cctgtagagc tgttgaatgt ttgcagctgg cccctcttag 3960 gcagttcaga attttgagta gttttcccat ccagcctctt aaaaattcct aagccttgca 4020 ccgatgggct ttcatgatgg gatagctaat aggcttttgc atcgtaaact tcaacacaaa 4080 agcctacatg attaatgcct actttaatta cattgcttac aagattaagg aatctttatc 4140 ttgaagaccc catgaaaggg atcattatgt gctgaaaatt agatgttcat attgctaaaa 4200 tttaaatgtg ctccaatgta cttgtgctta aaatcattaa attatacaa ttataaaat 4260 acttcactag agaatgtag tattagag gctgtctcct tattaata aagtcttgtt 4320 tgttgtctgt agttagtgtg ggcaatttg gggggatgtt cttctctaat cttttcagaa 4380 acttgacttc gaacacttaa gtggaccaga tcaggattg agccagaga ccgaattaa 4440 ctttaaggca ggaagacaa attttattct ccatgcagtg atgagcatttt addaattgca 4500 ggcctggcat agaggccgtc taactagga ctaagtacct taggcaggtg ggagatgatg 4560 gtcagagtaa aaggtaacta catattttgt ttccagaaag tcaggggtct aatttgacca 4620 tggctaaaca tctagggtaa vakactttc ccccattt ccaaatgc atgttgagtt 4680 taaatgctta cgatcatctc atccacttta gccttttgtc acccacttg agccacgagt 4740 ggggtcaggc atgtgggttt aaagagtttt cctttgcaga gcctcatttc atccttcatg 4800 gagctgctca ggactttgca tataagcgct tgcctctgtc ttctgttctg ctagtgagtg 4860 tgtgatgtga gaccttgcag tgagtttgtt tttcctggaa tgtggaggga gggggggatg 4920 gggcttactt gttctagctt tttttttaca gaccacacag aatgcaggtg tcttgacttc 4980 aggtcatgtc tgttctttgg cagtaatat gtgcagtact gttccaatct gctgctatta 5040 gaatgcattg tgacgcgact ggagtatgat taagaaagt tgtgtttccc caagtgtttg 5100 gagtagtggt tgttggagga aaagccatga gtaacaggct gagtgttgag gaaatggctc 5160 tctgcagctt tagtaaccc gtgtttgtga ttggagccga gtccctttgc tgtgctgcct 5220 taggtaaatg tttttgttca tttctggtga ggggggttgg gagcactgaa gcctttagtc 5280 tcttccagat tcaacttaaa atctgacaag aataaatca gacaagcaac attcttgaag 5340 aaattttaac tggcaagtgg aaatgttttg aacagttccg tggtctttag tgcattatct 5400 ttgtgtaggt gttctctctc ccctcccttg gtcttaattc ttacatgcag gaacattgac 5460 aacagcagac atctatctat tcaaggggcc agagaatcca gacccagtaa ggaaaaatag 5520 cccatttact ttaaatcgat aagtgaagca gacatgccat tttcagtgtg gggattggga 5580 agccctagtt ctttcagatg tacttcagac tgtagaagga gcttccagtt gaattgaaat 5640 tcaccagtgg acaaaatgag gacaacaggt gaacgagcct tttcttgttt aagattagct 5700 actggtaatc tagtgttgaa tcctctccag cttcatgctg gagcagctag catgtgatgt 5760 aatgttggcc ttggggtgga ggggtgaggt gggcgctaag ccttttttta agatttttca 5820 ggtacccctc actaaaggca ctgaaggctt aatgtaggac agcggagcct tcctgtgtgg 5880 ttgtatcgag accaaagtgg tatcatggtc ggttttgatt 5940 agcagtgggg actaccctac cgtaacacct tgttggaatt gaagcatcca aagaaaatac 6000 ttgagaggcc ctgggcttgt tttaacatct ggaaaaaagg ctgtttttat agcagcggtt 6060 accagcccaa acctcaagtt gtgcttgcag gggagggaaa agggggaaag cgggcaacca 6120 gtttccccag cttttccaga atcctgttac aaggtctccc cacaagtgat ttctctgcca 6180 catcgccacc atgggccttt ggcctaatca cagacccttc acccctcacc ttgatgcagc 6240 footgctgg atccttgagg tcacgttgca tatcggtttc aaggtaacca tggtgccaag 6300 gtcctgtggg ttgcaccaga aaaggccatc aattttcccc ttgcctgtaa tttaacatta 6360 aaaccatagc taagatgttt tatacatagc acctatgcag agtaaacaaa ccagtatggg 6420 tatagtatgt ttgataccag tgctgggtgg gaatgtagga agtcggatga aaagcaagcc 6480 tttgtaggaa gttgttgggg tgggattgca aaaattctct gctaagactt tttcaggtgg 6540 acataacaga cttggccaag ctagcatctt agtggaagca gattcgtcag tagggttgta 6600 aaggtttttc ttttcctgag aaaacaacct tttgttttct caggttttgc ttttggcct 6660 ttcctagct ttaaaaaaaa aaaagcaaaa gacgctggtg gctggcactc ctggtttcca 6720 ggacggggtt caagtccctg cggtgtcttt gcttgactct tatatcatga ggccattaca 6780 ttttcttgg agggttctaa aggctctggg tatggtagct gatatcactg gaacactccc 6840 cagcctcagt gttgaactct tgataattaa ctgcattgtc tttcaggtta tgcccaattc 6900 gtcttattac ctctgagtcg acacacctcc tactatttat tgaatacttt gatttatga 6960 aataaaaact aaatatctct ca 6982 <210> 4 <211> 8758 <212> DNA <213> Homo sapiens <220> <223> Malat1 mRNA <400> 4 gtaaaggact ggggccccgc aactggcctc tcctgccctc ttaagcgcag cgccatttta 60 gcaacgcaga agcccggcgc cgggaagcct cagctcgcct gaaggcaggt cccctctgac 120 gcctccggga gcccaggttt cccagagtcc ttgggacgca gcgacgagtt gtgctgctat 180 cttagctgtc cttataggct ggccattcca ggtggtggta tttagataaa accactcaaa 240 ctctgcagtt tggtcttggg gtttggagga aagcttttat ttttcttcct gctccggttc 300 agaaggtctg aagctcatac ctaaccaggc ataacacaga atctgcaaaa caaaaacccc 360 taaaaaagca gacccagagc agtgtaaaca cttctgggtg tgtccctgac tggctgccca 420 aggtctctgt gtttcggag acaaagccat tcgcttagtt ggtctacttt aaaaggccac 480 ttgaactcgc tttccatggc gatttgcctt gtgagcactt tcaggagagc ctggaagctg 540 aaaaacggta gaaaaatttc cgtgcgggcc gtggggggct ggcggcaact ggggggccgc 600 agatcagagt gggccactgg cagccaacgg cccccggggc tcaggcgggg agcagctctg 660 tggtgtggga ttgaggcgtt ttccaagagt gggttttcac gtttctaaga tttcccaagc 720 agacagcccg tgctgctccg atttctcgaa caaaaaagca aaacgtgtgg ctgtcttggg 780 agcaagtcgc aggactgcaa gcagttgggg gagaaagtcc gccattttgc cacttctcaa 840 ccgtccctgc aaggctgggg ctcagttgcg taatggaaag taaagccctg aactatcaca 900 ctttaatctt ccttcaaaag gtggtaaact atacctactg tccctcaaga gaacacaaga 960 agtgctttaa gaggtatttt aaaagttccg ggggttttgt gaggtgtttg atgacccgtt 1020 taaaatatga tttccatgtt tcttttgtct aaagtttgca gctcaaatct ttccacacgc 1080 tagtaattta agtatttctg catgtgtagt ttgcattcaa gttccataag ctgttaagaa 1140 aaatctagaa aagtaaaaact agaacctatt tttaaccgaa gaactacttt ttgcctccct 1200 cacaaaggcg gcggaaggtg atcgaattcc ggtgatgcga gttgttctcc gtctataaat 1260 acgcctcgcc cgagctgtgc ggtaggcatt gaggcagcca gcgcaggggc ttctgctgag 1320 ggggcaggcg gagcttgagg aaccgcaga taagtttt tctctttgaa agatagagat 1380 tatacaact acttaaaaaa tatagtcaat aggttacta gatttgctt agcgttaagt 1440 ttttaacgta attttatag cttaagattt tagagaaaaa tatgagact tagagagta 1500 gcatgaggaa ggaaagata aaagttctct aaaacatgac ggaggttgag atgaagcttc 1560 ttcatggagt aaaaatgta ttaaaagaaattgagaga aaggactaca gagccccgaa 1620 ttaataccaa tagagggca atgctttg attaaatga aggtgactta aacagcttaa 1680 agtttagttt aaagttgta ggtgattaa atatttgaa ggcgatcttt tAAaagaga 1740 ttaaaccgaa gtgattaaa agaccttgaa atccatgacg cagggaat tgcgtcattt 1800 aaagcctagt taacgcattt actaaacgca gacgaaatg gaagattaa ttgggagtgg 1860 Taggattgaa Caatttgaag Agathagaag tttgaagtgg aaactgaa Gagagagta 1920 cgggaaggcg aagaaaagaaagagagat aggaatta gagaaaaa acatactttt 1980 agagaaaaa agataattt aaacctgaaa agtaggaagc aggaaaaaa agataagcta 2040 ggaaaaaaaa agctaagggc aaatgtaca acttagaag aaattgaa gatagaaca 2100 agatagaaaa tgaaaatatt gtcaagagtt tcagatagaa atgaaaaac aagctaagac 2160 aagtattgga gaagtataga agatagaaa ataataagcc aaaaattgga taaatagca 2220 ctgaaaaaat gaggaaatta ttggtaacca atttatttta aaagcccatc atttaattt 2280 ctggtggtgc agaagttaga aggtaagct tgagagatg agggtgttta cgtagaccag 2340 aaccaattta gagaatact tgagctaga agggagtt ggttaaaaat cacatcaaaa 2400 agctactaaa aggactggtg taatttaaaaaaactaagg cagaaggctt ttggagt 2460 tagaagaatt tggaaggcct taaatatagt agcttagttt gaaaaatgtg aaggactttc 2520 gtaacggaag taattcaaga tcaagagtaa ttaccaactt aatgtttttg cattggactt 2580 tgagttaaga ttatttttta aatcctgagg actagcatta attgacagct gacccaggtg 2640 ctacacagaa gtggattcag tgaatctagg aagacagcag cagacaggat tccaggaacc 2700 agtgtttgat gaagctagga ctgaggagca agcgagcaag cagcagttcg tggtgaagat 2760 aggaaaagag tccaggagcc agtgcgattt ggtgaaggaa gctaggaaga aggaaggagc 2820 gctaacgatt tggtggtgaa gctaggaaaa aggattccag gaaggagcga gtgcaatttg 2880 gtgatgaagg tagcaggcgg cttggcttgg caaccacacg gaggaggcga gcaggcgttg 2940 tgcgtagagg atcctagacc agcatgccag tgtgccaagg ccacaggggaa agcgagtggt 3000 tggtaaaaat ccgtgaggtc ggcaatatgt tgtttttctg gaacttactt atggtaacct 3060 3120. tttatttatt ttctaatata atgggggagt ttcgtactga ggtgtaaagg gatttatatg gggacgtagg ccgatttccg ggtgttgtag gtttctcttt ttcaggctta tactcatgaa tcttgtctga agcttttgag ggcagactgc caagtcctgg agaaatagta gatggcaagt ttgtgggttt ttttttttta cacgaatttg aggaaaacca aatgaatttg atagccaaat tgagacaatt tcagcaaatc tgtaagcagt ttgtatgttt agttggggta atgaagtatt tcagttttgt gaatagatga cctgttttta cttcctcacc ctgaattcgt tttgtaaatg tagtttgg atgtgtaact gaggcggggg ggagttttca gtattttttt ttgtgggggt 3480. gggggcaaaa tatgttttca gttctttttc ccttaggtct gtctagaatc ctaaaggcaa atgactcaag gtgtaacaga aaacaagaaa atccaatatc aggataatca crack ggtttacagt ttatagaaac tagagcagtt ctcacgttga ggtctgtgga agagatgtcc attggagaaa tggctggtag ttactcttttt ttccccccac ccccttaatc agactttaaa 3720 agtgcttaac cccttaaact tgttattttt tacttgaagc attttgggat ggtcttaaca 3780 gggaagagag agggtggggg agaaaatgtt tttttctaag attttccaca gatgctatag 3840 tactattgac aaactgggtt agagaaggag tgtaccgctg tgctgttggc acgaacacct 3900 tcagggactg gagctgcttt tatccttgga agagtattcc cagttgaagc tgaaaagtac 3960 agcacagtgc agctttggtt catattcagt catctcagga gaacttcaga agagcttgag 4020 taggccaaat gttgaagtta agttttccaa taatgtgact tcttaaaagt tttattaaag 4080 gggaggggca aatattggca attagttggc agtggcctgt tacggttggg attggtgggg 4140 tgggtttagg taattgttta gtttatgatt gcagataaac tcatgccaga gaacttaaag 4200 tcttagaatg gaaaaagtaa agaaattca acttccaagt tggcaagtaa ctcccaatga 4260 tttagttttt ttccccccag tttgaattgg gaagctgggg gaagttaaat atgagccact 4320 gggtgtacca gtgcattaat ttgggcaagg aaagtgtcat aatttgatac tgtatctgtt 4380 ttccttcaaa gtatagagct tttgggggaag gaaagtattg aactggggt tggtctggcc 4440 tactgggctg acattaacta caattatggg aaatgcaaaa gttgtttgga tatggtagtg 4500 tgtggttctc ttttggaatt tttttcaggt gatttaataa taatttaaaa ctactataga 4560 aactgcagag caaaggaagt ggcttaatga tcctgaaggg attcttctg atggtagctt 4620 ttgtattatc aagtaagatt ctattttcag ttgtgtgtaa gcaagtttt ttttagtgta 4680 ggagaaatac ttttccattg tttaactgca aaacaagatg ttaaggtatg cttcaaaaat 4740 tttgtaaatt gtttattta aacttatctg tttgtaaatt gtaactgatt aagaattgtg 4800 atagttcagc ttgaatgtct cttagagggt gggcttttgt tgatgaggga ggggaaactt 4860 ttttttttc tatagacttt tttcagataa catcttctga gtcataacca gcctggcagt 4920 atgatggcct agatgcagag aaaacagctc cttggtgaat tgataagtaa aggcagaaaa 4980 gattatatgt catacctcca ttggggaata agcataaccc tgagattctt actactgatg 5040 agaacattat ctgcatatgc caaaaaattt taagcaaatg aaagctacca attaaagtt 5100 acggaatcta ccattttaaa gttaattgct tgtcaagcta taaccacaaa aataatgaat 5160 tgatgagaaa tacaatgaag aggcaatgtc catctcaaaa tactgctttt acaaaagcag 5220 aataaaagcg aaaagaaatg aaaatgttac actacattaa tcctggaata aaagaagccg 5280 aaataaatga gagatgagtt gggatcaagt ggattgagga ggctgtgctg tgtgccaatg 5340 ttcgtttgc ctcagacagg tatctcttcg tttcagaag agttgcttca ttcatctgg 5400 gagcagaaaa cagcaggcag ctgttaacag ataagtttaa cttgcatctg cagtattgca 5460 tgttagggat aagtgcttat ttttaagagc tgtggagttc ttaaatatca accatggcac 5520 tttctcctga ccccttccct aggggatttc aggattgaga aatttttcca tcgagccttt 5580 ttaaaattgt aggacttgtt cctgtgggct tcagtgatgg gatagtacac ttcactcaga 5640 ggcatttgca tctttaaata atttcttaaa agcctctaaa gtgatcagtg ccttgatgcc 5700 aactaaggaa atttgtttag cattgaatct ctgaaggctc tatgaaagga atagcatgat 5760 gtgctgttag aatcagatgt tactgctaaa atttacatgt tgtgatgtaa attgtgtaga 5820 aaaccattaa atcattcaaa ataataaact atttttatta gagaatgtat acttttagaa 5880 agctgtctcc ttatttaaat aaaatagtgt ttgtctgtag ttcagtgttg gggcaatctt 5940 gggggggatt cttctctaat ctttcagaaa ctttgtctgc gaacactctt taatggacca 6000 gatcaggatt tgagcggaag aacgaatgta actttaaggc aggaaagaca aattttattc 6060 ttcataaagt gatgagcata taataattcc aggcacatgg caatagaggc cctctaaata 6120 aggaataaat aacctcttag acaggtggga gattatgatc agagtaaaag gtaattacac 6180 attttatttc cagaaagtca ggggtctata aattgacagt gattagagta atactttttc 6240 acatttccaa agtttgcatg ttaactttaa atgcttacaa tcttagagtg gtaggcaatg 6300 ttttacacta ttgaccttat atagggaagg gagggggtgc ctgtggggtt ttaaagaatt 6360 ttcctttgca gaggcatttc atccttcatg aagccattca ggattttgaa ttgcatatga 6420 gtgcttggct cttccttctg ttctagtgag tgtatgagac cttgcagtga gtttatcagc 6480 atactcaaaa tttttttcct ggaatttgga gggatgggag gagggggtgg ggcttacttg 6540 ttgtagcttt tttttttttt acagacttca cagagaatgc agttgtcttg acttcaggtc 6600 tgtctgttct gttggcaagt aaatgcagta ctgttctgat cccgctgcta ttagaatgca 6660 ttgtgaaacg actggagtat gattaaagt tgtgttcccc aatgcttgga gtagtgattg 6720 ttgaaggaaa aaatccagct gagtgataaa ggctgagtgt tgaggaatt tctgcagttt 6780 tagcagtcg tatttgtgat tgaagctgag tacattttgc tggtgtattt ttaggtaaaa 6840 tgctttttgt cattctctgg tggtgggagg ggactgaagc ctttcttt tccagatgc 6900 aaccttaaaa tcagtgacaa gaacattcc aaacaagcaa cagtcttca gaaattaac 6960 tggcaagtgg aaatgtttaa acagttcagt gatctttagt gcattgttta tgtgtggtt 7020 tctctctcccc ctcccttggt cttaatttctt acatgcagga acaccagca gacacgta 7080 tgcgaagggc cagagaagcc agacccagta agaaaaaata gcctatttac tttaataaa 7140 ccaaacattc cattttaaat gtgggattg ggaaccacta gttctttcag atggtattct 7200 tcagactata gaaggagctt ccagttgaat tcaccagtgg acaaatgag gaaaacaggt 7260 gaacaagctt tttctgtatt tacatacaaa gtcagatcag ttatgggaca atagtattga 7320 atagatttca gctttatgct ggagtaactg gcatgtgagc aaactgtgtt ggcgtggggg 7380 tggaggggtg aggtgggcgc taagcctttt tttaagattt ttcaggtacc cctcactaaa 7440 ggcaccgaag gcttaaagta ggacaaccat ggagccttcc tgtggcagga gagacaacaa 7500 agcgctatta tcctaaggtc aagagaagtg tcagcctcac ctgattttta ttagtaatga 7560 ggacttgcct caactccctc tttctggagt gaagcatccg aaggaatgct tgaagtaccc 7620 ctgggcttct cttaacattt aagcaagctg ttttatagc agctcttaat aataaagccc 7680 aaatctcaag cggtgcttga aggggaggga aagggggaaa gcgggcaacc acttttccct 7740 agcttttcca gaagcctgtt aaaagcaagg tctccccaca agcaacttct ctgccacatc 7800 gccaccccgt gccttttgat ctagcacaga cccttcaccc ctcacctcga tgcagccagt 7860 agcttggatc cttgtgggca tgatccataa tcggtttcaa ggtaacgatg gtgtcgaggt 7920 ctttggtggg ttgaactatg ttagaaaagg ccattaattt gcctgcaaat tgttaacaga 7980 agggtattaa aaccacagct aagtagctct attataatac ttatccagtg actaaaacca 8040 acttaaacca gtaagtggag aaataacatg ttcaagaact gtaatgctgg gtgggaacat 8100 gtaacttgta gactggagaa gataggcatt tgagtggctg agagggcttt tgggtggggaa 8160 tgcaaaaatt ctctgctaag actttttcag gtgaacataa cagacttggc caagctagca 8220 tcttagcgga agctgatctc caatgctctt cagtagggtc atgaaggttt ttcttttcct 8280 gagaaaacaa cacgtattgt tttctcaggt tttgcttttt ggcctttttc tagcttaaaa 8340 aaaaaaaag caaaagatgc tggtggttgg cactcctggt ttccaggacg gggttcaaat 8400 ccctgcggcg tctttgcttt gactactaat ctgtcttcag gactctttct gtatttctcc 8460 ttttctctgc aggtgctagt tcttggagtt ttggggaggt gggaggtaac agcacaatat ctttgaacta tatacatcct tgatgtata tttgtcagga gcttgacttg attgtatatt catatttaca cgagaaccta fatheractgc cttgtctttt tcaggtaata gcctgcagct ggtgttttga gagccctac tgctgaaaac ttaacaattt tgtgtaataa aaatggagaa gctctaaatt gttgtggttc ttttgtgaat aaaaaaatct tgattgggga aaaaaaaa <210> 5 <211> 2761 <212> DNA <213> Mus musculus <220> <223> DMPK mRNA <400> 5 gaactggcca gagagaccca agggatagtc agggacgggc agcatgcag ctagggttct ggggcctgga caggggcagc caggccctgt gacggggaga ccccgagctc cggcccgggg 120 aggggccatg gtgttgcctg cccaacatgt cagccgaagt gcggctgagg cagctccagc 180 agctggtgct ggacccaggc ttcctgggac tggagcccct gctcgacctt ctcctgggcg 240 300. tccaccagga gctgggtgcc tctcacctag cccaggacaa gtatgtggcc gacttcttgc agtgggtgga gcccattgca gcaaggctta aggaggtccg actgcagagg gatgattttg 360 agttttgaa ggtgatcggg cgtggggcgt tcagcgaggt agcggtggtg aagatgaaac agacgggcca agtgtatgcc atgaagatta tgaatagtg ggacatgctg aagagaggcg aggtgtcgtg cttccggga gaaagggatg tattagtg aggggaccgg cgctggatca cacagctgca ctttgccttc caggatgaga actacctgta cctggtcatg gaatactacg tgggcgggga cctgctaacg ctgctgagca agttttgggga gcggatcccc gccgagatgg 660 ctcgcttcta cctggccgag attgtcatgg ccatagactc cgtgcaccgg ctgggctacg 720 780. tgcacaggga catcaaacca gataacattc tgctggaccg atgtgggcac attcgcctgg cagacttcgg ctcctgcctc aaactgcagc ctgatggaat ggtgaggtcg ctggtggctg 840 tgggcacccc ggactacctg tctcctgaga ttctgcaggc cgttggtgga gggcctgggg 900 caggcagcta cgggccagag tgtgactggt gggcactggg cgtgttcgcc tatgagatgt 960 tctatgggca gacccccttc tacgcggact ccacagccga gacatatgcc aagattgtgc 1020 actacaggga acacttgtcg ctgccgctgg cagacacagt tgtccccgag gaagctcagg 1080 acctcattcg tgggctgctg tgtcctgctg agataaggct aggtcgaggt ggggcaggtg 1140 atttccagaa acatcctttc ttctttggcc ttgattggga gggtctccga gacagtgtac 1200 ccccctttac accagacttc gagggtgcca cggacacatg caatttcgat gtggtggagg 1260 accggctcac tgccatggtg agcgggggcg gggagacgct gtcagacatg caggaagaca 1320 tgccccttgg ggtgcgcctg cccttcgtgg gctactccta ctgctgcatg gccttcagag 1380 acaatcaggt cccggacccc acccctatgg aactagaggc cctgcagttg cctgtgtcag 1440 acttgcaagg gcttgacttg cagcccccag tgtccccacc ggatcaagtg gctgaagagg 1500 ctgacctagt ggctgtccct gcccctgtgg ctgaggcaga gaccacggta acgctgcagc 1560 agctccagga agccctggaa gaagaggttc tcacccggca gagcctgagc cgcgagctgg 1620 aggccatccg gaccgccaac cagaacttct ccagccaact acaggaggcc gaggtccgaa 1680 accgagacct ggaggcgcat gttcggcagc tacaggaacg gatggagatg ctgcaggccc 1740 caggagccgc agccatcacg ggggtcccca gtccccgggc cacggatcca ccttcccatc 1800 tagatggccc cccggccgtg gctgtgggcc agtgcccgct ggtggggcca ggccccatgc 1860 accgccgtca cctgctgctc cctgccagga tccctaggcc tggcctatcc gaggcgcgtt 1920 gcctgctcct gttcgccgct gctctggctg ctgccgccac actgggctgc actgggttgg 1980 tggcctatac cggcggtctc accccagtct ggtgtttccc gggagccacc ttcgccccct 2040 gaaccctaag actccaagcc atctttcatt taggcctcct aggaaggtcg agcgaccagg 2100 gagcgaccca aagcgtctct gtgcccatcg cgcccccccc ccccccccac cgctccgctc 2160 cacacttctg tgagcctggg tccccaccca gctccgctcc tgtgatccag gcctgccacc 2220 tggcggccgg ggagggagga acagggctcg tgcccagcac ccctggttcc tgcagagctg 2280 gtagccaccg ctgctgcagc agctgggcat tcgccgacct tgctttactc agccccgacg 2340 tggatgggca aactgctcag ctcatccgat ttcacttttt cactctccca gccatcagtt 2400 acaagccata agcatgagcc ccctatttcc agggacatcc cattcccata gtgatggatc 2460 agcaagacct ctgccagcac acacggagtc tttggcttcg gacagcctca ctcctggggg 2520 ttgctgcaac tccttccccg tgtacacgtc tgcactctaa caacggagcc acagctgcac 2580 tcccccctcc cccaaagcag tgtgggtatt tattgatctt gttatctgac tcactgacag 2640 actccgggac ccacgtttta gatgcattga gactcgacat tcctcggtat ttattgtctg 2700 tccccaccta cgacctccac tcccgaccct tgcgaataaa atacttctgg tctgccctaa 2760 a 2761 <210> 6 <211> 3243 <212> DNA <213> Homo sapiens <220> <223> DMPK mRNA <400> 6 gccacaagcc tccaccccag ctggtccccc acccaggctg cccagtttaa cattcctagt 60 cataggacct tgacttctga gaggcctgat tgtcatctgt aaataagggg taggactaaa 120 gcactcctcc tggaggactg agagatgggc tggaccggag cacttgagtc tgggatatgt 180 gaccatgcta cctttgtctc cctgtcctgt tccttccccc agccccaaat ccagggtttt 240 ccaaagtgtg gttcaagaac cacctgcatc tgaatctaga ggtactggat acaaccccac 300 gtctgggccg ttacccagga cattctacat gagaacgtgg gggtggggcc ctggctgcac 360 ctgaactgtc acctggagtc agggtggaag gtggaagaac tgggtcttat ttccttctcc 420 ccttgttctt tagggtctgt ccttctgcag actccgttac cccaccctaa ccatcctgca 480 cacccttgga gccctctggg ccaatgccct gtcccgcaaa gggcttctca ggcatctcac 540 ctctatggga gggcattttt ggcccccaga accttacacg gtgtttatgt ggggaagccc 600 ctgggaagca gacagtccta gggtgaagct gagaggcaga gagaagggga gacagacaga 660 gggtggggct ttcccccttg tctccagtgc cctttctggt gaccctcggt tcttttcccc 720 caccaccccc ccagcggagc ccatcgtggt gaggcttaag gaggtccgac tgcagaggga 780 cgacttcgag attctgaagg tgatcggacg cggggcgttc agcgaggtag cggtagtgaa 840 gatgaagcag acgggccagg tgtatgccat gaagatcatg aacaagtggg acatgctgaa 900 gaggggcgag gtgtcgtgct tccgtgagga gagggacgtg ttggtgaatg gggaccggcg 960 gtggatcacg cagctgcact tcgccttcca ggatgagaac tacctgtacc tggtcatgga 1020 gtattacgtg ggcggggacc tgctgacact gctgagcaag tttggggagc ggattccggc 1080 cgagatggcg cgcttctacc tggcggagat tgtcatggcc atagactcgg tgcaccggct 1140 tggctacgtg cacagggaca tcaaacccga caacatcctg ctggaccgct gtggccacat 1200 ccgcctggcc gacttcggct cttgcctcaa gctgcgggca gatggaacgg tgcggtcgct 1260 ggtggctgtg ggcaccccag actacctgtc ccccgagatc ctgcaggctg tgggcggtgg 1320 gcctgggaca ggcagctacg ggcccgagtg tgactggtgg gcgctgggtg tattcgccta 1380 tgaaatgttc tatgggcaga cgcccttcta cgcggattcc acggcggaga cctatggcaa 1440 gatcgtccac tacaaggagc acctctctct gccgctggtg gacgaagggg tccctgagga 1500 ggctcgagac ttcattcagc ggttgctgtg tcccccggag acacggctgg gccggggtgg 1560 agcaggcgac ttccggacac atcccttctt ctttggcctc gactgggatg gtctccggga 1620 cagcgtgccc ccctttacac cggatttcga aggtgccacc gacacatgca acttcgactt 1680 ggtggaggac gggctcactg ccatggtgag cgggggcggg gagacactgt cggacattcg 1740 ggaaggtgcg ccgctagggg tccacctgcc ttttgtgggc tactcctact cctgcatggc 1800 cctcagggac agtgaggtcc caggccccac acccatggaa ctggaggccg agcagctgct 1860 tgagccacac gtgcaagcgc ccagcctgga gccctcggtg tccccacagg atgaaacagc 1920 tgaagtggca gttccagcgg ctgtccctgc ggcagaggct gaggccgagg tgacgctgcg 1980 ggagctccag gaagccctgg aggaggaggt gctcacccgg cagagcctga gccgggagat 2040 ggaggccatc cgcacggaca accagaactt cgccagtcaa ctacgcgagg cagaggctcg 2100 gaaccgggac ctagaggcac acgtccggca gttgcaggag cggatggagt tgctgcaggc 2160 agagggagcc acagctgtca cgggggtccc cagtccccgg gccacggatc caccttccca 2220 tctagatggc cccccggccg tggctgtggg ccagtgcccg ctggtggggc caggccccat 2280 gcaccgccgc cacctgctgc tccctgccag ggtccctagg cctggcctat cggaggcgct 2340 ttccctgctc ctgttcgccg ttgttctgtc tcgtgccgcc gccctgggct gcattgggtt 2400 ggtggcccac gccggccaac tcaccgcagt ctggcgccgc ccaggagccg cccgcgctcc 2460 ctgaacccta gaactgtctt cgactccggg gccccgttgg aagactgagt gcccggggca 2520 cggcacagaa gccgcgccca ccgcctgcca gttcacaacc gctccgagcg tgggtctccg 2580 cccagctcca gtcctgtgat ccgggcccgc cccctagcgg ccggggaggg aggggccggg 2640 tccgcggccg gcgaacgggg ctcgaagggt ccttgtagcc gggaatgctg ctgctgctgc 2700 tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgggg ggatcacaga 2760 ccatttcttt ctttcggcca ggctgaggcc ctgacgtgga tgggcaaact gcaggcctgg 2820 gaaggcagca agccgggccg tccgtgttcc atcctccacg cacccccacc tatcgttggt 2880 tcgcaaagtg caaagctttc ttgtgcatga cgccctgctc tggggagcgt ctggcgcgat 2940 ctctgcctgc ttactcggga aatttgcttt tgccaaaccc gctttttcgg ggatcccgcg 3000 cccccctcct cacttgcgct gctctcggag ccccagccgg ctccgcccgc ttcggcggtt 3060 tggatattta ttgacctcgt cctccgactc gctgacaggc tacaggaccc ccaacaaccc 3120 caatccacgt tttggatgca ctgagacccc gacattcctc ggtatttatt gtctgtcccc 3180 acctaggacc cccacccccg accctcgcga ataaaaggcc ctccatctgc ccaaagctct 3240 gga 3243 <210> 7 <211> 14 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(1), (13)..(13) <223> LNA <220> <221> misc_feature <222> (2)..(2), (14)..(14) <223> 5-methylcytosine LNA <400> 7 tcagtcatga cttc 14 <210> 8 <211> 14 <212> RNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(2), (13)..(14) <223> 2'-O-Me RNA <400> 8 gaagucauga cuga 14 <210> 9 <211> 16 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(1), (16)..(16) <223> 5-methylcytosine LNA <220> <221> misc_feature <222> (2)..(3), (14)..(15) <223> LNA <400> 9 ctagttcact gaatgc 16 <210> 10 <211> 16 <212> RNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (14)..(16) <223> 2'-O-Me RNA <400> 10 gcauucagug aacuag 16 <210> 11 <211> 16 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(1), (3)..(3), (14)..(16) <223> LNA <220> <221> misc_feature <222> (2)..(2) <223> 5-methylcytosine LNA <400> 11 acaataaata ccgagg 16 <210> 12 <211> 16 <212> RNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (14)..(16) <223> 2'-O-Me RNA <400> 12 ccucgguauu uauugu 16 <210> 13 <211> 16 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (14)..(16) <223> 2'-O-Me RNA <400> 13 gcattcagtg aactag 16 <210> 14 <211> 13 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (12)..(12) <223> LNA <220> <221> misc_feature <222> (13)..(13) <223> 5-methylcytosine LNA <400> 14 gttcactgaa tgc 13 <210> 15 <211> 13 <212> RNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (12)..(13) <223> 2'-O-Me RNA <400> 15 gcauucagug aac 13 <210> 16 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (5)..(5), (20)..(20) <223> 5-methylcytosine LNA <220> <221> misc_feature <222> (6)..(7), (18)..(19) <223> LNA <400> 16 cttcctagtt cactgaatgc 20 <210> 17 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(1), (16)..(16) <223> 5-methylcytosine LNA <220> <221> misc_feature <222> (2)..(3), (14)..(15) <223> LNA <400> 17 ctagttcact gaatgccttc 20 <210> 18 <211> 25 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(25) <223> Morpholino nucleic acid <400> 18 ggccaaacct cggcttacct gaaat 25 <210> 19 <211> 25 <212> RNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (23)..(25) <223> 2'-O-Me RNA <400> 19 auuucaggua agccgagguu uggcc 25 <210> 20 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 20 atccagcagt cagaaagcaa a 21 <210> 21 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 21 cagccatcca tttctgtaag g 21 <210> 22 <211> 13 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (2)..(2), (8)..(8), (12)..(12) <223> 5-methylcytosine LNA <220> <221> misc_feature <222> (4)..(4), (6)..(6), (10)..(10) <223> LNA <400> 22 acctcggctt acc 13 <210> 23 <211> 13 <212> RNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (11)..(13) <223> 2'-O-Me RNA <400> 23 ggtaagccga ggt 13 <210> 24 <211> 41 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(3), (14)..(16) <223> 2'-O-Me RNA <220> <221> misc_feature <222> (4)..(13), (17)..(25) <223> RNA <220> <221> misc_feature <222> (26)..(26), (41)..(41) <223> 5-methylcytosine LNA <220> <221> misc_feature <222> (27)..(28), (39)..(40) <223> LNA <400> 24 gcauucagug aacuaguuca agagactagt tcactgaatg c 41 <210> 25 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic <400> 25 ctccaacatc aaggaagatg gcatttctag 30 <210> 26 <211> 25 <212> DNA <213> Artificial <220> <223> Synthetic <400> 26 gttgcctccg gttctgaagg tgttc 25 <210> 27 <211> 21 <212> DNA <213> Artificial <220> <223> Synthetic <400> 27 cctccggttc tgaaggtgtt c 21 <210> 28 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic <400> 28 caatgccatc ctggagttcc tg 22 <210> 29 <211> 16 <212> DNA <213> Artificial <220> <223> Synthetic <400> 29 gcattcagtg aactag 16 <210> 30 <211> 16 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (4)..(13) <223> RNA <400> 30 gcauucagug aacuag 16

Claims

1. A double-stranded nucleic acid complex for suppressing or enhancing the expression level of a transcription product or translation product of a target gene in skeletal muscle or cardiac muscle of a subject, or for inhibiting the function of the transcription product or translation product of the target gene, comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand comprises a base sequence capable of hybridizing to all or a part of a transcription product of the target gene and has an antisense effect on the transcription product; the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and is bound to cholesterol or an analog thereof; The double-stranded nucleic acid complex, wherein the first nucleic acid strand is annealed to the second nucleic acid strand.

2. The double-stranded nucleic acid complex of claim 1, wherein the first nucleic acid strand comprises at least four consecutive deoxyribonucleosides.

3. The double-stranded nucleic acid complex of claim 2 , wherein the first nucleic acid strand is a gapmer.

4. The double-stranded nucleic acid complex according to claim 1 or 2, wherein the first nucleic acid strand is a mixmer.

5. The double-stranded nucleic acid complex according to any one of claims 1 to 4, wherein the second nucleic acid strand comprises at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the first nucleic acid strand.

6. The double-stranded nucleic acid complex according to any one of claims 1 to 5, wherein the second nucleic acid strand does not contain natural ribonucleosides.

7. The double-stranded nucleic acid complex according to any one of claims 1 to 6, wherein the nucleic acid portion of the second nucleic acid strand is composed of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside bonds.

8. The double-stranded nucleic acid complex according to any one of claims 1 to 7, wherein the second nucleic acid strand is bound to cholesterol or an analog thereof.

9. The double-stranded nucleic acid complex according to any one of claims 1 to 8, wherein the cholesterol or an analog thereof is bound to the 5' end and / or the 3' end of the second nucleic acid strand.

10. The double-stranded nucleic acid complex according to any one of claims 1 to 9, wherein a ligand is bound to the second nucleic acid strand via a cleavable or uncleavable linker.

11. The double-stranded nucleic acid complex according to any one of claims 1 to 10, wherein the first nucleic acid strand and the second nucleic acid strand are bound to each other via the linker.

12. The double-stranded nucleic acid complex according to claim 10 or 11, wherein the linker is made of a nucleic acid.

13. A pharmaceutical composition comprising the double-stranded nucleic acid complex according to any one of claims 1 to 12 as an active ingredient.

14. The pharmaceutical composition according to claim 13, which is for treating skeletal muscle dysfunction or cardiac dysfunction in a subject.

15. The pharmaceutical composition according to claim 13 or 14, wherein the skeletal muscle dysfunction or cardiac dysfunction is a disease selected from the group consisting of muscular dystrophy, myopathy, inflammatory myopathy, polymyositis, dermatomyositis, Danon disease, myasthenic syndrome, mitochondrial disease, myoglobinuria, glycogen storage disease, periodic paralysis, hereditary cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, hereditary arrhythmia, neurodegenerative disease, sarcopenia, and cachexia.

16. The pharmaceutical composition according to any one of claims 13 to 15, which is administered intravenously, intramuscularly or subcutaneously.

17. The pharmaceutical composition according to any one of claims 13 to 16, wherein the double-stranded nucleic acid complex is administered in a single dose of 0.1 mg / kg or more.

18. The pharmaceutical composition according to any one of claims 13 to 17, wherein the double-stranded nucleic acid complex is administered in a single dose of 0.01 mg / kg to 200 mg / kg.

19. The pharmaceutical composition according to any one of claims 13 to 18, wherein the transcription product of the target gene is any RNA selected from the group consisting of mRNA, microRNA, pre-mRNA, long non-coding RNA, and natural antisense RNA.

20. The pharmaceutical composition according to any one of claims 13 to 19, wherein the first nucleic acid strand is any RNA selected from the group consisting of steric blocking, splicing switch, exon skipping, and exon inclusion.

21. The pharmaceutical composition according to any one of claims 13 to 20, wherein the base sequence of the first nucleic acid strand in the double-stranded nucleic acid complex is represented by SEQ ID NO:

24.

22. A double-stranded nucleic acid complex for inducing RNA editing, exon skipping, or exon inclusion of a target gene in skeletal or cardiac muscle of a subject, or for sterically blocking a target RNA, comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand comprises a base sequence capable of hybridizing to all or a part of a transcription product of the target gene and has an antisense effect on the transcription product; the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand; The double-stranded nucleic acid complex, wherein the first nucleic acid strand is annealed to the second nucleic acid strand.

23. 23. The double-stranded nucleic acid complex of claim 22, wherein the first nucleic acid strand comprises at least one morpholino nucleic acid or ribose 2'-modified nucleic acid.

24. The double-stranded nucleic acid complex according to claim 22 or 23, wherein 50% or more of the bases in the first nucleic acid strand are morpholino nucleic acids or ribose 2'-modified nucleic acids.

25. The double-stranded nucleic acid complex according to any one of claims 22 to 24, wherein the first nucleic acid strand is a mixmer.

26. The double-stranded nucleic acid complex according to any one of claims 22 to 25, wherein 100% of the bases in the first nucleic acid strand are morpholino nucleic acids or ribose 2'-position-modified nucleic acids.

27. The double-stranded nucleic acid complex according to any one of claims 22 to 26, wherein the second nucleic acid strand does not contain natural ribonucleosides.

28. The double-stranded nucleic acid complex according to any one of claims 22 to 27, wherein the nucleic acid portion of the second nucleic acid strand consists of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside linkages.

29. The double-stranded nucleic acid complex according to any one of claims 22 to 28, wherein the second nucleic acid strand has a functional moiety attached thereto.

30. The double-stranded nucleic acid complex according to any one of claims 22 to 28, wherein the functional moiety is selected from the group consisting of cholesterol or an analogue thereof, tocopherol or an analogue thereof, phosphatidylethanolamine or an analogue thereof, a substituted or unsubstituted C1-30 alkyl group, a substituted or unsubstituted C2-30 alkenyl group, and a substituted or unsubstituted C1-30 alkoxy group.

31. 31. The double-stranded nucleic acid complex of claim 30, wherein the functional moiety is cholesterol or an analog thereof.

32. The double-stranded nucleic acid complex according to any one of claims 22 to 31, wherein the cholesterol or an analog thereof is bound to the 5' end and / or the 3' end of the second nucleic acid strand.

33. The double-stranded nucleic acid complex according to any one of claims 22 to 32, wherein a ligand is bound to the second nucleic acid strand via a cleavable or uncleavable linker.

34. A pharmaceutical composition comprising the double-stranded nucleic acid complex according to any one of claims 22 to 33 as an active ingredient.

35. 35. The pharmaceutical composition of claim 34 for treating muscular dystrophy in a subject.

36. 36. The pharmaceutical composition of claim 35, wherein the muscular dystrophy is myotonic dystrophy or Duchenne muscular dystrophy.

37. The pharmaceutical composition according to any one of claims 34 to 36, which is administered intravenously or subcutaneously.

38. The pharmaceutical composition according to any one of claims 34 to 37, wherein the double-stranded nucleic acid complex is administered in a single dose of 0.1 mg / kg or more.

39. The pharmaceutical composition according to any one of claims 34 to 38, wherein the double-stranded nucleic acid complex is administered in a single dose of 0.01 mg / kg to 200 mg / kg.

40. The pharmaceutical composition according to any one of claims 34 to 39, wherein the base sequence of the first nucleic acid strand in the double-stranded nucleic acid complex is represented by any one of SEQ ID NOs: 25 to 28.

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