Antisense oligomers for the treatment of conditions and diseases

Abstract

This invention provides a method for regulating the expression of the SCN1A protein in cells having mRNA containing a nonsense mutation-dependent RNA degradation mechanism-inducing exon (NMD exon mRNA) that encodes the SCN1A protein. [Solution] This method involves a step of contacting the cells with the therapeutic agent, thereby regulating the splicing of NMD exons from NMD exon mRNA encoding the SCN1A protein, and thereby regulating the level of processed mRNA encoding the SCN1A protein, and thus regulating the expression of the SCN1A protein in the cells.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the interests of U.S. Provisional Application No. 62 / 550,462 filed on 25 August 2017, U.S. Provisional Application No. 62 / 575,901 filed on 23 October 2017, U.S. Provisional Application No. 62 / 667,356 filed on 4 May 2018, and U.S. Provisional Application No. 62 / 671,745 filed on 15 May 2018, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002]

[0002] Nervous system disorders are often associated with channel diseases, characterized by disruptions in the function of ion channels that mediate neuronal excitability, neuronal interactions, and overall brain function. Mutations in the SCN1A gene, which is part of the SCN1A-SCN2A-SCN3A gene cluster encoding the pore-forming alpha subunit of voltage-gated sodium channels in neurons, are associated with the development of disease numbers for diseases and conditions such as Dravet syndrome (DS) (Miller et al., 1993-2015, GeneReviews, edited by Pagon RA et al., Seattle (WA): University of Washington, Seattle, Bookshelf ID: NBK1318, and Mulley et al., 2005, Hum. Mutat. 25: pp. 535-542). [Overview of the project] [Means for solving the problem]

[0003]

[0003] Disclosed herein is a method for regulating the expression of the SCN1A protein in cells having mRNA encoding the SCN1A protein, wherein in a particular embodiment the mRNA (NMD exon mRNA) containing a nonsense mutation-mediated RNA decay-inducing exon, comprising the step of contacting the cells with a therapeutic agent, thereby regulating the splicing of the NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of processed mRNA encoding the SCN1A protein, and regulating the expression of the SCN1A protein in the cells. In some embodiments the therapeutic agent is (a) binding to a targeting region of the NMD exon mRNA encoding SCN1A, (b) regulating the binding of factors involved in the splicing of the NMD exon mRNA, or (c) a combination of (a) and (b). In some embodiments the therapeutic agent interferes with the binding of factors involved in the splicing of the NMD exon from the region of the targeting region. In some embodiments the targeting region is proximal to the NMD exon. In some embodiments, the targeted region is located up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides upstream of the 5' end of the NMD exon. In some embodiments, the targeted region is located at least approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides, approximately 40 nucleotides, approximately 30 nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, approximately 2 nucleotides, and approximately 1 nucleotide upstream of the 5' end of the NMD exon.One. In some embodiments, the targeted region is located at least approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides downstream of the 3' end of the NMD exon. In some embodiments, the targeted region is located at least approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides, approximately 40 nucleotides, approximately 30 nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, approximately 2 nucleotides, and approximately 1 nucleotide downstream of the 3' end of the NMD exon. In some embodiments, the targeted portion is located in an intron region between two canonical exon regions of the NMD exon mRNA encoding SCN1A, and the intron region contains an NMD exon. In some embodiments, the targeted portion at least partially overlaps with an NMD exon. In some embodiments, the targeted portion at least partially overlaps with an upstream intron of the NMD exon. In some embodiments, the targeted portion includes a 5' NMD exon-intron junction or a 3' NMD exon-intron junction. In some embodiments, the targeted portion is located within an NMD exon. In some embodiments, the targeted portion contains approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of the NMD exon. In some embodiments, the NMD exon mRNA encoding SCN1A contains a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with one of sequence numbers 2 or 7-10.In some embodiments, the NMD exon mRNA encoding SCN1A is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with SEQ ID NOs: 1 or 3-6. In some embodiments, the targeted region is located up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the targeted region is located approximately 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, 40 nucleotides, 30 nucleotides, 20 nucleotides, 10 nucleotides, 5 nucleotides, 4 nucleotides, 2 nucleotides, and 1 nucleotide upstream of the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the targeted region is located up to approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of the genomic site GRCh37 / hg19:chr2:166,863,740.In some embodiments, the targeted region is approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides, approximately 40 nucleotides, and approximately 30 nucleotides at the genomic site GRCh37 / hg19:chr2:166,863,740. The targeting region of the NMD exon mRNA encoding SCN1A is located downstream of nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, approximately 2 nucleotides, and approximately 1 nucleotide. In some embodiments, the targeting region of the NMD exon mRNA encoding SCN1A contains a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least 8 consecutive nucleic acids of SEQ ID NOs. 2 or 7-10. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) containing a sequence that is at least approximately 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 21-67, 210-256, or 304-379. In some embodiments, the targeting region of the NMD exon mRNA encoding SCN1A is located within the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) containing a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 42-50 or 231-239. In some embodiments, the targeting portion of the NMD exon mRNA encoding SCN1A is located upstream or downstream of the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) containing a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 21-38, 53-67, 210-227, or 242-256. In some embodiments, the targeting portion of the NMD exon mRNA contains the exon-intron junction of exon 20x of SCN1A. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 39-41, 51, 52, 228-230, 240, or 241. In some embodiments, the therapeutic agent facilitates the elimination of NMD exons from processed mRNA encoding the SCN1A protein.In some embodiments, the elimination of NMD exons from processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, and 1 The levels increase by approximately 0.1 to 8 times, approximately 1.1 to 9 times, approximately 2 to 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. In some embodiments, the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in cells. In some embodiments, the amount of processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is approximately 1.1 to 10 times, approximately 1.5 to 10 times, approximately 2 to 10 times, approximately 3 to 10 times, approximately 4 to 10 times, approximately 1.1 to 5 times, approximately 1.1 to 6 times, approximately 1.1 to 7 times, approximately 1.1 to 8 times, and approximately 1. The increase is approximately 1 to 9 times, approximately 2 to 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. In some embodiments, the therapeutic agent increases SCN1A protein expression in cells.In some embodiments, the amount of SCN1A produced in cells contacted with the therapeutic agent is about 1.1 to about 10 times, or about 1.5 to about 10 times, compared to the total amount of SCN1A produced in control cells. The increase is approximately 2 to 10 times, approximately 3 to 10 times, approximately 4 to 10 times, approximately 1.1 to 5 times, approximately 1.1 to 6 times, approximately 1.1 to 7 times, approximately 1.1 to 8 times, approximately 1.1 to 9 times, approximately 2 to 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. In some embodiments, the therapeutic agent inhibits the elimination of NMD exons from processed mRNA encoding the SCN1A protein. In some embodiments, the elimination of NMD exons from processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent was approximately 1 / 1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1 to 1 / 5, 1 / 1 to 1 / 6, 1 / 1 to 1 / 7, 1 / 1 to 1 / 8, and 1 / 1 to 9 minutes, compared to the elimination of NMD exons from processed mRNA encoding the SCN1A protein in control cells. The levels are reduced to approximately 1 / 2 to 1 / 5, approximately 1 / 2 to 1 / 6, approximately 1 / 2 to 1 / 7, approximately 1 / 2 to 1 / 8, approximately 1 / 2 to 1 / 9, approximately 1 / 3 to 1 / 6, approximately 1 / 3 to 1 / 7, approximately 1 / 3 to 1 / 8, approximately 1 / 3 to 1 / 9, approximately 1 / 4 to 1 / 7, approximately 1 / 4 to 1 / 8, approximately 1 / 4 to 1 / 9, at least approximately 1 / 1.1, at least approximately 1 / 1.5, at least approximately 1 / 2, at least approximately 1 / 2.5, at least approximately 1 / 3, at least approximately 1 / 3.5, at least approximately 1 / 4, at least approximately 1 / 5, or at least approximately 1 / 10. In some embodiments, the therapeutic agent reduces the level of processed mRNA encoding the SCN1A protein in cells.In some embodiments, the amount of processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is approximately 1 / 1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1 to 1 / 5, 1 / 1 to 1 / 6, 1 / 1 to 1 / 7, 1 / 1 to 1 / 8, 1 / 1 to 1 / 9, and 1 / 2 compared to the total amount of processed mRNA encoding the SCN1A protein in control cells. It is reduced to about 1 / 5, about 1 / 2 to about 1 / 6, about 1 / 2 to about 1 / 7, about 1 / 2 to about 1 / 8, about 1 / 2 to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1.1, at least about 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 5, or at least about 1 / 10. In some embodiments, the therapeutic agent reduces the expression of the SCN1A protein in cells. In some embodiments, the amount of SCN1A produced in cells contacted with the therapeutic agent is approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, 1 / 1.1 to 1 / 9, 1 / 2 to 1 / 5, 1 / 2 to 1 / 6, 1 / 2 to 1 / 7, 1 / 2 to 1 / 8, 1 / 2 to 1 / 9, 1 / 3 to 1 / 6, 1 / 3 to 1 / 7, and 1 / 3 to 1 / 8. 1 of, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1 / 1.1, at least about 1 / 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least The amount is reduced to approximately 1 / 3.5, at least 1 / 4, at least 1 / 5, or at least 1 / 10. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a skeletal modification including a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is composed of 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, and 10 The nucleic acid bases consist of up to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or up to 12 to 15 nucleic acid bases. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeting portion of the protein-coding NMD exon mRNA. In some embodiments, the method further includes the step of assessing SCN1A mRNA or protein expression.In some embodiments, the cells are ex vivo.

[0004]

[0004] Disclosed herein is a method of treating a disease or condition in a subject requiring such treatment by modulating the expression of the SCN1A protein in cells of the subject, comprising the step of contacting the cells of the subject with a therapeutic agent containing a nonsense mutation-dependent mRNA degradation mechanism-inducing exon (NMD exon) that modulates the splicing of the NMD exon from mRNA in the cell encoding SCN1A, thereby modulating the level of processed mRNA encoding the SCN1A protein and modulating the expression of the SCN1A protein in the cells of the subject. In some embodiments, the therapeutic agent is (a) binding to a targeting region of the NMD exon mRNA encoding SCN1A, (b) modulating the binding of factors involved in the splicing of the NMD exon mRNA, or (c) a combination of (a) and (b). In some embodiments, the therapeutic agent interferes with the binding of factors involved in the splicing of the NMD exon from the region of the targeting region. In some embodiments, the targeting region is proximal to the NMD exon. In some embodiments, the targeted region is located upstream of the 5' end of the NMD exon, at a maximum of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides. In some embodiments, the targeted region is located upstream of the 5' end of the NMD exon, at least approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 40 nucleotides, approximately 30 nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, and approximately It is located 2 nucleotides upstream, or about 1 nucleotide upstream. In some embodiments, the targeted region is located at the 3' end of the NMD exon, up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream. In some embodiments, the targeted region is located at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, and about 1 nucleotide downstream of the 3' end of the NMD exon. In some embodiments, the targeted region is located in an intron region between two canonical exon regions of the NMD exon mRNA encoding SCN1A, and the intron region contains the NMD exon. In some embodiments, the targeted region at least partially overlaps with the NMD exon. In some embodiments, the targeted region at least partially overlaps with an upstream intron of the NMD exon. In some embodiments, the targeted portion includes a 5' NMD exon-intron junction or a 3' NMD exon-intron junction. In some embodiments, the targeted portion is located within an NMD exon. In some embodiments, the targeted portion includes approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides in an NMD exon. In some embodiments, the NMD exon mRNA encoding SCN1A includes a sequence having at least approximately 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with one of sequence numbers 2 or 7-10.In some embodiments, the NMD exon mRNA encoding SCN1A is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with SEQ ID NOs: 1 or 3-6. In some embodiments, the targeting region is located up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the targeted region is located approximately 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, 40 nucleotides, 30 nucleotides, 20 nucleotides, 10 nucleotides, 5 nucleotides, 4 nucleotides, 2 nucleotides, and 1 nucleotide upstream of the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the targeted region is located approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of the genomic site GRCh37 / hg19:chr2:166,863,740.In some embodiments, the targeted region is approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides, and approximately . The targeting region of the NMD exon mRNA encoding SCN1A is located downstream of 40 nucleotides, approximately 30 nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, approximately 2 nucleotides, and approximately 1 nucleotide. In some embodiments, the targeting region of the NMD exon mRNA encoding SCN1A contains a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least 8 consecutive nucleic acids of SEQ ID NOs. 2 or 7-10. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and the ASO contains a sequence that is at least approximately 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 21-67, 210-256, or 304-379. In some embodiments, the targeting region of the NMD exon mRNA encoding SCN1A is located within the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) containing a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 42-50 or 231-239. In some embodiments, the targeting portion of the NMD exon mRNA encoding SCN1A is located upstream or downstream of the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) containing a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 21-38, 53-67, 210-227, or 242-256. In some embodiments, the targeting portion of the NMD exon mRNA contains the exon-intron junction of exon 20x of SCN1A. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 39-41, 51, 52, 228-230, 240, or 241. In some embodiments, the therapeutic agent facilitates the elimination of NMD exons from processed mRNA encoding the SCN1A protein.In some embodiments, the elimination of NMD exons from processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, and 1 The levels increase by approximately 0.1 to 8 times, approximately 1.1 to 9 times, approximately 2 to 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. In some embodiments, the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in cells. In some embodiments, the amount of processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is approximately 1.1 to 10 times, approximately 1.5 to 10 times, approximately 2 to 10 times, approximately 3 to 10 times, approximately 4 to 10 times, approximately 1.1 to 5 times, approximately 1.1 to 6 times, approximately 1.1 to 7 times, approximately 1.1 to 8 times, and approximately 1. The increase is approximately 1 to 9 times, approximately 2 to 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. In some embodiments, the therapeutic agent increases SCN1A protein expression in cells.In some embodiments, the amount of SCN1A produced in cells contacted with the therapeutic agent is about 1.1 to about 10 times the total amount of SCN1A produced in control cells. It increases by a factor of 2, approximately 1.5 to 10, approximately 2 to 10, approximately 3 to 10, approximately 4 to 10, approximately 1.1 to 5, approximately 1.1 to 6, approximately 1.1 to 7, approximately 1.1 to 8, approximately 1.1 to 9, approximately 2 to 5, approximately 2 to 6, approximately 2 to 7, approximately 2 to 8, approximately 2 to 9, approximately 3 to 6, approximately 3 to 7, approximately 3 to 8, approximately 3 to 9, approximately 4 to 7, approximately 4 to 8, approximately 4 to 9, at least approximately 1.1, at least approximately 1.5, at least approximately 2, at least approximately 2.5, at least approximately 3, at least approximately 3.5, at least approximately 4, at least approximately 5, or at least approximately 10 times. In some embodiments, the therapeutic agent inhibits the elimination of NMD exons from processed mRNA encoding the SCN1A protein. In some embodiments, the elimination of NMD exons from processed mRNA encoding the SCN1A protein in cells exposed to the therapeutic agent is approximately 1 / 1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1 to 1 / 5, 1 / 1 to 1 / 6, 1 / 1 to 1 / 7, 1 / 1 to 1 / 8, and 1 / 1 to 9. The levels are reduced to approximately 1 / 2 to 1 / 5, approximately 1 / 2 to 1 / 6, approximately 1 / 2 to 1 / 7, approximately 1 / 2 to 1 / 8, approximately 1 / 2 to 1 / 9, approximately 1 / 3 to 1 / 6, approximately 1 / 3 to 1 / 7, approximately 1 / 3 to 1 / 8, approximately 1 / 3 to 1 / 9, approximately 1 / 4 to 1 / 7, approximately 1 / 4 to 1 / 8, approximately 1 / 4 to 1 / 9, at least approximately 1 / 1.1, at least approximately 1 / 1.5, at least approximately 1 / 2, at least approximately 1 / 2.5, at least approximately 1 / 3, at least approximately 1 / 3.5, at least approximately 1 / 4, at least approximately 1 / 5, or at least approximately 1 / 10. In some embodiments, the therapeutic agent reduces the level of processed mRNA encoding the SCN1A protein in cells.In some embodiments, the amount of processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is approximately 1 / 1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1 to 1 / 5, 1 / 1 to 1 / 6, 1 / 1 to 1 / 7, 1 / 1 to 1 / 8, 1 / 1 to 1 / 9, and 1 / 2 compared to the total amount of processed mRNA encoding the SCN1A protein in control cells. It is reduced to about 1 / 5, about 1 / 2 to about 1 / 6, about 1 / 2 to about 1 / 7, about 1 / 2 to about 1 / 8, about 1 / 2 to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1.1, at least about 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 5, or at least about 1 / 10. In some embodiments, the therapeutic agent reduces the expression of the SCN1A protein in cells. In some embodiments, the amount of SCN1A produced in cells contacted with the therapeutic agent is approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, and 1 / 1.1 to 1 / 7, compared to the total amount of SCN1A produced in control cells. Approximately 1 / 1.1 to approximately 1 / 8, approximately 1 / 1.1 to approximately 1 / 9, approximately 1 / 2 to approximately 1 / 5, approximately 1 / 2 to approximately 1 / 6, approximately 1 / 2 to approximately 1 / 7, approximately 1 / 2 to approximately 1 / 8, approximately 1 / 2 to approximately 1 / 9, approximately 1 / 3 to approximately 1 / 6, approximately 1 / 3 to approximately 1 / 7, approximately 1 / 3 to approximately 1 / 8, approximately 1 / 3 to approximately 1 / 9, approximately 1 / 4 to approximately 1 / 7, approximately 1 / 4 to approximately 1 / 8, approximately 1 / 4 to approximately 1 / 9, at least approximately 1 / 1.1, at least approximately 1 / 1.5, at least approximately 1 / 2, at least approximately 1 / 2.5, at least The amount is reduced to about one-third, at least one-third-fifth, at least one-quarter, at least one-fifth, or at least one-tenth. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a skeletal modification including a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is composed of 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, and 10 The nucleic acid bases consist of up to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or up to 12 to 15 nucleic acid bases. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeting portion of the protein-coding NMD exon mRNA. In some embodiments, the method further includes the step of assessing SCN1A mRNA or protein expression.In some embodiments, the disease or condition is Na. v It is triggered by loss-of-function mutations in 1.1. In some embodiments, the disease or condition is associated with haploinsufficiency of the SCN1A gene, and the subject has a first allele encoding functional SCN1A and a second allele in which SCN1A is not produced or is produced at reduced levels, or a second allele encoding non-functional SCN1A or partially functional SCN1A. In some embodiments, the disease or condition is encephalopathy. In some embodiments, the encephalopathy is epileptic encephalopathy. In some embodiments, the disease or condition is Dravet syndrome (DS); severe myoclonic epilepsy of infants (SMEI) borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures (epilepsy, generalized, with febrile Seizures plus (GEFS+); Early infantile epileptic encephalopathy13; Cryptogenic generalized epilepsy; Cryptogenic focal epilepsy; Myoclonic ataxia; Lennox-Gastaut syndrome; West syndrome; Idiopathic spasm; Early myoclonic encephalopathy; Progressive myoclonic epilepsy; Childhood alternating hemiplegia; Unclassified epileptic encephalopathy; Sudden unexpected death in epilepsy (SUDEP); Sick sinus syndrome1; Autism; or Infant malignant migratory partial seizures. In some embodiments, GEFS+ is generalized epilepsy with febrile seizures plus type 2. In some embodiments, febrile seizures are familial febrile seizures3A. In some embodiments, SMEB is SMEB without generalized spikes (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more SMEI features (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). In some embodiments, the therapeutic agent promotes the elimination of NMD exons from processed mRNA encoding the SCN1A protein, thereby increasing SCN1A expression in cells. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) comprising a sequence that is at least approximately 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs: 22-24, 26, 27, 29-35, 37-62, 64-67, or 304-379. In the embodiment of the part, the disease or condition is Na v It is induced by gain-of-function mutations in 1.1. In some embodiments, the target is an allele produced at increased levels of SCN1A, or Na in cells. v 1.1 has an allele encoding a mutant SCN1A that induces increased activity. In some embodiments, the disease or condition is migraine. In some embodiments, the migraine is familial hemiplegic migraine. In some embodiments, the disease or condition is Na v1.1 The patient has predisposed epilepsy. In some embodiments, the therapeutic agent inhibits the elimination of NMD exons from processed mRNA encoding the SCN1A protein, thereby reducing SCN1A expression in cells. In some embodiments, the therapeutic agent is an antisense oligomer (ASO), and the ASO contains a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs. 21, 25, 28, 36, or 63. In some embodiments, the subject is human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, embryo, or child. In some embodiments, the therapeutic agent is administered by intrathecal, intraventricular, intraperitoneal, intramuscular, subcutaneous, intravitreous, or intravenous injection of the subject. In some embodiments, the method further includes the step of administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is a small molecule. In some embodiments, the second therapeutic agent is an ASO. In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs: 115-161. In some embodiments, the second therapeutic agent corrects intron retention. In some embodiments, the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy, or SCN5A arrhythmia. In some embodiments, the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy, or SCN5A arrhythmia. In some embodiments, the cells are ex vivo. Embedding by reference

[0005] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.

[0005]

[0006] The novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which describes illustrative embodiments in which the principles of the invention are utilized, and to the appended drawings. This specification includes the following disclosure of the invention: [1] A method for regulating the expression of SCN1A protein in a cell having mRNA encoding SCN1A protein, the method comprising contacting the cell with a therapeutic agent, whereby the therapeutic agent regulates the splicing of a nonsense mutation-dependent RNA decay mechanism-inducing exon (NMD exon) from the NMD exon mRNA encoding SCN1A protein, thereby regulating the level of processed mRNA encoding SCN1A protein and regulating the expression of SCN1A protein in the cell. [2] A method for treating a disease or condition in a subject that requires treatment of the disease or condition by regulating the expression of SCN1A protein in the subject's cells, the method comprising contacting the subject's cells with a therapeutic agent that regulates the splicing of a nonsense mutation-dependent mRNA decay mechanism-inducing exon (NMD exon) from mRNA in cells containing the NMD exon and encoding SCN1A, thereby regulating the level of processed mRNA encoding SCN1A protein and regulating the expression of SCN1A protein in the subject's cells. [3] The therapeutic agent is (a) binds to a targeting portion of the NMD exon mRNA encoding SCN1A, (b) regulates the binding of factors involved in the splicing of the NMD exon mRNA, and also (c) a combination of (a) and (b) The method according to [1] or [2], wherein the method is as described above. [4] The method according to [3], wherein the therapeutic agent interferes with the binding of factors involved in the splicing of the NMD exon from the region of the targeting portion. [5] The method according to [3], wherein the targeting portion is proximal to the NMD exon. [6] The method according to [5], wherein the targeting portion is upstream of the 5' end of the NMD exon by at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides. [7] The method according to [5], wherein the targeting portion is upstream of the 5' end of the NMD exon by at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide. [8] The method according to [5], wherein the targeting portion is downstream of the 3' end of the NMD exon by at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides. [9] The method according to [5], wherein the targeting portion is downstream of the 3' end of the NMD exon by at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide.

[10] The method according to [3], wherein the targeting region is located in an intron region between two canonical exon regions of an NMD exon mRNA encoding SCN1A, and the intron region contains an NMD exon.

[11] The method according to [3], wherein the targeting portion at least partially overlaps with the NMD exon.

[12] The method according to [3], wherein the targeted portion at least partially overlaps with an intron upstream of an NMD exon.

[13] The method according to [3], wherein the targeting portion comprises a 5'NMD exon-intron junction or a 3'NMD exon-intron junction.

[14] The method described in [3], in which the target portion is located within an NMD exon.

[15] The method according to [3], wherein the targeting portion comprises approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of an NMD exon.

[16] The method according to [1] or [2], wherein the NMD exon mRNA encoding SCN1A comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any one of sequence numbers 2 or 7-10.

[17] The method according to [1] or [2], wherein the NMD exon mRNA encoding SCN1A is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with SEQ ID NOs. 1 or 3–6.

[18] The method according to [5], wherein the target region is located up to approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides upstream of the genomic site GRCh37 / hg19:chr2:166,863,803.

[19] The method according to [5], wherein the target region is located approximately 1,000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, 40 nucleotides, 30 nucleotides, 20 nucleotides, 10 nucleotides, 5 nucleotides, 4 nucleotides, 2 nucleotides, and 1 nucleotide upstream of the genomic site GRCh37 / hg19:chr2:166,863,803.

[20] The method according to [5], wherein the target region is located up to approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of genomic site GRCh37 / hg19:chr2:166,863,740.

[21] The method according to [5], wherein the target region is located approximately 1,000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, 40 nucleotides, 30 nucleotides, 20 nucleotides, 10 nucleotides, 5 nucleotides, 4 nucleotides, 2 nucleotides, and 1 nucleotide downstream of the genomic site GRCh37 / hg19:chr2:166,863,740.

[22] The method according to [3], wherein the targeting portion of the NMD exon mRNA encoding SCN1A comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to a region comprising at least eight consecutive nucleic acids of sequence number 2 or 7-10.

[23] The method according to [1] or [2], wherein the therapeutic agent is an antisense oligomer (ASO) comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 21-67, 210-256, or 304-379.

[24] The method described in [3], wherein the targeting region of the NMD exon mRNA encoding SCN1A is located within the nonsense mutation-dependent RNA degradation mechanism inducing exon 20x of SCN1A.

[25] The method according to

[24] , wherein the therapeutic agent is an antisense oligomer (ASO), the ASO comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 42-50 or 231-239.

[26] The method according to [3], wherein the targeting region of the NMD exon mRNA encoding SCN1A is located upstream or downstream of the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A.

[27] The method according to

[26] , wherein the therapeutic agent is an antisense oligomer (ASO) and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 21-38, 53-67, 210-227, or 242-256.

[28] The method according to [3], wherein the targeting portion of the NMD exon mRNA includes the exon-intron junction of exon 20x of SCN1A.

[29] The therapeutic agent is an antisense oligomer (ASO), and the ASO is sequence number 39 The method according to

[28] , comprising a sequence that is at least approximately 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of ~41, 51, 52, 228~230, 240, or 241.

[30] The method according to [1] or [2], wherein the therapeutic agent promotes the elimination of NMD exons from processed mRNA encoding the SCN1A protein.

[31] In cells exposed to the therapeutic agent, the elimination of NMD exons from processed mRNA encoding the SCN1A protein was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, and 1 The method described in

[30] , which increases by approximately 0.1 to 9 times, approximately 2 to 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

[32] The method thereof, wherein the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in cells,

[30] .

[33] The amount of processed mRNA encoding the SCN1A protein in cells exposed to the therapeutic agent was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, and 2 times compared to the total amount of processed mRNA encoding the SCN1A protein in control cells. The method described in

[30] , which increases by approximately 5 times, approximately 2 to approximately 6 times, approximately 2 to approximately 7 times, approximately 2 to approximately 8 times, approximately 2 to approximately 9 times, approximately 3 to approximately 6 times, approximately 3 to approximately 7 times, approximately 3 to approximately 8 times, approximately 3 to approximately 9 times, approximately 4 to approximately 7 times, approximately 4 to approximately 8 times, approximately 4 to approximately 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

[34] The method thereof, wherein the therapeutic agent increases the expression of the SCN1A protein in cells,

[30] .

[35] The amount of SCN1A produced in cells exposed to the therapeutic agent was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, and 2 to 7 times compared to the total amount of SCN1A produced in control cells. The method described in

[30] , which increases by a factor of two, approximately two to eight, approximately two to nine, approximately three to six, approximately three to seven, approximately three to eight, approximately three to nine, approximately four to seven, approximately four to eight, approximately four to nine, at least approximately 1.1, at least approximately 1.5, at least approximately 2, at least approximately 2.5, at least approximately 3, at least approximately 3.5, at least approximately 4, at least approximately 5, or at least approximately 10.

[36] A disease or condition in which Na v The method described in [2], which is induced by loss-of-function mutations in 1.1.

[37] The method according to

[36] , wherein the disease or condition is associated with a haploinsufficiency of the SCN1A gene, and the subject has a first allele encoding a functional SCN1A and a second allele in which SCN1A is not produced or is produced at reduced levels, or a second allele encoding a non-functional SCN1A or a partially functional SCN1A.

[38] The method according to

[36] , wherein the disease or condition is encephalopathy.

[39] The method relating to

[38] , wherein the encephalopathy is epileptic encephalopathy.

[40] A disease or condition is Dravet syndrome (DS); severe myoclonic epilepsy of infants. The method described in

[36] , which includes SMEI) borderline epilepsy (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic ataxia; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; autism; or infant malignant migratory partial seizures.

[41] The method described in

[40] , wherein GEFS+ is generalized epilepsy with febrile seizures plus type 2.

[42] The method according to

[40] , wherein the febrile seizure is familial febrile seizure 3A.

[43] The method according to

[40] , wherein SMEB is SMEB without generalized spikes (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more SMEI features (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[44] The method thereof, wherein the therapeutic agent promotes the elimination of NMD exons from processed mRNA encoding the SCN1A protein, thereby increasing the expression of SCN1A in cells,

[36] .

[45] The method according to

[36] , wherein the therapeutic agent is an antisense oligomer (ASO) and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 22-24, 26, 27, 29-35, 37-62, 64-67, or 304-379.

[46] The method according to [1] or [2], wherein the therapeutic agent inhibits the elimination of NMD exons from processed mRNA encoding the SCN1A protein.

[47] In cells exposed to the therapeutic agent, the elimination of NMD exons from processed mRNA encoding the SCN1A protein was approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, 1 / 1.1 to 1 / 9, and 1 / 2 compared to the elimination of NMD exons from processed mRNA encoding the SCN1A protein in control cells. The method described in

[46] , which reduces the amount to approximately 1 / 5, 1 / 2 to 1 / 6, 1 / 2 to 1 / 7, 1 / 2 to 1 / 8, 1 / 2 to 1 / 9, 1 / 3 to 1 / 6, 1 / 3 to 1 / 7, 1 / 3 to 1 / 8, 1 / 3 to 1 / 9, 1 / 4 to 1 / 7, 1 / 4 to 1 / 8, 1 / 4 to 1 / 9, at least 1 / 1.1, at least 1 / 1.5, at least 1 / 2, at least 1 / 2.5, at least 1 / 3, at least 1 / 3.5, at least 1 / 4, at least 1 / 5, or at least 1 / 10.

[48] ​​The method according to

[46] , wherein the therapeutic agent reduces the level of processed mRNA encoding the SCN1A protein in cells.

[49] The amount of processed mRNA encoding the SCN1A protein in cells exposed to the therapeutic agent was approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, and 1.1 min 1 to about 1 / 9, about 1 / 2 to about 1 / 5, about 1 / 2 to about 1 / 6, about 1 / 2 to about 1 / 7, about 1 / 2 to about 1 / 8, about 1 / 2 to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1 / 1.1, at least about 1 / 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5 The method described in

[46] , which reduces the amount to at least about one-quarter, at least about one-fifth, or at least about one-tenth.

[50] The method thereof, wherein the therapeutic agent reduces the expression of the SCN1A protein in cells,

[46] .

[51] The amount of SCN1A produced in cells exposed to the therapeutic agent was approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, 1 / 1.1 to 1 / 9, 1 / 2 to 1 / 5, 1 / 2 to 1 / 6, and 1 / 2 to 1 / 7. 1. The method described in

[46] for reducing to approximately 1 / 2 to approximately 1 / 8, approximately 1 / 2 to approximately 1 / 9, approximately 1 / 3 to approximately 1 / 6, approximately 1 / 3 to approximately 1 / 7, approximately 1 / 3 to approximately 1 / 8, approximately 1 / 3 to approximately 1 / 9, approximately 1 / 4 to approximately 1 / 7, approximately 1 / 4 to approximately 1 / 8, approximately 1 / 4 to approximately 1 / 9, at least approximately 1 / 1.1, at least approximately 1 / 1.5, at least approximately 1 / 2, at least approximately 1 / 2.5, at least approximately 1 / 3.5, at least approximately 1 / 4, at least approximately 1 / 5, or at least approximately 1 / 10.

[52] A disease or condition in which Na v The method described in [2], which is induced by a gain-of-function mutation as described in 1.1.

[53] The target is an allele produced at an increased level of SCN1A, or Na in cells. v The method according to

[52] , having an allele encoding a mutant SCN1A that induces increased activity of 1.1.

[54] The method according to

[52] , wherein the disease or condition is migraine.

[55] The method according to

[54] , wherein the migraine is familial hemiplegic migraine3.

[56] Disease or condition is Na v 1.1 Predisposing epilepsy, as described in [2].

[57] The method thereof, wherein the therapeutic agent inhibits the elimination of NMD exons from processed mRNA encoding the SCN1A protein, thereby reducing the expression of SCN1A in cells,

[52] .

[58] The method according to

[52] , wherein the therapeutic agent is an antisense oligomer (ASO) and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 21, 25, 28, 36, or 63.

[59] The method according to [1] or [2], wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a skeletal modification including a phosphorothioate linkage or a phosphorodiamidate linkage.

[60] The method according to [1] or [2], wherein the therapeutic agent is an antisense oligomer (ASO), the antisense oligomer comprising a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[61] The method according to [1] or [2], wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

[62] The method according to

[61] , wherein each sugar portion is a modified sugar portion.

[63] The therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is composed of 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 The method according to [1] or [2], comprising a nucleic acid base, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or up to 12 to 15 nucleic acid bases.

[64] The method according to [3], wherein the therapeutic agent is an antisense oligomer (ASO) which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeting portion of the protein-coding NMD exon mRNA.

[65] The method according to [1], further comprising the step of assessing SCN1A mRNA or protein expression.

[66] The method described in [2], wherein the subject is a human.

[67] The method according to [2], wherein the subject is a non-human animal.

[68] The method according to [2], wherein the subject is a fetus, embryo, or child.

[69] The method according to [1] or [2], wherein the cells are ex vivo.

[70] The method according to [2], wherein the therapeutic agent is administered by target intrathecal, intraventricular, intraperitoneal, intramuscular, subcutaneous, intravitreous, or intravenous injection.

[71] The method according to [2], further comprising the step of administering a second therapeutic agent.

[72] The method according to

[71] , wherein the second therapeutic agent is a low molecular weight agent.

[73] The method according to

[71] , wherein the second therapeutic agent is an ASO.

[74] The method according to

[73] , wherein the ASO comprises a sequence that is at least approximately 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 115–161.

[75] The method according to

[71] , wherein a second therapeutic agent corrects intron retention.

[76] The method according to [2], wherein the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy, or SCN5A arrhythmia.

[77] The method according to

[30] ,

[32] , or

[34] , wherein the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy, or SCN5A arrhythmia. [Brief explanation of the drawing]

[0006] [Figure 1A]

[0007] This is a schematic diagram of the therapeutic-mediated elimination of nonsense mutation-dependent mRNA degradation mechanism-inducing exons, which increase the expression of nonsense mutation-dependent mRNA degradation mechanism-inducing exons (NMD exon mRNA) and full-length target proteins or functional RNAs. The diagram shows a cell divided into nuclear and cytoplasmic compartments. In the nucleus, the mRNA precursor transcript of the target gene is spliced ​​to produce mRNA, which is then transported to the cytoplasm and translated into the target protein. With respect to this target gene, a fraction of the mRNA contains nonsense mutation-dependent mRNA degradation mechanism-inducing exons (NMD exon mRNA) that are degraded in the cytoplasm and therefore do not result in target protein production. [Figure 1B] This is a schematic diagram of the therapeutic elimination of nonsense mutation-dependent mRNA degradation mechanism-inducing exons, which increase the expression of target mRNA containing nonsense mutation-dependent mRNA degradation mechanism-inducing exons (NMD exon mRNA) and full-length target proteins or functional RNA. An example of the same cell divided into nuclear and cytoplasmic compartments is shown. Treatment with a therapeutic agent, such as an antisense oligomer (ASO), promotes the elimination of nonsense mutation-dependent mRNA degradation mechanism-inducing exons, resulting in an increase in mRNA, which is then translated into higher levels of target proteins. [Figure 1C] This diagram illustrates the therapeutic elimination of nonsense mutation-dependent mRNA degradation mechanism-inducing exons, which increase the expression of target mRNA containing nonsense mutation-dependent mRNA degradation mechanism-inducing exons (NMD exon mRNA) and full-length target proteins or functional RNA. It also shows a schematic diagram of therapeutic ASO-mediated elimination of nonsense mutation-dependent mRNA degradation mechanism-inducing exons, which converts non-productive mRNA into productive mRNA and increases the expression of full-length target proteins derived from the productive mRNA. [Figure 2]

[0008] This figure illustrates the identification of an exemplary nonsense mutation-dependent mRNA degradation mechanism (NMD)-inducing exon in the SCN1A gene. The identification of the NMD-inducing exon in the SCN1A gene using comparative genomics is visualized in the UCSC Genome Browser. The top panel shows a scaled-to-size graphic representation of the SCN1A gene. Conservation levels across 100 vertebrate species are shown as peaks. The highest peaks correspond to exons (black squares), while no peaks are observed for most introns (arrows). A conservation peak was identified in intron 20 (NM_006920), shown in the middle panel. Examination of the conserved sequence identified a 64 bp exon-like sequence (lower panel, highlighted in gray) flanked by the 3' and 5' splice sites (underlined sequences). Inclusion of this exon results in a frameshift and the introduction of an immature stop codon into exon 21, making the transcript a target for NMD. [Figure 3A]

[0009] Figure 3A is a graph showing the confirmation of NMD-inducing exons via cycloheximide treatment. The presence of a band corresponding to the NMD-inducing exon (21x) was confirmed by RT-PCR analysis using cytoplasmic RNA from DMSO- (CHX-) or cycloheximide-treated (CHX+) Neuro 2A (mouse neural progenitor cells) and primers for exon 21 and downstream exons. Product identity was confirmed by sequencing. Band density analysis was performed to calculate the exon 21x content percentage of the total SCN1A transcript. Treatment of Neuro 2A with cycloheximide (CHX+) to inhibit NMD resulted in a twofold increase in the product corresponding to the NMD-inducing exon 21x in the cytoplasmic fraction (see light gray bars, i.e., CHX- vs. dark gray bars, i.e., CHX+). [Figure 3B]

[0010] Figure 3B is a graph showing the confirmation of NMD-inducing exons via cycloheximide treatment. The presence of bands corresponding to NMD-inducing exons (20x) was confirmed by RT-PCR analysis using cytoplasmic RNA from DMSO- (CHX-) or cycloheximide-treated (CHX+) RenCell VM (human neural progenitor cells), as well as primers for exons 20 and 23. Product identity was confirmed by sequencing. Band density analysis was performed to calculate the exon 20x content percentage of total SCN1A transcripts. Treatment of RenCell VM with cycloheximide (CHX+) to inhibit NMD resulted in a twofold increase in the product corresponding to NMD-inducing exon 20x in the cytoplasmic fraction (see light gray bars, i.e., CHX- vs. dark gray bars, i.e., CHX+). [Figure 4]

[0011] This figure illustrates exemplary ASO gait in the SCN1A exon 20x region. It shows a graphical representation of ASO gait performed using a PS-based 2'-MOE ASO, targeting the sequence upstream of the 3' splice site, the sequence traversing the 3' splice site, exon 20x, the sequence traversing the 5' splice site, and the sequence downstream of the 5' splice site in the SCN1A exon 20x region. The ASO was designed to cover these regions by moving 5 nucleotides at a time. [Figure 5A]

[0012] This figure shows the ASO gait of the SCN1A exon 20x region as evaluated by RT-PCR. Representative PAGEs show SYBR-safe stained RT-PCR products of SCN1A treated with a 20 μM concentration in RenCell VM cells using a simulated treatment (Sham), SMN-controlled ASO (SMN), or 2'-MOE ASO targeting the exon 20x region as described herein in the examples and Figure 4, using reagent-free uptake. Two types of products corresponding to exon 20x inclusion (upper band) and full-length exon 20x exclusion (lower band) were quantified. [Figure 5B]

[0013] This graph plots the exon-20x coverage percentage from the data in Figure 5A. The black line indicates no change for Sham. [Figure 5C]

[0014] This graph shows the full-length product normalized to the internal control RPL32, plotting the change factor compared to Sham. The black line indicates a ratio of 1, and that there is no change for Sham. [Figure 6]

[0015] This graph shows exemplary ASO gait in the SCN1A exon 20x region, as evaluated by RT-qPCR. The SYBR-green RT-qPCR SCN1A amplification results, normalized for RPL32 and obtained using the same ASO uptake experiment shown in Figure 5 and evaluated by SYBR-safe RT-PCR, are plotted as the change factor compared to Sham to confirm the SYBR-safe RT-PCR results. The black line indicates a ratio of 1 (no change for Sham). [Figure 7A]

[0016] Figure 7A is a table showing some members of the voltage-gaited sodium channel alpha subunit. The arrows correspond to the bar colors in Figure 7B. X indicates that no expression was detected. [Figure 7B]

[0017] Figure 7B is a graph showing the selected ASOs assessed by Taqman qPCR of SCN1A, SCN2A, SCN3A, SCN8A, and SCN9A to evaluate target selectivity. Taqman-qPCR amplification results normalized to RPL32, obtained using Ex20x+1, IVS20x+18, and IVS20x+33 ASOs, are plotted as the change factor compared to Sham. The black line indicates a ratio of 1 (no change for Sham). [Figure 8A]

[0018] Figure 8A shows an exemplary dose-dependent effect of selected ASOs in CXH-treated cells. Representative pages are shown showing SYBR-safe stained RT-PCR products of mouse Scn1a cells treated with SYBR-safe staining at concentrations of 30 nM, 80 nM, and 200 nM in Neuro 2A (mouse neuroblastoma) cells, either simulated by RNAiMAX transfection (Sham, RNAiMAX only) or treated with Ex21x+1 2'-MOE ASO targeting exon 21x (mouse nomenclature, corresponding to human exon 20x). Ex21x+1 (mouse nomenclature) and Ex20x+1 (human nomenclature) are identical. Two types of products corresponding to exon 20x inclusion (upper band) and full-length exon 20x exclusion (lower band) were quantified. [Figure 8B]

[0019] Figure 8B is a graph plotting the inclusion of exon-20x from the data in Figure 7A. The black line indicates that there is no change for Sham. [Figure 8C]

[0020] Figure 8C is an exemplary graph of the full-length product normalized to the internal control, Hprt, plotting the multiplier of change compared to Sham. The black line indicates a multiplier of 1, and no change for Sham. [Figure 9A]

[0021] Figure 9A shows exemplary results of intravitreal (IVT) injection of selected ASOs in C57BL6J mice (male, 3 months old). It shows PAGE gels of SYBR-safe stained RT-PCR products of mouse Scn1a derived from the left eye (-) injected with PBS (1 μL), or the right eye (+) injected with 10 mM IVS20x-21, Ex21x+1, IVS21x+18, IVS21x+33, or Cep290 (negative control ASO, Gerard et al., Mol.Ther.Nuc.Ac., 2015) 2'-MOE ASO (1 μL). Ex21x+1, IVS21x+18, and IVS21x+33 (mouse nomenclature) are identical to Ex20x+1, IVS20x+18, and IVS20x+33 (human nomenclature). Two types of products were quantified: one containing exon-21x (upper band) and another excluding the entire length (lower band). [Figure 9B]

[0022] Figure 9B is a graph plotting the exon 21x inclusion percentage from the data in Figure 9A. White bars correspond to eyes injected with ASO, and gray bars correspond to eyes injected with PBS, with n=5 in each group. [Figure 9C]

[0023] Figure 9C is a graph of the full-length product normalized to the internal control Gapdh, plotting the change ratio of eyes injected with ASO compared to eyes injected with PBS. The black line indicates a ratio of 1, no change for PBS, and n=5 in each group. [Figure 10A]

[0024] Figure 10A shows exemplary results of intraventricular (ICV) injection of selected ASOs in C57BL6J mice (male, 3 months old). It shows PAGE gels of SYBR-safe stained RT-PCR products of mouse Scn1a derived from brains injected with uninjected (-, no-ASO control) or 300 μg of Cep290 (negative control ASO, Gerard et al., Mol.Ther.Nuc.Ac., 2015), Ex21x+1, IVS21x+18, IVS21x+33 2'-MOE ASO. Ex21x+1, IVS21x+18, and IVS21x+33 (mouse nomenclature) are identical to Ex20x+1, IVS20x+18, and IVS20x+33 (human nomenclature). Two types of products were quantified: one containing exon-21x (upper band) and another excluding the entire length (lower band). [Figure 10B]

[0025] Figure 10B is a graph plotting the exon 21x inclusion percentage from the data in Figure 10A, showing n=6 (each target ASO), n=5 (Cep290 ASO), and n=1 (no injection, no ASO control). [Figure 10C]

[0026] Figure 10C is a graph of the results of a Taqman qPCR assay performed using two different probes spanning the junction of exons 21 and 22. The products are normalized to an internal control, Gapdh, and the change multipliers of ASO-injected brains compared to Cep290-injected brains are plotted. The black lines indicate a ratio of 1, and no change for Cep290, n=6 (each target ASO), n=5 (Cep290 ASO), and n=1 (uninjected, no-ASO control). [Figure 11A]

[0027] Figure 11A shows exemplary results of intraventricular (ICV) injection of selected ASOs in C57BL6J mice (male, 3 months old). PAGE gels of SYBR-safe stained RT-PCR products of mouse Scn1a derived from brains injected with 300ug of Cep290 (negative control ASO, Gerard et al., Mol.Ther.Nuc.Ac., 2015) or 33ug, 100ug, and 300ug of Ex21x+1 2'-MOE ASO. Ex21x+1 (mouse nomenclature) and Ex20x+1 (human nomenclature) are identical. Two types of products corresponding to exon 21x inclusion (upper band) and full-length exon 21x exclusion (lower band) were quantified. [Figure 11B]

[0028] Figure 11B is a graph plotting the exon-21x inclusion percentage from the data in Figure 11A, with n=5 (for each group). [Figure 11C]

[0029] Figure 11C is a graph of the results of a Taqman qPCR assay performed using two different probes spanning the junction of exons 21 and 22. The products are normalized to the internal control Gapdh, and the change ratios of ASO-injected brains compared to Cep290-injected brains are plotted. The black line indicates a ratio of 1, and no change for Cep290, n=5 (each group). [Figure 12A]

[0030] Figure 12A shows exemplary results of intraventricular (ICV) injection of selected ASO in C57BL6J mice (2 days postnatally). PAGE gels of SYBR-safe stained RT-PCR products of mouse Scn1a derived from brains of uninjected (-, no-ASO control) or injected with 20 μg of Ex21x+1 2'-MOE ASO are shown. Two types of products corresponding to exon 21x inclusion (upper band) and full-length exon 21x exclusion (lower band) were quantified. Ex21x+1 (mouse nomenclature) and Ex20x+1 (human nomenclature) are identical. [Figure 12B]

[0031] Figure 12B is a graph plotting the exon-21x inclusion percentage from the data in Figure 12A, showing n=4 (for each group). [Figure 12C]

[0032] Figure 12C is a graph of the results of a Taqman qPCR assay performed using two different probes spanning the junction of exons 21 and 22. The products are normalized to the internal control, Gapdh, and the change multiplier of ASO-injected brains compared to non-ASO control brains is plotted. The black line indicates a ratio of 1, and no change for the non-ASO control, n=4 (each group). [Figure 13A]

[0033] Figure 13A is a graph plotting the exon 21x content percentage in the indicated mouse CNS sample. [Figure 13B]

[0034] Figure 13B is a graph plotting the exon 20x content percentage in the indicated human CNS sample. [Figure 14A]

[0035] Figure 14A is a graph plotting the percentage decrease in exon 21x inclusion at the indicated dose. [Figure 14B]

[0036] Figure 14B is a graph plotting the percentage increase in Scn1a mRNA at the indicated dose. [Figure 14C]

[0037] Figure 14C is a graph plotting the percentage increase in Nav1.1 protein levels at the indicated dose. [Figure 15A]

[0038] Figure 15A is a graph plotting the percentage decrease in exon 21x inclusion at the indicated dose. [Figure 15B]

[0039] Figure 15B is a graph plotting the percentage increase in Scn1a mRNA at the indicated dose. [Figure 16]

[0040] This graph shows the selected Scn1a-targeted ASOs, which were administered to mice 2 days postnatally via ICV injection at a dose of 10 ug and evaluated 5 days post-injection by Taqman qPCR on SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A, and SCN11A to assess target selectivity. Taqman-qPCR amplification results normalized to Gapdh, obtained using Ex20x+1 ASO, are plotted as a change factor compared to mice injected with PBS. [Figure 17A]

[0041] This graph shows exemplary results three days after intraventricular (ICV) injection of selected ASO at the indicated dose into wild-type (WT) or heterozygous Dravet mice (HET) F1 mice resulting from a 129S-Scn1atm1Kea×C57BL / 6J cross, on postnatal day 2.

[0042] Figure 17A is a graph of the results of a Taqman qPCR assay performed using a probe spanning exons 21 and 22. The product is normalized to the internal control Gapdh, and the change multiplier of ASO-injected brains compared to PBS-injected brains is plotted. [Figure 17B]

[0043] Figure 17B is a graph of the results of Western blotting performed using an anti-Nav1.1 antibody. The product is normalized to the Ponceau staining band, and the change factor of ASO-injected brains compared to PBS-injected brains is plotted. [Figure 18]

[0044] This graph shows exemplary results of SCN1A exon 20x region ASO microwalking in RenCell via free uptake. The ASOs were designed to cover the regions surrounding the three target ASOs (indicated by asterisks) previously identified in Figure 6, either by moving one nucleotide at a time (6–41) or by reducing the length of ASO17 (1–5). The graph shows the exon 20x inclusion percentage, measured by SYBR green qPCR. The black line indicates no change for ASO-free(-). [Figure 19]

[0045] This graph plots the time-dependent increase in Scn1a mRNA levels in mouse coronal brain sections after injection of an SCN1A-targeted ASO. As shown, the increase in Scn1a mRNA levels was maintained for at least 80 days after injection. [Figure 20]

[0046] This is an exemplary survival curve graph demonstrating the 100% survival benefit provided by SCN1A-targeted ASO in the Dravet mouse model. + / + represents the WT genotype, + / - represents the 129S-scn1atm1Kea heterozygous genotype (Dravet mouse model), A represents PBS treatment, and B represents ASO treatment. As shown, mice in group A+ / - (PBS-treated Dravet mice) began dying around 16 days postnatally, while all mice in the other three groups, including group B+ / - (ASO-treated Dravet mice), survived at least until 35 days postnatally. [Modes for carrying out the invention]

[0007] Splicing and nonsense mutation-dependent mRNA degradation mechanisms

[0047] Intervening sequences or introns are removed by large, highly dynamic RNA-protein complexes called spliceosomes, which regulate the complex interactions between the primary transcript, nuclear small RNA (snRNA), and numerous proteins. Spliceosomes assemble regularly on each intron, starting with recognition of the 5' splice site (5'ss) by U1 snRNA or the 3' splice site (3'ss) by the U2 pathway. In the U2 pathway, the U2 cofactor (U2AF) binds to the 3'ss region. U2 facilitates the binding of U2 to branching point sequences (BPS). U2AF is a stable heterodimer consisting of a 65kD subunit (U2AF65) encoded by U2AF2, which binds to polypyrimidine tracts (PPT), and a 35kD subunit (U2AF35) encoded by U2AF1, which interacts with the highly conserved AG dinucleotide at 3'ss and stabilizes U2AF65 binding. Accurate splicing requires, in addition to the BPS / PPT unit and 3'ss / 5'ss, auxiliary sequences or structures known as introns or exon splicing enhancers or silencers that activate or suppress splice site recognition. These elements allow the true splice site to be recognized from among a very excessive number of potential or false sites in the genome of higher eukaryotes, which have the same sequence but are 10 times more numerous than the original site. While the elements often have a regulatory function, the exact mechanisms of their activation or suppression are not well understood.

[0008]

[0048] The decision of whether or not to splice can typically be modeled as a stochastic rather than a deterministic process, as even the clearest splicing signals can occasionally lead to missplicing. However, under normal conditions, mRNA precursor splicing occurs with surprisingly high fidelity. This is thought to be partly due to the activity of adjacent cis-active accessory exons and introns, which act as splicing regulatory elements (ESRs or ISRs). Typically, these functional elements are classified as either exon or intron splicing enhancers (ESEs or ISEs) or silencers (ESSs or ISSs), respectively, based on their ability to stimulate or inhibit splicing. Currently, there is evidence that some accessory cis-active elements may act by influencing the dynamics of spliceosome assembly, for example, the arrangement of the complex between U1 snRNP and 5'ss, but it seems very likely that many elements function in coordination with trans-active RNA-binding proteins (RBPs). For example, the serine and arginine-rich family of RBPs (SR proteins) is a conserved family of proteins that play a crucial role in defining exons. SR proteins facilitate exon recognition by recruiting components of the press-splicosome to adjacent splice sites or by weakening the effect of ESS in their vicinity. The repressive effect of ESS may be mediated by members of the heteronuclear ribonucleoprotein (hnRNP) family, which can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their role in splicing regulation, silencer elements are suggested to play a role in repressing pseudoexons, which are several pairs of decoy intron splice sites that have typical exon spacing but lack a functional open reading frame. ESEs and ESSs, along with their transactive RBP relatives, represent key components of a set of splicing regulators that specify how, where, and when mRNA is assembled from its precursor.

[0009]

[0049] Sequences indicating exon-intron boundaries are degenerate signals of varying strengths that can occur frequently within human genes. In polyexon genes, different pairs of splice sites can link to each other in many different combinations, allowing a wide variety of transcripts to be produced from a single gene. This is generally referred to as alternative mRNA precursor splicing. While most mRNA isoforms produced by alternative splicing can be transported from the nucleus and translated into functional polypeptides, different mRNA isoforms originating from a single gene can exhibit significant variability in their translation efficiency. These mRNA isoforms, which have an immature stop codon (PTC) at least 50 bp upstream of the exon junction complex, are likely to be targeted for degradation by the nonsense mutation-dependent mRNA degradation (NMD) pathway. Mutations in traditional (BPS / PPT / 3'ss / 5'ss) and auxiliary splicing motifs can lead to abnormal splicing, e.g., exon splicing Kipping, or latent (or false) exon inclusion or splice site activation, can occur and be a significant contributing factor to human morbidity and mortality. Both abnormal and alternative splicing patterns can be influenced by native DNA variants in exons and introns.

[0010]

[0050] Given that exon-intron boundaries can occur at any of the three codon positions, it is clear that only a subset of alternative splicing events can maintain a canonical open reading frame. For example, only exons divisible by 3 can be skipped or incorporated into the mRNA without any alteration to the reading frame. Splicing events that do not have a suitable phase can induce a frameshift. Unless canceled by a downstream event, a frameshift will almost certainly result in one or more PTCs, and likely subsequently degraded by an NMD. The NMD is a translation-coupled mechanism that excludes mRNA containing PTCs. The NMD can function as a surveillance pathway present in all eukaryotes. By excluding mRNA transcripts containing immature stop codons, the NMD can reduce failures in gene expression. Translation of these abnormal mRNAs can, in some cases, result in harmful gain-of-function or dominant-negative activity of the resulting protein. NMD targets not only transcripts containing PTCs but also a wide range of mRNA isoforms expressed from many endogenous genes, suggesting that NMD is a major regulator driving both fine and coarse-scale regulation at the steady-state RNA level in cells.

[0011]

[0051] NMD-inducing exons (NIEs) are pseudoexons, which are regions within exons or introns, that can activate the NMD pathway when included in mature RNA transcripts. In constitutive splicing events, introns containing NIEs are typically excised, but during alternative or aberrant splicing events, the intron or its portion (e.g., NIE) may be retained. Mature mRNA transcripts containing such NIEs may be unproductive due to frameshifts that induce the NMD pathway. Inclusion of NIEs in mature RNA transcripts can downregulate gene expression. mRNA transcripts containing NIEs may be referred to in this disclosure as "NIE-containing mRNA" or "NMD exon mRNA."

[0012]

[0052] Potential (or false splice) sites have the same splicing recognition sequence as true splice sites but are not used in the splicing reaction. They are 10 times more numerous than true splice sites in the human genome and are usually repressed by molecular mechanisms that are not yet fully understood. Potential 5' splice sites have a consensus NNN / GUNNNN or NNN / GCNNNN, where N is any nucleotide and / N is the exon-intron boundary. Potential 3' splice sites have a consensus NAG / N. Their activation is positively influenced by surrounding nucleotides that make them more similar to the optimal consensus of the original splice site, i.e., MAG / GURAGU and YAG / G, respectively, where M is C or A, R is G or A, and Y is C or U.

[0013]

[0053] The splice site and its control sequences can be identified by those skilled in the art, for example, by Kralovicova, J. and Vorechovsky, I. (2007) Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res., 35, pp. 6399-6413, (http: / / www.ncbi.nlm.nih.gov / pmc / articl They can be easily identified using the publicly available and suitable algorithms listed in es / PMC2095810 / pdf / gkm680.pdf.

[0014]

[0054] Potential splice sites or splicing regulatory sequences may compete with NIE splice sites for RNA-binding proteins such as U2AF. In one embodiment, a drug may bind to a potential splice site or splicing regulatory sequence to prevent the binding of RNA-binding proteins, thereby favoring the utilization of the NIE splice site.

[0015]

[0055] In one embodiment, the potential splice site may not include the 5' or 3' splice site of the NIE. The potential splice site may be at least 10 nucleotides upstream of the NIE 5' splice site. The potential splice site may be at least 20 nucleotides upstream of the NIE 5' splice site. The potential splice site may be at least 50 nucleotides upstream of the NIE 5' splice site. The potential splice site may be at least 100 nucleotides upstream of the NIE 5' splice site. The potential splice site may be at least 200 nucleotides upstream of the NIE 5' splice site.

[0016]

[0056] The potential splice site may be at least 10 nucleotides downstream of the NIE3' splice site. The potential splice site may be at least 20 nucleotides downstream of the NIE3' splice site. The potential splice site may be at least 50 nucleotides downstream of the NIE3' splice site. The potential splice site may be at least 100 nucleotides downstream of the NIE3' splice site. The potential splice site may be at least 200 nucleotides downstream of the NIE3' splice site. Target transcript

[0057] In some embodiments, the methods of this disclosure utilize the presence of NIE in mRNA precursors transcribed from the SCN1A gene. Splicing of identified SCN1A NIE mRNA precursor species that produce functional mature SCN1A mRNA can be induced using therapeutic agents such as ASO that stimulate exon skipping of NIE. Induction of exon skipping can result in inhibition of the NMD pathway. The resulting mature SCN1A mRNA is translated, usually without activating the NMD pathway, thereby increasing the amount of SCN1A protein in patient cells and alleviating the symptoms of conditions associated with SCN1A deficiency, such as Dravet syndrome (DS); generalized febrile epilepsy plus type 2; familial febrile seizures; autism; early infantile epileptic encephalopathy; sick sinus syndrome; Alzheimer's disease; or SUDEP.

[0017]

[0058] In various embodiments, the Disclosure provides therapeutic agents that can target SCN1A mRNA transcripts to modulate, for example, enhance or inhibit splicing or protein expression levels. The therapeutic agents may be small molecules, polynucleotides, or polypeptides. In some embodiments, the therapeutic agent is an ASO. Various regions or sequences of the SCN1A mRNA precursor may be targeted by the therapeutic agent, e.g., the ASO. In some embodiments, the ASO targets an SCN1A mRNA precursor transcript containing an NIE. In some embodiments, the ASO targets a sequence within the NIE of the SCN1A mRNA precursor transcript. In some embodiments, the ASO targets a sequence upstream (or 5') of the NIE of the SCN1A mRNA precursor transcript from the 5' end (3'ss). In some embodiments, the ASO targets a sequence downstream (or 3') of the NIE of the SCN1A mRNA precursor transcript from the 3' end (5'ss). In some embodiments, the ASO targets a sequence within an intron adjacent to the 5' end of the NIE of the SCN1A mRNA precursor transcript. In some embodiments, the ASO targets a sequence in the intron adjacent to the 3' end of the NIE of the SCN1A mRNA precursor transcript. In some embodiments, the ASO targets the NIE-intron of the SCN1A mRNA precursor transcript. The ASO targets sequences that include NIE boundaries. The NIE-intron boundary may refer to the junction between an intron sequence and an NIE region. The intron sequence may be adjacent to the 5' end or the 3' end of the NIE. In some embodiments, the ASO targets sequences within exons of the SCN1A mRNA precursor transcript. In some embodiments, the ASO targets sequences within introns of the SCN1A mRNA precursor transcript. In some embodiments, the ASO targets sequences that include both a portion of an intron and a portion of an exon.

[0018]

[0059] In some embodiments, the therapeutic agents described herein modulate the binding of factors involved in the splicing of NMD exon mRNA.

[0060] In some embodiments, the therapeutic agents described herein interfere with the binding of factors involved in the splicing of NMD exon mRNA.

[0019]

[0061] In some embodiments, the therapeutic agents described herein interfere with the binding of factors involved in the splicing of NMD exon mRNA.

[0062] In some embodiments, the therapeutic agent targets a targeting region located in the intron region between two canonical exon regions of the NMD exon mRNA encoding SCN1A, where the intron region contains an NMD exon.

[0020]

[0063] In some embodiments, the therapeutic agent targets a targeting region that at least partially overlaps with the NMD exon.

[0064] In some embodiments, the therapeutic agent targets a targeting region that at least partially overlaps with an intron upstream of an NMD exon.

[0021]

[0065] In some embodiments, the therapeutic agent targets a targeting region within the NMD exon.

[0066] In some embodiments, the therapeutic agent targets a targeting region of the NMD exon that contains at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides. In some embodiments, the therapeutic agent targets a targeting region of the NMD exon that contains up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides. In some embodiments, the therapeutic agent targets a targeting region of the NMD exon that contains approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides.

[0022]

[0067] In some embodiments, the therapeutic agent targets a targeting region located proximal to the NMD exon.

[0068] In some embodiments, the ASO targets a sequence (or 5') approximately 4 to 300 nucleotides upstream from the 5' end of the NIE. In some embodiments, the ASO is located approximately 1 to 20 nucleotides from the 5' end of the NIE region, approximately 20 to 50 nucleotides, approximately 50 to 100 nucleotides, approximately 100 to 150 nucleotides, approximately 150 to 200 nucleotides, approximately 200 to 250 nucleotides, approximately 250 to 300 nucleotides, approximately 250 to 300 nucleotides, approximately 350 to 400 nucleotides, approximately 450 to 500 nucleotides, approximately 550 to 600 nucleotides, approximately 650 to 700 nucleotides, approximately 750 to 800 nucleotides, approximately 850 to 900 nucleotides, approximately 950 to 1000 nucleotides, approximately 1050 to 1100 nucleotides, approximately 1150 to 1200 nucleotides, approximately 1250 to 1300 nucleotides, and approximately 1350 nucleotides. The target is approximately 1400 nucleotides upstream, or approximately 1450 to 1500 nucleotides upstream (or 5'). In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5' end of the NIE. In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides downstream from the 3' end of the NIE (or 3'). In some embodiments, the ASO targets a sequence downstream of approximately 1 to approximately 20 nucleotides, approximately 20 to approximately 50 nucleotides, approximately 50 to approximately 100 nucleotides, approximately 100 to approximately 150 nucleotides, approximately 150 to approximately 200 nucleotides, approximately 200 to approximately 250 nucleotides, approximately 250 to approximately 300 nucleotides, approximately 350 to approximately 400 nucleotides, approximately 450 to approximately 500 nucleotides, approximately 550 to approximately 600 nucleotides, approximately 650 to approximately 700 nucleotides, approximately 750 to approximately 800 nucleotides, approximately 850 to approximately 900 nucleotides, approximately 950 to approximately 1000 nucleotides, approximately 1050 to approximately 1100 nucleotides, approximately 1150 to approximately 1200 nucleotides, approximately 1250 to approximately 1300 nucleotides, approximately 1350 to approximately 1400 nucleotides, or approximately 1450 to approximately 1500 nucleotides from the 3' end of the NIE. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3' end of the NIE.

[0023]

[0069] In some embodiments, the ASO targets a sequence (or 5') approximately 4 to 300 nucleotides upstream from the 5' end of the NIE. In some embodiments, the ASO targets a sequence (or 5') at least about 1 nucleotide, at least about 10 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 96 nucleotides, at least about 97 nucleotides, at least about 98 nucleotides, at least about 99 nucleotides, at least about 100 nucleotides, at least about 101 nucleotides, at least about 102 nucleotides, at least about 103 nucleotides, at least about 104 nucleotides, at least about 105 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, or at least about 1000 nucleotides upstream from the 5' end of the NIE region. In some embodiments, the ASO targets a sequence (or 3') approximately 4 to 300 nucleotides downstream from the 3' end of the NIE.In some embodiments, the ASO targets a sequence at least about 1 nucleotide, at least about 10 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 96 nucleotides, at least about 97 nucleotides, at least about 98 nucleotides, at least about 99 nucleotides, at least about 100 nucleotides, at least about 101 nucleotides, at least about 102 nucleotides, at least about 103 nucleotides, at least about 104 nucleotides, at least about 105 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, or at least about 1000 nucleotides downstream from the 3' end of the NIE. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3' end of the NIE.

[0024]

[0070] In some embodiments, the ASO targets a sequence (or 5') approximately 4 to 300 nucleotides upstream from the 5' end of the NIE. In some embodiments, the ASO targets up to approximately 10 nucleotides, up to approximately 20 nucleotides, and up to 5' from the 5' end of the NIE region. The target is the upstream sequence (or 5') of approximately 50 nucleotides, up to approximately 80 nucleotides, up to approximately 85 nucleotides, up to approximately 90 nucleotides, up to approximately 95 nucleotides, up to approximately 96 nucleotides, up to approximately 97 nucleotides, up to approximately 98 nucleotides, up to approximately 99 nucleotides, up to approximately 100 nucleotides, up to approximately 101 nucleotides, up to approximately 102 nucleotides, up to approximately 103 nucleotides, up to approximately 104 nucleotides, up to approximately 105 nucleotides, up to approximately 110 nucleotides, up to approximately 120 nucleotides, up to approximately 150 nucleotides, up to approximately 200 nucleotides, up to approximately 300 nucleotides, up to approximately 400 nucleotides, up to approximately 500 nucleotides, up to approximately 600 nucleotides, up to approximately 700 nucleotides, up to approximately 800 nucleotides, up to approximately 900 nucleotides, up to approximately 1000 nucleotides, up to approximately 1100 nucleotides, up to approximately 1200 nucleotides, up to approximately 1300 nucleotides, up to approximately 1400 nucleotides, or up to approximately 1500 nucleotides. In some embodiments, the ASO targets a sequence (or 3') approximately 4 to 300 nucleotides downstream from the 3' end of the NIE. In some embodiments, the ASO targets up to approximately 10 nucleotides, up to approximately 20 nucleotides, up to approximately 50 nucleotides, up to approximately 80 nucleotides, up to approximately 85 nucleotides, up to approximately 90 nucleotides, up to approximately 95 nucleotides, up to approximately 96 nucleotides, up to approximately 97 nucleotides, up to approximately 98 nucleotides, up to approximately 99 nucleotides, up to approximately 100 nucleotides, up to approximately 101 nucleotides, up to approximately 102 nucleotides, up to approximately 103 nucleotides, up to approximately 104 nucleotides, up to approximately 105 nucleotides, up to approximately 1 The target is a sequence downstream of 10 nucleotides, up to approximately 120 nucleotides, up to approximately 150 nucleotides, up to approximately 200 nucleotides, up to approximately 300 nucleotides, up to approximately 400 nucleotides, up to approximately 500 nucleotides, up to approximately 600 nucleotides, up to approximately 700 nucleotides, up to approximately 800 nucleotides, up to approximately 900 nucleotides, or up to approximately 1000 nucleotides, up to approximately 1100 nucleotides, up to approximately 1200 nucleotides, up to approximately 1300 nucleotides, up to approximately 1400 nucleotides, or up to approximately 1500 nucleotides.In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3' end of the NIE.

[0025]

[0071] In some embodiments, the NIE described herein is located between GRCh37 / hg19:chr2:166,863,740 and GRCh37 / hg19:chr2:166,863,803, as shown in Figure 2. In some embodiments, the 5' end of the NIE is located at GRCh37 / hg19:chr2:166,863,803. In some embodiments, the 3' end of the NIE is located at GRCh37 / hg19:chr2:166,863,740.

[0026]

[0072] In some embodiments, the ASO targets a sequence (or 5') approximately 4 to 300 nucleotides upstream from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 250 to 300 nucleotides, 350 to 400 nucleotides, and 450 to 500 nucleotides upstream from the genomic site GRCh37 / hg19:chr2:166,863,803. Otide targets sequences (or 5') approximately 550 to 600 nucleotides upstream, approximately 650 to 700 nucleotides, approximately 750 to 800 nucleotides, approximately 850 to 900 nucleotides, approximately 950 to 1000 nucleotides upstream, approximately 1050 to 1100 nucleotides, approximately 1150 to 1200 nucleotides upstream, approximately 1250 to 1300 nucleotides upstream, approximately 1350 to 1400 nucleotides upstream, or approximately 1450 to 1500 nucleotides upstream. In some embodiments, the ASO may target sequences more than 300 nucleotides upstream from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets sequences approximately 4 to 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740. The sequence (or 3') of the flow is targeted. In some embodiments, the ASO is derived from GRCh37 / hg19:chr2:166,863,740 to approximately 1 to approximately 20 nucleotides, approximately 20 to approximately 50 nucleotides, approximately 50 to approximately 100 nucleotides, approximately 100 to approximately 150 nucleotides, approximately 150 to approximately 200 nucleotides, approximately 200 to approximately 250 nucleotides, approximately 250 to approximately 300 nucleotides, approximately 350 to approximately 400 nucleotides, approximately 450 to approximately 500 nucleotides, The ASO targets sequences approximately 550 to 600 nucleotides downstream, approximately 650 to 700 nucleotides, approximately 750 to 800 nucleotides, approximately 850 to 900 nucleotides, approximately 950 to 1000 nucleotides downstream, approximately 1050 to 1100 nucleotides, approximately 1150 to 1200 nucleotides downstream, approximately 1250 to 1300 nucleotides, approximately 1350 to 1400 nucleotides downstream, or approximately 1450 to 1500 nucleotides downstream. In some embodiments, the ASO targets sequences more than 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.

[0027]

[0073] In some embodiments, the ASO targets a sequence (or 5') approximately 4 to 300 nucleotides upstream from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence (or 5') at least approximately 1 nucleotide, at least approximately 10 nucleotides, at least approximately 20 nucleotides, at least approximately 50 nucleotides, at least approximately 80 nucleotides, at least approximately 85 nucleotides, at least approximately 90 nucleotides, at least approximately 95 nucleotides, at least approximately 96 nucleotides, at least approximately 97 nucleotides, at least approximately 98 nucleotides, at least approximately 99 nucleotides, at least approximately 100 nucleotides, at least approximately 101 nucleotides, at least approximately 102 nucleotides, at least approximately 103 nucleotides, at least approximately 104 nucleotides, at least approximately 105 nucleotides, at least approximately 110 nucleotides, at least approximately 120 nucleotides, at least approximately 150 nucleotides, at least approximately 200 nucleotides, at least approximately 300 nucleotides, at least approximately 400 nucleotides, at least approximately 500 nucleotides, at least approximately 600 nucleotides, at least approximately 700 nucleotides, at least approximately 800 nucleotides, at least approximately 900 nucleotides, or at least approximately 1000 nucleotides upstream of the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence (or 3') approximately 4 to 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.In some embodiments, the ASO targets a sequence downstream of at least about 1 nucleotide, at least about 10 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 96 nucleotides, at least about 97 nucleotides, at least about 98 nucleotides, at least about 99 nucleotides, at least about 100 nucleotides, at least about 101 nucleotides, at least about 102 nucleotides, at least about 103 nucleotides, at least about 104 nucleotides, at least about 105 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, or at least about 1000 nucleotides from GRCh37 / hg19:chr2:166,863,740. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.

[0028]

[0074] In some embodiments, the ASO targets a sequence (or 5') approximately 4 to 300 nucleotides upstream from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets the genomic site GRCh37 / hg From 19:chr2:166,863,803, up to approximately 10 nucleotides, up to approximately 20 nucleotides, up to approximately 50 nucleotides, up to approximately 80 nucleotides, up to approximately 85 nucleotides, up to approximately 90 nucleotides, up to approximately 95 nucleotides, up to approximately 96 nucleotides, up to approximately 97 nucleotides, up to approximately 98 nucleotides, up to approximately 99 nucleotides, up to approximately 100 nucleotides, up to approximately 101 nucleotides, up to approximately 102 nucleotides, up to approximately 103 nucleotides, up to approximately 104 nucleotides, up to approximately 105 nucleotides, up to approximately 110 nucleotides The rheotide targets a sequence (or 5') up to approximately 120 nucleotides, 150 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 1100 nucleotides, 1200 nucleotides, 1300 nucleotides, 1400 nucleotides, or up to approximately 1500 nucleotides upstream. In some embodiments, the ASO targets a sequence (or 3') up to approximately 4 to 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.In some embodiments, ASO is derived from GRCh37 / hg19:chr2:166,863,740 in a range of up to approximately 10 nucleotides, up to approximately 20 nucleotides, up to approximately 50 nucleotides, up to approximately 80 nucleotides, up to approximately 85 nucleotides, up to approximately 90 nucleotides, up to approximately 95 nucleotides, up to approximately 96 nucleotides, up to approximately 97 nucleotides, up to approximately 98 nucleotides, up to approximately 99 nucleotides, up to approximately 100 nucleotides, up to approximately 101 nucleotides, up to approximately 102 nucleotides, up to approximately 103 nucleotides, up to approximately 104 nucleotides, and up to approximately 105 nucleotides. The ASO targets sequences downstream of nucleotides, up to approximately 110 nucleotides, up to approximately 120 nucleotides, up to approximately 150 nucleotides, up to approximately 200 nucleotides, up to approximately 300 nucleotides, up to approximately 400 nucleotides, up to approximately 500 nucleotides, up to approximately 600 nucleotides, up to approximately 700 nucleotides, up to approximately 800 nucleotides, up to approximately 900 nucleotides, or up to approximately 1000 nucleotides, up to approximately 1100 nucleotides, up to approximately 1200 nucleotides, up to approximately 1300 nucleotides, up to approximately 1400 nucleotides, or up to approximately 1500 nucleotides. In some embodiments, the ASO targets sequences more than 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.

[0029]

[0075] As described in the examples herein, the SCN1A gene (SEQ ID NO: 1) was analyzed for NIE and the inclusion of a portion of intron 20 (SEQ ID NO: 4) (this portion is referred to as exon 20x throughout this disclosure) was observed. In some embodiments, the ASO disclosed herein targets an NIE-containing mRNA precursor (SEQ ID NO: 2) transcribed from the SCN1A genomic sequence. In some embodiments, the ASO targets an NIE-containing mRNA precursor transcript derived from the SCN1A genomic sequence containing a portion of intron 20. In some embodiments, the ASO targets an NIE-containing mRNA precursor transcript derived from the SCN1A genomic sequence containing exon 20x (SEQ ID NO: 6). In some embodiments, the ASO targets an NIE-containing mRNA precursor transcript of SEQ ID NO: 2 or 12. In some embodiments, the ASO targets an NIE-containing mRNA precursor transcript of SEQ ID NO: 2 or 12 containing NIE. In some embodiments, the ASO targets an NIE-containing mRNA precursor transcript of SEQ ID NO: 2 containing exon 20x (SEQ ID NO: 10). In some embodiments, the ASO disclosed herein targets an SCN1A mRNA precursor sequence (SEQ ID NO: 2 or 12). In some embodiments, the ASO targets an SCN1A mRNA precursor sequence containing an NIE (SEQ ID NO: 10 or 20). In some embodiments, the ASO targets an SCN1A mRNA precursor sequence described in any one of SEQ ID NOs: 7-10 or 17-20. In some embodiments, the ASO has any one of the sequences described in SEQ ID NOs: 21-67. In some embodiments, the ASO has any one of the sequences described in SEQ ID NOs: 68-114. In some embodiments, the ASO has any one of the sequences described in SEQ ID NOs: 115-209. In some embodiments, the ASO has the sequence described in any one of sequence numbers 210 to 256. In some embodiments, the ASO has the sequence described in any one of sequence numbers 257 to 303. In some embodiments, the ASO has the sequence described in any one of sequence numbers 304 to 341. In some embodiments, the ASO has the sequence described in any one of sequence numbers 342 to 379.

[0030]

[0076] In some embodiments, the SCN1A NIE-containing mRNA precursor transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 1 or 11. In some embodiments, the SCN1A NIE mRNA precursor transcript contains a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs. 2-10 and 12-20.

[0031]

[0077] In some embodiments, the SCN1A NIE-containing mRNA precursor transcript (or NMD exon mRNA) contains a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 2, 7-10, 12, and 17-20. In some embodiments, the SCN1A NIE-containing mRNA precursor transcript (or NMD exon mRNA) is encoded by a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs: 1, 3-6, 11, and 13-16. In some embodiments, the targeted portion of the NMD exon mRNA contains a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least eight consecutive nucleic acids of SEQ ID NOs: 2, 7-10, 12, and 17-20.

[0032]

[0078] In some embodiments, the ASO targets exon 20 of the SCN1A NIE-containing mRNA precursor containing NIE exon 20x. In some embodiments, the ASO targets the exon 21 sequence (or 3') downstream of NIE exon 20x. In some embodiments, the ASO targets the sequence (or 5') about 4 to 300 nucleotides upstream from the 5' end of exon 20x. In some embodiments, the ASO targets the sequence (or 3') about 4 to 300 nucleotides downstream from the 3' end of exon 20x. In some embodiments, the ASO has the sequence described in any one of SEQ ID NOs. 210 to 67. In some embodiments, the ASO has the sequence described in any one of SEQ ID NOs. 210 to 256.

[0033]

[0079] In some embodiments, the ASO targets a sequence upstream of the 5' end of the NIE. For example, an ASO targeting a sequence upstream of the 5' end of an NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any one of sequence numbers 21-38. In another example, an ASO targeting a sequence upstream of the 5' end of an NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any one of sequence numbers 68-85. In some embodiments, the ASO targets a sequence containing an exon-intron boundary (or junction). For example, an ASO targeting a sequence containing an exon-intron boundary may include a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 39-41, 51, 52, 228-230, 240, or 241. In another example, an ASO targeting a sequence containing an exon-intron boundary may have at least one sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 86-88 and 98-99. The ASO may also include sequences having 80%, 85%, 90%, 95%, 97%, or 100% sequence identity. In some embodiments, the ASO targets sequences downstream from the 3' end of the NIE. For example, an ASO targeting sequences downstream from the 3' end of an NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include sequences having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 53-67. In another example, an ASO targeting sequences downstream from the 3' end of an NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include sequences having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 100-114. In some embodiments, the ASO targets a sequence within the NIE. For example, an ASO targeting a sequence within the NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any one of sequence numbers 42-50 or 231-239. In another example, an ASO targeting a sequence within the NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any one of sequence numbers 89-97.

[0034]

[0080] In some embodiments, the ASO targets exon 20x in the SCN1A NIE-containing mRNA precursor containing exon 20x. In some embodiments, the ASO targets the exon 20x sequence (or 3') downstream from the 5' end of exon 20x in the SCN1A mRNA precursor. In some embodiments, the ASO targets SCN1A The target is the exon 20x sequence (or 5') upstream from the 3' end of exon 20x of the mRNA precursor.

[0035]

[0081] In some embodiments, the targeting region of the SCN1A NIE-containing mRNA precursor is located in introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 (the intron numbering corresponds to the mRNA sequence of NM_006920). In some embodiments, hybridization of the ASO to the targeting region of the NIE mRNA precursor results in exon skipping of at least one NIE within introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, which subsequently increases SCN1A protein production. In some embodiments, hybridization of the ASO to the targeted region of the NIE mRNA precursor inhibits or blocks the exon skipping of at least one NIE within introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and subsequently reduces SCN1A protein production. In some embodiments, the targeted region of the SCN1A NIE-containing mRNA precursor is located in intron 20. Those skilled in the art can determine the corresponding intron number in any isoform based on the intron sequences provided herein, or using the numbers provided for the mRNA sequences NM_006920, NM_001202435, NM_001165964, or NM_001165963. Those skilled in the art can also determine the sequences of adjacent exons in any SCN1A isoform to be targeted using the method of the present invention, based on the intron sequences provided herein, or using the intron numbers provided for the mRNA sequences NM_006920, NM_001202435, NM_001165964, or NM_001165963.

[0036]

[0082] In some embodiments, the methods and compositions of the present disclosure are used to regulate, e.g., increase or decrease, the expression of SCN1A by inducing or inhibiting exon skipping of a pseudoexon of an SCN1A NIE-containing mRNA precursor. In some embodiments, the pseudoexon is an internal sequence within any of introns 1-25. In some embodiments, the pseudoexon is an internal sequence within any of introns 2, 4, 6, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, and 25. In some embodiments, the pseudoexon is an internal sequence within any of introns 15, 18, and 19. In some embodiments, the pseudoexon can be any SCN1A intron or a portion thereof. In some embodiments, the pseudoexon is within intron 20. The numbering of SCN1A introns used herein corresponds to the mRNA sequence of NM_006920. It is understood that the intron numbering can vary when referring to different SCN1A isoform sequences. SCN1A protein

[0083] The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel, Na v 1.1, also sometimes referred to as SCN1A (sodium channel, voltage-gated, type I, alpha subunit) protein. As also described above, SCN1A mutations in DS spread throughout the protein. Over 100 novel mutations have been identified throughout the gene, and more debilitating mutations are de novo. These include those of the truncated type (47%), missense (43%), deletion (3%), and splice site mutations (7%). The percentage of subjects carrying SCN1A mutations varies between 33 and 100%. Most of the mutations are novel changes (88%).

[0037]

[0084] In some embodiments, the methods described herein are used to modulate, for example, increase or decrease, the production of functional SCN1A protein. As used herein, the term “functional” refers to the amount of activity or function of the SCN1A protein required to eliminate one or more symptoms of the condition being treated, e.g., Dravet syndrome; generalized epileptic febrile seizures plus type 2; familial febrile seizures; autism; early infantile epileptic encephalopathy; sick sinus syndrome; Alzheimer’s disease; or SUDEP. In some embodiments, the methods are used to increase the production of partially functional SCN1A protein. As used herein, the term “partially functional” refers to any amount of activity or function of the SCN1A protein less than the amount of activity or function required to eliminate or prevent one or more symptoms of the disease or condition. In some embodiments, a partially functional protein or RNA may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower activity compared to a fully functional protein or RNA.

[0038]

[0085] In some embodiments, the method is a method for increasing the expression of SCN1A protein by target cells having an NIE-containing mRNA precursor encoding the SCN1A protein, the target having Dravet syndrome caused by a deficiency in SCN1A protein activity, the deficiency of SCN1A protein caused by SCN1A protein haploinsufficiency. In such embodiments, the target has a first allele encoding a functional SCN1A protein and a second allele that does not produce SCN1A protein. In another such embodiment, the target has a first allele encoding a functional SCN1A protein and a second allele encoding a non-functional SCN1A protein. In yet another such embodiment, the target has a first allele encoding a functional SCN1A protein and a second allele encoding a partially functional SCN1A protein. In any of these embodiments, the antisense oligomer is the second It binds to the targeting region of the NIE-containing mRNA precursor transcribed from the allele, thereby inducing exon skipping of pseudoexons from the mRNA precursor, leading to an increase in the level of mature mRNA encoding the functional SCN1A protein and an increase in the expression of the SCN1A protein in the target cells.

[0039]

[0086] In a related embodiment, the method is a method for increasing the expression of a protein or functional RNA using an ASO. In some embodiments, ASO is used to increase the expression of the SCN1A protein in target cells having an NIE-containing mRNA precursor encoding the SCN1A protein, the target having a deficiency in the amount or function of the SCN1A protein, e.g., Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infants (SMEI) borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic apoptosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; pediatric alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; early infant SCN1A encephalopathy; early infant epileptic encephalopathy (EIEE); or autism. In some embodiments, ASO is used to increase the expression of SCN1A protein in target cells, and the subject has a deficiency in the amount or function of SCN8A protein, for example, early infant epileptic encephalopathy 13. In some embodiments, ASO is used to increase the expression of SCN1A protein in target cells, and the subject has a deficiency in the amount or function of SCN5A protein, for example, sick sinus syndrome 1.

[0040]

[0087] In some embodiments, NIE-containing mRNA precursor transcripts encoding a disease-causing protein are targeted by the ASO described herein. In some embodiments, NIE-containing mRNA precursor transcripts encoding a non-disease-causing protein are targeted by the ASO. For example, a disease resulting from a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting an NIE-containing mRNA precursor encoding a second protein, thereby increasing the production of the second protein. In some embodiments, the function of the second protein can compensate for a mutation or deficiency of the first protein (which causes the disease or condition).

[0041]

[0088] In some embodiments, the subject is (a) (i) SCN1A protein is produced at reduced levels compared to production from the wild-type allele. (ii) The SCN1A protein is produced in a form with reduced function compared to an equivalent wild-type protein, or (iii) SCN1A protein or functional RNA is not produced. The first variant allele, and (b) (i) The SCN1A protein is produced at a reduced level compared to production from the wild-type allele. (ii) The SCN1A protein is produced in a form with reduced function compared to an equivalent wild-type protein, or (iii) SCN1A protein is not produced Second mutant allele The NIE-containing mRNA precursor is transcribed from a first allele and / or a second allele. In these embodiments, the ASO binds to a targeting region of the NIE-containing mRNA precursor transcribed from the first or second allele, thereby inducing exon skipping of pseudoexons from the NIE-containing mRNA precursor and the SCN1A protein This results in an increase in the level of encoding mRNA and an increase in the expression of the target protein or functional RNA in the target cells. In these embodiments, the target protein or functional RNA having increased expression levels resulting from exon skipping of pseudoexons from the NIE-containing mRNA precursor is in a form that has reduced function (partial functionality) compared to an equivalent wild-type protein, or has full functionality (full functionality) compared to an equivalent wild-type protein.

[0042]

[0089] In some embodiments, the level of mRNA encoding the SCN1A protein is increased 1.1 to 10 times compared to the amount of mRNA encoding the SCN1A protein produced in control cells, for example, cells not treated with an antisense oligomer, or cells treated with an antisense oligomer that does not bind to the targeting region of the SCN1A NIE-containing mRNA precursor.

[0043]

[0090] In some embodiments, subjects treated using the methods of the present disclosure express a partially functional SCN1A protein from a single allele, and the partially functional SCN1A protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, subjects treated using the methods of the present invention express a nonfunctional SCN1A protein from a single allele, and the nonfunctional SCN1A protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion in a single allele. In some embodiments, subjects treated using the methods of the present invention have a total SCN1A gene deletion in a single allele.

[0044]

[0091] In some embodiments, the method is a method for reducing the expression of the SCN1A protein by target cells having an NIE-containing mRNA precursor encoding the SCN1A protein, wherein the target is Na v1.1 has a gain-of-function mutation. In such embodiments, the target is a mass-produced allele with elevated SCN1A, or a cell with Na v 1.1 has an allele encoding a mutant SCN1A that induces increased activity. In some embodiments, Na v 1.1 The increased activity is due to the mutant Na v 1.1 The channel is characterized by an extended or nearly persistent sodium current mediated by the channel, slowing of rapid inactivation, a positive shift to steady-state inactivation, higher channel utilization between repeated stimuli, increased non-inactivating depolarization-inducible persistent sodium current, delayed transition to inactivation, rapid recovery from rapid inactivation, and / or rescue of folding defects by incubation at lower temperatures or co-expression of interacting proteins. In any of these embodiments, the antisense oligomer binds to a targeting region of an NIE-containing mRNA precursor transcribed from a second allele, thereby inhibiting or blocking exon skipping of pseudoexons from the mRNA precursor, resulting in a decrease in the level of mature mRNA encoding a functional SCN1A protein and a decrease in the expression of the SCN1A protein in the target cells.

[0045]

[0092] In related embodiments, the method is a method for reducing the expression of a protein or functional RNA using an ASO. In some embodiments, the ASO is used to reduce the expression of the SCN1A protein in target cells having an NIE-containing mRNA precursor encoding the SCN1A protein. In some embodiments, the target is Na v Gain-of-function mutations in 1.1, e.g., migraine. In some embodiments, ASO is used to reduce the expression of the SCN1A protein in the target cells, and the target is Na v They have a gain-of-function variant as described in 1.1, for example, familial hemiplegic migraine 3.

[0046]

[0093] In some embodiments, the level of mRNA encoding the SCN1A protein is compared to control cells, e.g., cells not treated with antisense oligomers, or SC1A. Compared to the amount of mRNA encoding the SCN1A protein produced in cells treated with an antisense oligomer that does not bind to the targeting region of the N1A NIE-containing mRNA precursor, the amount of mRNA produced is reduced to 1 / 1.1 to 1 / 10.

[0047]

[0094] In some embodiments, the subject treated using the method of the present disclosure expresses a mutant SCN1A protein from a single allele, the mutant SCN1A protein being caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion, and the mutant SCN1A protein is Na v 1.1 This results in elevated activity levels. In some embodiments, subjects treated using the methods of the present disclosure express elevated amounts of SCN1A protein from a single allele due to frameshift mutations, nonsense mutations, missense mutations, or partial gene deletions.

[0048]

[0095] In embodiments of the present invention, the subject may have mutations in SCN1A. Mutations in SCN1A may spread throughout the entire gene. The SCN1A protein may consist of four domains. The SCN1A domain may have a transmembrane segment. Mutations in the SCN1A protein may occur throughout the entire protein. The SCN1A protein may consist of at least two isoforms. Mutations in SCN1A may include R931C, R946C, M934I, R1648C, or R1648H. In some cases, the mutation may be observed at the C-terminus of the SCN1A protein. Mutations in the SCN1A protein may also be found in the loop between segments 5 and 6 of the first three domains of the SCN1A protein. In some cases, the mutation may be observed at the N-terminus of the SCN1A protein. Examples of mutations within SCN1A include, but are not limited to, R222X, R712X, I227S, R1892X, W952X, R1245X, R1407X, W1434R, c.4338+1G>A, S1516X, L1670fsX1678, or K1846fsX1856. Mutations that can be targeted by the present invention may also encode ion channel pores.

[0049]

[0096] In some embodiments, the methods and compositions described herein can be used to treat DS. In other embodiments, the methods and compositions described herein can be used to treat severe myoclonic epilepsy of infants (SMEI). In other embodiments, the methods and compositions described herein can be used to treat borderline Dravet syndrome; generalized febrile epilepsy plus type 2; familial febrile seizures 3A; familial hemiplegic migraine 3; autism; early infantile epileptic encephalopathy 13; sick sinus syndrome 1; Alzheimer's disease, or SUDEP. The methods and compositions described herein can also be used to treat borderline SMEI. Furthermore, the methods and compositions described herein can be used to treat generalized febrile epilepsy plus (GEFS+). GEFS+ may be associated with mutations in epilepsy-related ion channel subunits such as SCN1B or GABRG2. The methods and compositions described herein can also be used to treat sodium channel disease. Sodium channel disease may be associated with mutations in SCN1A. Sodium channel disorders may also be associated with subunits of SCN1A, such as the beta subunit, SCN1B. In some cases, further diseases associated with SCN1A mutations may also be treated using the present disclosure. Related SCN1A diseases associated with SCN1A mutations include, but are not limited to, atypical congenital myotonia, hyperkalemic periodic paralysis, and congenital myotonia.

[0050]

[0097] In some embodiments, subjects having any SCN1A mutation known to those skilled in the art and described in the literature referenced above (e.g., by Hamdan et al., 2009, Mulley et al., 2005) are treated using the methods and compositions described herein. This is possible. In some embodiments, the mutation is located within any SCN1A intron or exon. Exon inclusion

[0098] As used herein, “NIE-containing mRNA precursor” is an mRNA precursor transcript containing at least one pseudoexon. Alternative or aberrant splicing can result in the inclusion of at least one pseudoexon in the mature mRNA transcript. The terms “mature mRNA” and “fully spliced ​​mRNA” are used interchangeably herein to refer to fully processed mRNA. Inclusion of at least one pseudoexon results in unproductive mRNA and can lead to NMD of mature mRNA. NIE-containing mature mRNA may occasionally result in abnormal protein expression.

[0051]

[0099] In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of NIE-containing mRNA precursors transcribed from a gene encoding a target protein in a cell. In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of NIE-containing mRNA precursors transcribed from a gene encoding a target protein in a cell, and the population of NIE-containing mRNA precursors contains two or more included pseudo-exons. In some embodiments, an antisense oligomer that targets the most abundant pseudo-exon in a population of NIE-containing mRNA precursors encoding a target protein induces exon skipping of one or more pseudo-exons in the population, including the pseudo-exon that the antisense oligomer targets or binds to. In embodiments, the targeting region is in a pseudo-exon that is the most abundant pseudo-exon in an NIE-containing mRNA precursor encoding the SCN1A protein.

[0052]

[0100] The degree of exon inclusion is the exon inclusion rate, for example, the degree to which a given pseudoexon is inclusion. It can be expressed as a percentage of the transcripts that are exon-containing. In short, the exon-containing percentage can be calculated as the percentage of the amount of RNA transcripts with exon-containing exons relative to the sum of the average amount of RNA transcripts with exon-containing exons and the average amount of RNA transcripts with exon exclusion exons.

[0053]

[0101] In some embodiments, the included pseudoexons are at least about 5%, at least Exons identified as containing pseudoexons are those with an inclusion rate of approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, or at least approximately 50%. In an embodiment, containing pseudoexons are those with an inclusion rate of approximately 5% to approximately 100%, approximately 5% to approximately 95%, approximately 5% to approximately 90%, approximately 5% to approximately 85%, approximately 5% to approximately 80%, approximately 5% to approximately 75%, approximately 5% to approximately 70%, approximately 5% to approximately 65%, approximately 5% to approximately 60%, approximately 5% to approximately 55%, approximately 5% to approximately 50%, approximately 5% to approximately 45%, approximately 5% to approximately 40%, or approximately 5% to approximately 35%. Approximately 5% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15%, approximately 10% to 100%, approximately 10% to 95%, approximately 10% to 90%, approximately 10% to 85%, approximately 10% to 80%, approximately 10% to 75%, approximately 10% to 70%, approximately 10% to 65%, approximately 10% to 60%, approximately 10% to 55%, approximately 10% to 50%, approximately 10% to approximately 45%, approximately 10% to approximately 40%, approximately 10% to approximately 35%, approximately 10% to approximately 30%, approximately 10% to approximately 25%, approximately 10% to approximately 20%, approximately 15% to approximately 100%, approximately 15% to approximately 95%, approximately 15% to approximately 90%, approximately 15% to approximately 85%, approximately 15% to approximately 80%, approximately 15% to approximately 75%, approximately 15% to approximately 70%, approximately 15% to approximately 65%, Approximately 15% to 60%, approximately 15% to 55%, approximately 15% to 50%, approximately 15% to 45%, approximately 15% to 40%, approximately 15% to 35%, approximately 15% to 30%, approximately 15% to 25%, approximately 20% to 100%, approximately 20% to 95%, approximately 20% to 90%, approximately 20% to 85%, approximately 20% to 80%, approximately 20% to 75%, approximately 20% These are exons identified as inclusion pseudoexons based on a determination of inclusion rates of approximately 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 25% to 100%, 25% to 95%, 25% to 90%, 25% to 85%, 25% to 80%, 25% to 75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 50%, 25% to 45%, 25% to 40%, or 25% to 35%. ENCODE data (for example, as described by Tilgner et al., 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs," Genome Research 22(9):16 pp. 1616-25) can be used to assist in the identification of exon inclusion.

[0054]

[0102] In some embodiments, cells are targeted by the SCN1A mRNA precursor transcript. Contact with an ASO complementary to the compounding moiety results in an increase in the amount of SCN1A protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence of ASO / treatment. In some embodiments, the total amount of SCN1A protein produced by cells contacted with the antisense oligomer is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, and about 2 to about The increase is 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. The control compound may be, for example, an oligonucleotide that is not complementary to the targeting region of the mRNA precursor.

[0055]

[0103] In some embodiments, cells are targeted by the SCN1A mRNA precursor transcript. Contact with an ASO complementary to the compounding moiety results in a reduction in the amount of SCN1A protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence of ASO / treatment. In some embodiments, the total amount of SCN1A protein produced by cells contacted with the antisense oligomer is approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, 1 / 1.1 to 1 / 9, 1 / 2 to 1 / 5, or 1 / 2 compared to the amount of target protein produced by the control compound. It decreases to about 1 / 6, about 1 / 2 to about 1 / 7, about 1 / 2 to about 1 / 8, about 1 / 2 to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1 / 1.1, at least about 1 / 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 5, or at least about 1 / 10. The substance could be, for example, an oligonucleotide that is not complementary to the targeting region of the mRNA precursor.

[0056]

[0104] In some embodiments, cells are targeted by the SCN1A mRNA precursor transcript. Contact with an ASO complementary to the compounding moiety results in an increase in the amount of mRNA encoding SCN1A, including mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding the SCN1A protein, or mature mRNA encoding the SCN1A protein, increases by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence of ASO / absence of treatment. In some embodiments, the total amount of mature mRNA encoding the SCN1A protein produced in cells contacted with mRNA encoding the SCN1A protein, or with an antisense oligomer, is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, and 1.5 times, respectively, compared to the amount of mature RNA produced in untreated cells, e.g., untreated cells or cells treated with a control compound. The increase is 1 to approximately 7 times, approximately 1.1 to approximately 8 times, approximately 1.1 to approximately 9 times, approximately 2 to approximately 5 times, approximately 2 to approximately 6 times, approximately 2 to approximately 7 times, approximately 2 to approximately 8 times, approximately 2 to approximately 9 times, approximately 3 to approximately 6 times, approximately 3 to approximately 7 times, approximately 3 to approximately 8 times, approximately 3 to approximately 9 times, approximately 4 to approximately 7 times, approximately 4 to approximately 8 times, approximately 4 to approximately 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. The control compound may be, for example, an oligonucleotide that is not complementary to the targeting portion of the SCN1A NIE-containing mRNA precursor.

[0057]

[0105] In some embodiments, cells are targeted by the SCN1A mRNA precursor transcript. Contact with an ASO complementary to the compounding moiety results in a reduction in the amount of SCN1A-encoding mRNA, including mature mRNA encoding the target protein. In some embodiments, the amount of SCN1A protein-encoding mRNA, or mature mRNA encoding SCN1A protein, is reduced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence of ASO / absence of treatment. In some embodiments, the total amount of mature mRNA encoding the SCN1A protein produced in cells contacted with mRNA encoding the SCN1A protein, or with an antisense oligomer, is approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, and 1 / 1. The amount decreases from 1 / 1 to about 1 / 9, from about 1 / 2 to about 1 / 5, from about 1 / 2 to about 1 / 6, from about 1 / 2 to about 1 / 7, from about 1 / 2 to about 1 / 8, from about 1 / 2 to about 1 / 9, from about 1 / 3 to about 1 / 6, from about 1 / 3 to about 1 / 7, from about 1 / 3 to about 1 / 8, from about 1 / 3 to about 1 / 9, from about 1 / 4 to about 1 / 7, from about 1 / 4 to about 1 / 8, from about 1 / 4 to about 1 / 9, at least about 1.1, at least about 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 5, or at least about 1 / 10. The control compound may be, for example, an oligonucleotide that is not complementary to the targeting portion of the SCN1A NIE-containing mRNA precursor.

[0058]

[0106] NIE can be of any length. In some embodiments, the NIE is an introduction. It contains the complete sequence of the intron, in which case it can be called intron retention. In some embodiments, the NIE may be a portion of an intron. In some embodiments, the NIE may be the 5' terminal portion of an intron containing a 5'ss sequence. In some embodiments, the NIE may be the 3' terminal portion of an intron containing a 3'ss sequence. In some embodiments, the NIE may be a portion within an intron that does not include a 5'ss sequence. In some embodiments, the NIE may be a portion within an intron that does not include a 3'ss sequence. In some embodiments, the NIE may be a portion within an intron that does not include either a 5'ss or a 3'ss sequence. In some embodiments, the NIE may be 5 to 10 nucleotides long, 10 to 15 nucleotides long, 15 to 20 nucleotides long, 20 to 25 nucleotides long, 25 to 30 nucleotides long, 30 to 35 nucleotides long, 35 to 40 nucleotides long, 40 to 45 nucleotides long, 45 to 50 nucleotides long, 50 to 55 nucleotides long, 55 to 60 nucleotides long, 60 to 65 nucleotides long, 65 to 70 nucleotides long, 70 to 75 nucleotides long, 75 to 80 nucleotides long, 80 to 85 nucleotides long, 85 to 90 nucleotides long, 90 to 95 nucleotides long, or 95 to 100 nucleotides long. In some embodiments, the NIE may be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleoids, at least 70 nucleotides, at least 80 nucleotides in length, at least 90 nucleotides, or at least 100 nucleotides in length.In some embodiments, the NIE may be 100 to 200 nucleotides long, 200 to 300 nucleotides long, 300 to 400 nucleotides long, 400 to 500 nucleotides long, 500 to 600 nucleotides long, 600 to 700 nucleotides long, 700 to 800 nucleotides long, 800 to 900 nucleotides long, or 900 to 1,000 nucleotides long. In some embodiments, the NIE may be longer than 1,000 nucleotides long.

[0059]

[0107] Pseudoexon inclusion leads to frameshift and immature mRNA transcripts. This can lead to the introduction of a stop codon (PIC), making the transcript a target for NMD. Mature mRNA transcripts containing an NIE may be non-productive mRNA transcripts that do not result in protein expression. The PIC can be located at any position downstream of the NIE. In some embodiments, the PIC can be located in any exon downstream of the NIE. In some embodiments, the PIC can be located within the NIE. For example, inclusion of exon 20x into an mRNA transcript encoded by the SCN1A gene can induce the PIC in the mRNA transcript, for example, in exon 21 of the mRNA transcript. Therapeutic drugs

[0108] In various embodiments of this disclosure, the protein expression level of SCN1A is regulated. A composition and method comprising a therapeutic agent is provided. In some embodiments, SCNA1 Compositions and methods for modulating alternative splicing of mRNA precursors are provided herein. In some embodiments, compositions and methods for inducing exon skipping in the splicing of SCN1A mRNA precursors, for example, for inducing pseudoexon skipping during the splicing of SCN1A mRNA precursors, are provided herein. In other embodiments, therapeutic agents may be used to induce exon inclusion and reduce protein expression levels.

[0060]

[0109] In some embodiments, the therapeutic agents disclosed herein are low molecular weight, polypeptide It is a cytoplasm, or polynucleic acid polymer. In some cases, the therapeutic agent is a small molecule. In some cases, the therapeutic agent is a polypeptide. In some cases, the therapeutic agent is a polynucleic acid polymer. In some cases, the therapeutic agent is an inhibitor. In even more cases, the therapeutic agent is an accelerator.

[0061]

[0110] The therapeutic agents disclosed herein may be NIE inhibitors. The therapeutic agents are polynucleic acids. It may contain polymers.

[0111] According to one aspect of this disclosure, conditions associated with functional SCN1A protein deficiency Methods for treating or preventing a condition are provided herein, comprising the step of administering an NIE inhibitor to a target to increase the level of functional SCN1A protein, wherein the agent binds to a region of the mRNA precursor transcript and reduces the inclusion of NIE in the mature transcript. For example, methods for treating or preventing a condition associated with functional SCN1A protein deficiency are provided herein, comprising the step of administering an NIE inhibitor to a target to increase the level of functional SCN1A protein, wherein the agent binds to a region of an intron containing NIE in the mRNA precursor transcript (e.g., intron 20 in the human SCN1A gene), or to an NIE activation regulatory sequence in the same intron.

[0062]

[0112] When referring to reducing NIE inclusion in mature mRNA, the reduction is The reduction may be complete, e.g., 100%, or partial. The reduction may be clinically significant. The reduction / correction may be compared to the level of NIE inclusion in subjects without treatment, or to the amount of NIE inclusion in a similar population of subjects. The reduction / correction may be at least 10% less NIE inclusion than the average subject or subjects before treatment. The reduction may be at least 20% less NIE inclusion than the average subject or subjects before treatment. The reduction may be at least 40% less NIE inclusion than the average subject or subjects before treatment. The reduction may be at least 50% less NIE inclusion than the average subject or subjects before treatment. The reduction may be at least 60% less NIE inclusion than the average subject or subjects before treatment. The reduction may be at least 80% less NIE inclusion than the average subject or subjects before treatment. The reduction may be at least 90% less NIE inclusion than the average subject or subjects before treatment.

[0063]

[0113] When referring to increasing the level of active SCN1A protein, The increase may be clinically significant. The increase may be compared to the level of active SCN1A protein in untreated subjects or to the amount of active SCN1A protein in a similar population of subjects. The increase may be at least 10% more active SCN1A protein than the average subject or the pre-treatment subject. The increase may be at least 20% more active SCN1A protein than the average subject or the pre-treatment subject. The increase may be at least 40% more active SCN1A protein than the average subject or the pre-treatment subject. The increase may be at least 50% more active SCN1A protein than the average subject or the pre-treatment subject. The increase may be at least 80% more active SCN1A protein than the average subject or the pre-treatment subject. The increase may be at least 100% more active SCN1A protein than the average subject or the pre-treatment subject. The increase may be at least 200% more active SCN1A protein than the average subject or the pre-treatment subject. The increase may be at least 500% higher than that of the average subject or the subject before treatment, in terms of active SCN1A protein.

[0064]

[0114] In embodiments in which the NIE inhibitor contains a polynucleic acid polymer, It can be approximately 50 nucleotides long. A polynucleotide polymer can be approximately 45 nucleotides long. A polynucleotide polymer can be approximately 40 nucleotides long. A polynucleotide polymer can be approximately 35 nucleotides long. A polynucleotide polymer can be approximately 30 nucleotides long. A polynucleotide polymer can be approximately 24 nucleotides long. A polynucleotide polymer can be approximately 25 nucleotides long. Polynucleotide polymers can be approximately 20 nucleotides long. Polynucleotide polymers can be approximately 19 nucleotides long. Polynucleotide polymers can be approximately 18 nucleotides long. Polynucleotide polymers can be approximately 17 nucleotides long. Polynucleotide polymers can be approximately 16 nucleotides long. Polynucleotide polymers can be approximately 15 nucleotides long. Polynucleotide polymers can be approximately 14 nucleotides long. Polynucleotide polymers can be approximately 13 nucleotides long. Polynucleotide polymers can be approximately 12 nucleotides long. Polynucleotide polymers can be approximately 11 nucleotides long. Polynucleotide polymers can be approximately 10 nucleotides long. Polynucleotide polymers can be between approximately 10 and approximately 50 nucleotides long. Polynucleotide polymers can be between approximately 10 and approximately 45 nucleotides long. Polynucleotide polymers can be between approximately 10 and approximately 40 nucleotides long. Polynucleotide polymers can be between approximately 10 and approximately 35 nucleotides long. Polynucleotide polymers can be between approximately 10 and approximately 30 nucleotides long. Polynucleotide polymers can be between approximately 10 and approximately 25 nucleotides long. Polynucleotide polymers can be between approximately 10 and approximately 20 nucleotides long. Polynucleotide polymers can be between approximately 15 and approximately 25 nucleotides in length. Polynucleotide polymers can be between approximately 15 and approximately 30 nucleotides in length. Polynucleotide polymers can be between approximately 12 and approximately 30 nucleotides in length.

[0065]

[0115] The sequence of polynucleotide polymers is an mRNA transcript, for example, partially processed The sequence of the polynucleic acid polymer may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to the target sequence of the mRNA transcript.

[0066]

[0116] The sequence of the polynucleic acid polymer is four or fewer relative to the target sequence of the mRNA precursor transcript. The following mismatches may be present: The sequence of the polynucleic acid polymer may have three or fewer mismatches with respect to the target sequence of the mRNA precursor transcript. The sequence of the polynucleic acid polymer may have two or fewer mismatches with respect to the target sequence of the mRNA precursor transcript. The sequence of the polynucleic acid polymer may have one or fewer mismatches with respect to the target sequence of the mRNA precursor transcript. The sequence of the polynucleic acid polymer may not have any mismatches with respect to the target sequence of the mRNA precursor transcript.

[0067]

[0117] Polynucleic acid polymers specifically hybridize target sequences in mRNA precursor transcripts. For example, polynucleic acid polymers may have sequence complementarity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% to the target sequence of the mRNA precursor transcript. Hybridization can be carried out under highly stringent hybridization conditions.

[0068]

[0118] The polynucleic acid polymer is paired with a sequence selected from the group consisting of SEQ ID NOs. 21-67. The polynucleic acid polymer may have sequences with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity. The polynucleic acid polymer may have sequences with 100% sequence identity to sequences selected from the group consisting of SEQ ID NOs. 21 to 67. In some examples, the polynucleic acid polymer may have sequences with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to sequences selected from the group consisting of SEQ ID NOs. 68 to 114. In some cases, the polynucleic acid polymer may have a sequence that has 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 68 to 114.

[0069]

[0119] When referring to polynucleic acid polymer sequences, a person skilled in the art will know one or more positions Substitutions may be permitted, and two substitutions may be optionally permitted in the sequence, and it will be understood that the substitutions maintain the ability of the polynucleic acid polymer sequence to hybridize to the target sequence, or, if the substitutions are present in the target sequence, to be recognized as the target sequence. The criterion for sequence identity may be determined by BLAST sequence alignment using standard / initial parameters. For example, a sequence may have 99% identity and still function according to the disclosure. In another embodiment, a sequence may have 98% identity and still function according to the disclosure. In yet another embodiment, a sequence may have 95% identity and still function according to the disclosure. In yet another embodiment, a sequence may have 90% identity and still function according to the disclosure. Antisense oligomers

[0120] By binding to the target region of the SCN1A NIE-containing mRNA precursor, Compositions comprising antisense oligomers that induce exon skipping are provided herein. As used herein, the terms “ASO” and “antisense oligomer” are interchangeable and refer to oligomers, such as polynucleotides, containing nucleic acid bases that hybridize to a target nucleic acid sequence (e.g., an SCN1A NIE-containing mRNA precursor) by Watson-Crick base pairing or fluctuation base pairing (GU). ASOs may have a very close sequence complementary to the target sequence or very high complementarity (e.g., sufficient to bind to the target sequence and enhance splicing at the splice site). ASOs are designed to bind (hybridize) to a target nucleic acid (e.g., a targeted portion of an mRNA precursor transcript) and remain hybridized under physiological conditions. Typically, when an ASO hybridizes to a site other than the intended (targeted) nucleic acid sequence, it hybridizes to a limited number of non-target nucleic acid sequences (a small number of non-target nucleic acid sites). The design of the ASO may take into account the presence of the nucleic acid sequence of the targeted portion of the mRNA precursor transcript, or a sufficiently similar nucleic acid sequence, at other locations in the genome or cellular mRNA precursor or transcriptome, so as to limit the possibility of the ASO binding to other sites and causing “off-target” effects. Any antisense oligomer known in the art, for example, in PCT application no. PCT / US2014 / 054151, published as WO2015 / 035091, named “Reducing Nonsense-Mediated mRNA Decay,” which is incorporated herein by reference, can be used to carry out the method described herein.

[0070]

[0121] In some embodiments, the ASO is a target nucleic acid or NIE-containing mRNA precursor. It "specifically hybridizes" or is "specific" to the targeted part of the body. Typically, such hybridization occurs at temperatures substantially higher than 37°C, preferably at least 50°C, typically between 60°C and approximately 90°C. mThis is carried out. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, T m This is the temperature at which 50% of the target sequence hybridizes to complementary oligonucleotides.

[0071]

[0122] Oligomers, such as oligonucleotides, undergo hybridization with two strands. Single-stranded polynucleotides are "complementary" to each other when hybridization occurs in an antiparallel configuration. Double-stranded polynucleotides can be "complementary" to another polynucleotide if hybridization can occur between one strand of the first polynucleotide and the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion (e.g., percentage) of bases in the opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base pairing rules. The sequence of an antisense oligomer (ASO) does not need to be 100% complementary to the sequence of the target nucleic acid to hybridize. In certain embodiments, the ASO is at least 70%, at least 75%, and less complementary to the target region in the target nucleic acid sequence to be targeted. All can include sequence complementarity of 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. For example, an ASO in which 18 of the 20 nucleic acid bases of an oligomeric compound are complementary to the target region and therefore can specifically hybridize represents 90 percent complementarity. In this example, the remaining non-complementary nucleic acid bases may cluster together or have complementary nucleic acid bases scattered among them, and do not need to be continuous with respect to each other or to the complementary nucleic acid bases. The percentage of complementarity of an ASO with the region of the target nucleic acid can conventionally be determined using the BLAST program (basic local alignment search tool) and the PowerBLAST program (Altschul et al., J.Mol.Biol., 1990, pp. 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, pp. 649-656), which are known in the art.

[0072]

[0123] ASO does not need to hybridize to all nucleic acid bases in the target sequence. The nucleic acid bases that the ASO hybridizes may be continuous or discontinuous. The ASO may hybridize across one or more segments of the mRNA precursor transcript so that intervening or adjacent segments do not participate in the hybridization event (e.g., a loop or hairpin structure may be formed). In certain embodiments, the ASO hybridizes to discontinuous nucleic acid bases in the target mRNA precursor transcript. For example, the ASO may hybridize to nucleic acid bases in the mRNA precursor transcript that are separated by one or more nucleic acid bases that the ASO does not hybridize.

[0073]

[0124] The ASOs described herein are located in the target region of NIE-containing mRNA precursors. It includes nucleic acid bases that are complementary to the nucleic acid bases present. The term ASO includes oligonucleotides and any other oligomeric molecules that include nucleic acid bases that can hybridize to complementary nucleic acid bases of target mRNA, but do not include a sugar moiety, such as peptide nucleic acid (PNA). An ASO may include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding ones. The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides having modified or substituted sugar groups and / or a modified skeleton. In some embodiments, all nucleotides in the ASO are modified nucleotides. Chemical modifications of ASO or components of ASO that are compatible with the methods and compositions described herein may be obvious to those skilled in the art and can be found, for example, in U.S. Patent No. 8,258,109B2, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012 / 0190728, and Dias and Stein, Mol. Cancer Ther. 2002, pp. 347-355, which are incorporated herein by whole reference.

[0074]

[0125] One or more nucleic acid bases of ASO are adenine, guanine, cytosine, thyme The modified nucleic acid bases may be any naturally occurring unmodified nucleic acid base such as 5,6-dihydrouracil, or any synthetic or modified nucleic acid base that is sufficiently similar to the unmodified nucleic acid base to be able to form hydrogen bonds with the nucleic acid base present in the target mRNA precursor. Examples of modified nucleic acid bases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine (hydroxymethoylcytosine).

[0075]

[0126] The ASOs described herein also have a skeletal structure that connects the components of the oligomer. Includes. The terms "skeletal structure" and "oligomer linkage" can be used interchangeably and refer to the linkage between monomers in ASOs. In naturally occurring oligonucleotides, The skeleton includes a 3'-5' phosphodiester linkage connecting the sugar moieties of the oligomer. Examples of the skeleton structure or oligomer linkage of ASOs described herein include (but are not limited to) phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, and phosphoramidates. For example, LaPlanche et al., Nucleic Acids Res.14:9081 page (1986); Stec et al., J.Am.Chem.Soc.106:6077 page (1984), Stein et al., Nucleic Acids See Res.16:3209 (1988), Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the ASO skeleton does not contain phosphorus, but rather contains peptide bonds in linking groups, such as peptide nucleic acids (PNA), or carbamates, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the skeleton modification is phosphothioate linkage. In some embodiments, the skeleton modification is phosphoramidate linkage.

[0076]

[0127] In one embodiment, the phosphorus nucleotide linkage of the ASO backbone (phosphor The stereochemistry at each of the internucleotide linkages is random. In embodiments, the stereochemistry at each of the phosphonucleotide linkages of the ASO backbone is controlled and not random. For example, U.S. Patent Application Publication 2014 / 0194610, “Methods for the Synthesis of Functionalized Nucleic Acids,” incorporated herein by reference, describes a method for independently selecting the chirality at each phosphorus atom in a nucleic acid oligomer. In embodiments, the ASOs used in the methods of the present invention include ASOs having non-random phosphonucleotide linkages, including but not limited to any ASOs listed herein in Tables 5 and 6. In embodiments, the composition used in the methods of the present invention includes a pure diastereomer ASO. In an embodiment, the composition used in the method of the present invention comprises an ASO having a diastereomer purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0077]

[0128] In this embodiment, ASO has Rp and It has a non-random mixture of Rp and Sp configurations. For example, it has been suggested that a mixture of Rp and Sp is necessary in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan et al., 2014, "Synthesis, biophysical properties and biological activity of second "Generation antisense oligonucleotides containing chiral phosphorothioate linkages," Nucleic Acids Research 42(22): pp. 13456-13468). In embodiments, any ASO used in the method of the present invention may be, but is not limited to, those described herein in SEQ ID NOs. 21-114. SO includes approximately 5-100% Rp, at least approximately 5% Rp, at least approximately 10% Rp, at least approximately 15% Rp, at least approximately 20% Rp, at least approximately 25% Rp, at least approximately 30% Rp, at least approximately 35% Rp, at least approximately 40% Rp, at least approximately 45% Rp, at least approximately 50% Rp, at least approximately 55% Rp, at least approximately 60% Rp, at least approximately 65% ​​Rp, at least approximately 70% Rp, at least approximately 75% Rp, at least approximately 80% Rp, at least approximately 85% Rp, at least approximately 90% Rp, or at least approximately 95% Rp plus the remainder Sp, or approximately 100% Rp. Examples of ASOs used in the methods of the present invention include, but are not limited to, any ASOs described herein in Sequence IDs 21-114, and include ASOs with Rp values ​​of about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, and about 55% Including approximately 100% Rp, approximately 60% to approximately 100% Rp, approximately 65% ​​to approximately 100% Rp, approximately 70% to approximately 100% Rp, approximately 75% to approximately 100% Rp, approximately 80% to approximately 100% Rp, approximately 85% to approximately 100% Rp, approximately 90% to approximately 100% Rp, or approximately 95% to approximately 100% Rp, approximately 20% to approximately 80% Rp, approximately 25% to approximately 75% Rp, approximately 30% to approximately 70% Rp, approximately 40% to approximately 60% Rp, or approximately 45% to approximately 55% Rp and the remainder Sp.

[0078]

[0129] In embodiments, any AS described herein in Sequence IDs 21 to 114 ASO used in the method of the present invention includes, but is not limited to, O, about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp and the remainder Rp, or about 100% Sp. Examples of ASOs used in the methods of the present invention include, but are not limited to, any ASOs described herein in Sequence IDs 21-114, and include ASOs with approximately 10% to approximately 100% Sp, approximately 15% to approximately 100% Sp, approximately 20% to approximately 100% Sp, approximately 25% to approximately 100% Sp, approximately 30% to approximately 100% Sp, approximately 35% to approximately 100% Sp, approximately 40% to approximately 100% Sp, approximately 45% to approximately 100% Sp, approximately 50% to approximately 100% Sp, and approximately 55% to Including approximately 100% Sp, approximately 60% to approximately 100% Sp, approximately 65% ​​to approximately 100% Sp, approximately 70% to approximately 100% Sp, approximately 75% to approximately 100% Sp, approximately 80% to approximately 100% Sp, approximately 85% to approximately 100% Sp, approximately 90% to approximately 100% Sp, or approximately 95% to approximately 100% Sp, approximately 20% to approximately 80% Sp, approximately 25% to approximately 75% Sp, approximately 30% to approximately 70% Sp, approximately 40% to approximately 60% Sp, or approximately 45% to approximately 55% Sp and the remainder Rp.

[0079]

[0130] Any ASOs described herein are present in naturally occurring nucleotides. The modified sugar moiety may contain a sugar moiety containing ribose or deoxyribose, or a modified sugar moiety or sugar analog containing a morpholine ring. Non-limiting examples of modified sugar moieties include 2' substituents, e.g., 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. In some embodiments, the sugar modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar modification is, for example, additional crosslinking in locked nucleic acid (LNA). In some embodiments, the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety includes ribofuransyl or 2'-deoxyribofuransyl modification. In some embodiments, the sugar moiety includes 2'-4'-restricted 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety includes cEt 2',4'-restricted 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety includes tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety includes ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety includes MCE modification. The modifications are known in the art and are incorporated herein by reference, e.g., Jarver et al., 2014, "A Chemical View." This is described in "of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1): pp. 37-47.

[0080]

[0131] In some embodiments, each monomer of the ASO is similarly modified, for example, Each linkage in the ASO skeleton contains a phosphorothioate linkage, or each ribose sugar moiety contains a 2'-O-methyl modification. Such modifications present in each monomer component of ASO are referred to as “homogeneous modifications.” In some examples, combinations of different modifications may be desired; for example, ASO may contain a combination of phosphorodiamidate linkages and sugar moieties containing a morpholine ring (morpholino). Combinations of different modifications to ASO are referred to as “mixed modifications” or “mixed chemistry.”

[0081]

[0132] In some embodiments, the ASO includes one or more skeletal modifications. In some embodiments, the ASO comprises one or more sugar moieties. In some embodiments, the ASO comprises one or more skeletal modifications and one or more sugar moieties. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate skeleton. In some embodiments, the ASO comprises phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of an ASO (e.g., nucleic acid bases, sugar moieties, skeletons), may be modified to achieve desired properties or activities of the ASO, or to reduce undesirable properties or activities of the ASO. For example, one or more components of an ASO or any ASO may be modified to enhance the binding affinity of an mRNA precursor transcript to a target sequence, to reduce binding to any non-target sequence, to reduce degradation by cellular nucleases (i.e., RNase H), to improve the uptake of the ASO into the cell and / or nucleus, to alter the pharmacokinetics or pharmacodynamics of the ASO, and / or to modulate the half-life of the ASO.

[0082]

[0133] In some embodiments, ASO is 2'-O-(2-methoxyethyl)(M The ASO is composed of phosphorothioate-modified nucleotides (OE). ASOs composed of such nucleotides are particularly well suited to the methods disclosed herein, and oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, which makes them suitable for oral delivery, for example, in some embodiments described herein. See, for example, Geary et al., J Pharmacol Exp Ther. 2001;296(3):890-897 and Geary et al., J Pharmacol Exp Ther. 2001;296(3):898-904.

[0083]

[0134] Methods for synthesizing ASO are known to those skilled in the art. Alternatively or additionally, A SO can be obtained from commercial suppliers.

[0135] Unless otherwise specified, single-stranded nucleic acids (e.g., mRNA precursor transcripts, oligonucleotides) The left-hand end of a nucleotide (ASO, etc.) sequence is the 5' end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single- or double-stranded) is the 3' end or direction. Generally, the region or sequence 5' to the reference point in a nucleic acid is referred to as "upstream," and the region or sequence 3' to the reference point in a nucleic acid is referred to as "downstream." Generally, the 5' direction or end of mRNA is where the start or departure codon is located, and the 3' end or direction is where the stop codon is located. In some embodiments, nucleotides upstream of a reference point in a nucleic acid may be indicated by negative numbers, and nucleotides downstream of a reference point may be indicated by positive numbers. For example, a reference point (e.g., an exon-exon junction in mRNA) may be explicitly represented as a "zero" site. In this case, the upstream nucleotide directly adjacent to the reference point is explicitly represented as "minus 1," for example, "-1," and the downstream nucleotide directly adjacent to the reference point is explicitly represented as "plus 1," for example, "+1."

[0084]

[0136] In some embodiments, ASO is an SCN1A NIE-containing mRNA precursor. The ASO is complementary to (and binds to) the targeting region of the SCN1A NIE-containing mRNA precursor located downstream (3' direction) of the 5' splice site (or 3' end of the NIE) of the containing exon (e.g., in the direction indicated by a positive number relative to the 5' splice site). In some embodiments, the ASO is complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located in the region approximately +1 to approximately +500 nucleotides relative to the 5' splice site (or 3' end) of the containing exon. In some embodiments, the ASO may be complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located in the region between +6 and +496 nucleotides relative to the 5' splice site (or 3' end) of the containing exon. In some embodiments, the ASO is approximately +1 to +500, approximately +1 to +490, approximately +1 to +480, approximately +1 to +470, approximately +1 to +460, approximately +1 to +450, approximately +1 to +440, approximately +1 to +430, approximately +1 to +420, and approximately +1 to + 410, approximately +1 to approximately +400, approximately +1 to approximately +390, approximately +1 to approximately +380, approximately +1 to approximately +370, approximately +1 to approximately +360, approximately +1 to approximately +350, approximately +1 to approximately +340, approximately +1 to approximately +330, approximately +1 to approximately +320, approximately +1 to approximately +310, approximately +1 to approximately +300, approximately +1 to approximately +290, approximately +1 to approximately +280, approximately +1 From approximately +270, from approximately +1 to approximately +260, from approximately +1 to approximately +250, from approximately +1 to approximately +240, from approximately +1 to approximately +230, from approximately +1 to approximately +220, from approximately +1 to approximately +210, from approximately +1 to approximately +200, from approximately +1 to approximately +190, from approximately +1 to approximately +180, from approximately +1 to approximately +170, from approximately +1 to approximately +160, from approximately +1 to approximately +150, from approximately +1 to approximately +140 It is complementary to the targeting portion located within the ranges of approximately +1 to +130, approximately +1 to +120, approximately +1 to +110, approximately +1 to +100, approximately +1 to +90, approximately +1 to +80, approximately +1 to +70, approximately +1 to +60, approximately +1 to +50, approximately +1 to +40, approximately +1 to +30, or approximately +1 to +20.In some embodiments, the ASO is complementary to the targeting portion located in the region approximately +1 to +100, approximately +100 to +200, approximately +200 to +300, approximately +300 to +400, or approximately +400 to +500 relative to the 5' splice site (or 3' end) of the containing exon.

[0085]

[0137] In some embodiments, ASO is an SCN1A NIE-containing mRNA precursor. Upstream (5' direction) of the 5' splice site (or 3' end) of the containing exon in (for example, the direction indicated by a negative number relative to the 5' splice site), SCN1A The ASO is complementary to (or binds to) the targeting region of the NIE-containing mRNA precursor. In some embodiments, the ASO is complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located in the region approximately -4 to approximately -270 nucleotides relative to the 5' splice site (or 3' end) of the containing exon. In some embodiments, the ASO is between -1 and -264 nucleotides relative to the 5' splice site (or 3' end) of the containing exon. The targeting region of the SCN1A NIE-containing mRNA precursor within the region may be complementary. In some embodiments, the ASO is approximately -1 to approximately -270, approximately -1 to approximately -260, approximately -1 to approximately -250, approximately -1 to approximately -240, approximately -1 to approximately -230, approximately -1 to approximately -220, approximately -1 to approximately -210, approximately -1 to approximately -200, approximately -1 to approximately -190, approximately -1 to approximately -180, approximately -1 to approximately -170, and approximately -1 to approximately -1 The ASO is complementary to the targeting portion located in the regions of 60, approximately -1 to approximately -150, approximately -1 to approximately -140, approximately -1 to approximately -130, approximately -1 to approximately -120, approximately -1 to approximately -110, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -150 to approximately -150, approximately -150 to approximately -200, or approximately -200 to approximately -250 relative to the 5' splice site (or 3' end) of the containing exon.

[0086]

[0138] In some embodiments, ASO is an SCN1A NIE-containing mRNA precursor. The ASO is complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located upstream (5' direction) (e.g., direction indicated by a negative number) of the 3' splice site (or 5' end) of the containing exon. In some embodiments, the ASO is complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located in the region from approximately -1 to approximately -500 relative to the 3' splice site (or 5' end) of the containing exon. In some embodiments, the ASO is complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located in the region from -1 to -496 relative to the 3' splice site of the containing exon. In some embodiments, the ASO is approximately -1 to -500, approximately -1 to -490, approximately -1 to -480, approximately -1 to -470, approximately -1 to -460, approximately -1 to -450, approximately -1 to -440, approximately -1 to -430, approximately -1 to -420, approximately -1 to -410, Approximately -1 to approximately -400, approximately -1 to approximately -390, approximately -1 to approximately -380, approximately -1 to approximately -370, approximately -1 to approximately -360, approximately -1 to approximately -350, approximately -1 to approximately -340, approximately -1 to approximately -330, approximately -1 to approximately -320, approximately -1 to approximately -310, approximately -1 to approximately -300, approximately -1 to approximately -290, approximately -1 to approximately -280, approximately - From 1 to approximately -270, from approximately -1 to approximately -260, from approximately -1 to approximately -250, from approximately -1 to approximately -240, from approximately -1 to approximately -230, from approximately -1 to approximately -220, from approximately -1 to approximately -210, from approximately -1 to approximately -200, from approximately -1 to approximately -190, from approximately -1 to approximately -180, from approximately -1 to approximately -170, from approximately -1 to approximately -160, from approximately -1 to approximately -150, from approximately -1 The ASO is complementary to targeting regions within the ranges of approximately -140, approximately -1 to approximately -130, approximately -1 to approximately -120, approximately -1 to approximately -110, approximately -1 to approximately -100, approximately -1 to approximately -90, approximately -1 to approximately -80, approximately -1 to approximately -70, approximately -1 to approximately -60, approximately -1 to approximately -50, approximately -1 to approximately -40, or approximately -1 to approximately -30 relative to the 3' splice site of the containing exon. In some embodiments, the ASO is complementary to targeting regions within the ranges of approximately -1 to approximately -100, approximately -100 to approximately -200, approximately -200 to approximately -300, approximately -300 to approximately -400, or approximately -400 to approximately -500 relative to the 3' splice site of the containing exon.

[0087]

[0139] In some embodiments, ASO is an SCN1A NIE-containing mRNA precursor. The ASO is complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located downstream (3' direction) (e.g., direction indicated by a positive number) of the 3' splice site (5' end) of the containing exon. In some embodiments, the ASO is complementary to the targeting region of the SCN1A NIE-containing mRNA precursor located in a region approximately +1 to approximately +100 relative to the 3' splice site of the containing exon. In some embodiments, the ASO is complementary to the region approximately +1 to approximately +90, approximately +1 to approximately +80, approximately +1 to approximately +70, approximately +1 to approximately +60, approximately +1 to approximately +50, approximately +1 to approximately +40, approximately + It is complementary to target regions within the ranges of 1 to approximately +30, approximately +1 to approximately +20, or approximately +1 to approximately +10.

[0088]

[0140] In some embodiments, the targeting portion of the SCN1A NIE-containing mRNA precursor The fraction is located within the region from +100 to -100 relative to the 5' splice site (3' end) of the containing exon. In some embodiments, the targeting region of the SCN1A NIE-containing mRNA precursor is located within the NIE. In some embodiments, the targeting region of the SCN1A NIE-containing mRNA precursor includes the boundary between the pseudoexon and the intron.

[0089]

[0141] ASO is suitable for any length that effectively enhances specific binding and splicing. In some embodiments, the ASO consists of 8 to 50 nucleic acid bases. For example, the ASO may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleic acid bases long. In some embodiments, the ASO consists of more than 50 nucleic acid bases. Some mechanisms, ASOs, are available for 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, and 12 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, 12 to 15 nucleic acid bases, 13 to 50 nucleic acid bases, 13 to 40 nucleic acid bases, 13 to 35 nucleic acid bases, 13 to 30 nucleic acid bases, 13 to 25 nucleic acid bases, 13 to 20 nucleic acid bases, 14 to 50 nucleic acid bases, 14 to 40 nucleic acid bases, 14 to 35 nucleic acid bases, 14 to 30 nucleic acid bases, 14 to 25 nucleic acid bases, 14 to 20 nucleic acid bases, The ASO is 15 to 50 nucleotides long, 15 to 40 nucleotides long, 15 to 35 nucleotides long, 15 to 30 nucleotides long, 15 to 25 nucleotides long, 15 to 20 nucleotides long, 20 to 50 nucleotides long, 20 to 40 nucleotides long, 20 to 35 nucleotides long, 20 to 30 nucleotides long, 20 to 25 nucleotides long, 25 to 50 nucleotides long, 25 to 40 nucleotides long, 25 to 35 nucleotides long, or up to 25 to 30 nucleotides long. In some embodiments, the ASO is 18 nucleotides long. In some embodiments, the ASO is 15 nucleotides long. In some embodiments, the ASO is 25 nucleotides long.

[0090]

[0142] In some embodiments, NIE-containing mRNA has different chemical properties. Two or more ASOs complementary to the same targeting region of the precursor are used. In some embodiments, two or more ASOs complementary to different targeting regions of the NIE-containing mRNA precursor are used.

[0091]

[0143] In embodiments, the antisense oligonucleotide of the present invention is one or Multiple moieties or conjugates are chemically linked to a target moiety or other conjugate, for example, to enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, cholesterol moieties, lipid moieties as cholesteryl moieties, aliphatic chains such as dodecanediol or undecyl residues, polyamine or polyethylene glycol chains, or adamantane acetate. Oligonucleotides containing lipophilic moieties and methods for their preparation are described in the published literature. In embodiments, the antisense oligonucleotide is a debasalized nucleotide, poly(I / O) The conjugate may be attached to a moiety such as tel, polyamine, polyamide, peptide, carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipid, or polyhydrocarbon compound. The conjugate may be linked to one or more nucleotides, including antisense oligonucleotides, at any of several positions of a sugar, base, or phosphate group, for example, using a linker, as understood in the art and described in the literature. Examples of linkers include divalent or trivalent branched linkers. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, “Carbohydrate conjugates as delivery agents for oligonucleotides,” which is incorporated herein by reference.

[0092]

[0144] In some embodiments, nucleic acids targeted by ASO are cells, for example This is an SCN1A NIE-containing mRNA precursor expressed in eukaryotic cells. In some embodiments, the term "cells" may refer to a population of cells. In some embodiments, the cells are in a sample. In some embodiments, the cells are isolated from the sample. In some embodiments, the cells are ex vivo. In some embodiments, the cells are state or disease-associated cells or cell lines. In some embodiments, the cells are in vitro (e.g., in a cell culture). Pharmaceutical composition

[0145] For use of the compositions described and in any of the methods described Pharmaceutical compositions or formulations containing drugs, such as antisense oligonucleotides, can be prepared according to conventional techniques well known in the pharmaceutical industry and described in published literature. In embodiments, a pharmaceutical composition or formulation for treating a target comprises an effective amount of any antisense oligomer, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof, as described herein. A pharmaceutical formulation containing an antisense oligomer may further comprise a pharmaceutically acceptable excipient, diluent, or carrier.

[0093]

[0146] Pharmaceutically acceptable salts do not cause excessive toxicity, irritation, or allergic reactions. Therefore, it is suitable for use in contact with human and lower animal tissues and corresponds to a reasonable benefit / risk ratio. (See, for example, SMBerge et al., J. Pharmaceutical Sciences, 66: pp. 1-19 (1977), incorporated herein by reference for this purpose.) Salts can be prepared in situ during the final isolation and purification of the compound, or separately by reacting the free basic functional group with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other documented methodologies such as ion exchange. Other pharmaceutically acceptable salts include adipines, alginates, ascorbic acid, aspartates, benzenesulfonates, benzoates, bisulfates, borates, and butyrates. Camphor salt, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate Examples include thiocyanates, p-toluenesulfonates, undecanoates, and valersates. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfons, and aryl sulfons, where appropriate.

[0094]

[0147] In embodiments, the composition is not limited to these, but includes tablets, capsules, The composition may be formulated into one of many possible dosage forms, such as gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the composition may be formulated as a suspension in an aqueous, non-aqueous, or mixed medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers. In embodiments, pharmaceutical formulations or compositions of the present invention may include, but are not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0095]

[0148] The pharmaceutical compositions or formulations described herein may, as appropriate, be well known to those skilled in the art. The present invention may include one or more penetration enhancers, carriers, excipients, or other active or inactive components as described in the open literature. In embodiments, liposomes may also be sterically stabilized liposomes, such as liposomes containing one or more specialized lipids. These specialized lipids result in liposomes with enhanced cyclic life. In embodiments, the sterically stabilized liposomes may contain one or more glycolipids or be derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. In embodiments, surfactants are included in the pharmaceutical formulation or composition. The use of surfactants in drug products, formulations, and emulsions is well known in the art. In embodiments, the present invention uses penetration enhancers to provide efficient delivery of antisense oligonucleotides, for example, by facilitating diffusion across cell membranes and / or enhancing the permeability of lipophilic drugs. In embodiments, the penetration enhancer is a surfactant, a fatty acid, a bile salt, a chelating agent, or a non-chelating non-surfactant.

[0096]

[0149] In this embodiment, the pharmaceutical formulation comprises a plurality of antisense oligonucleotides. In some embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. Combination therapy

[0150] In some embodiments, the ASO disclosed herein is one or more types It can be used in combination with other therapeutic agents. In some embodiments, one or more further therapeutic agents may include small molecules. For example, one or more further therapeutic agents may include small molecules described in WO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1, WO201524876A2, WO2013119916A2, and WO2014209841A2, which are incorporated herein by reference in their entirety. In some embodiments, one or more further therapeutic agents include ASOs that can be used to correct intron retention. In some embodiments, one or more other agents may be selected from the ASOs listed in Table 1a or Table 1b.

[0097] [Table 1-1]

[0098] [Table 1-2]

[0099] [Table 2-1]

[0100] [Table 2-2]

[0101] Target treatment

[0151] Any of the compositions provided herein can be administered to an individual. The term "body" can be used interchangeably with "subject" or "patient." The individual may be a mammal, such as a human, or an animal such as a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In some embodiments, the individual is a human. In some embodiments, the individual is a fetus, embryo, or child. In other embodiments, the individual may be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered ex vivo to cells.

[0102]

[0152] In some embodiments, the compositions provided herein treat a disease or disorder. The method of treatment involves administering the substance to an individual. In some embodiments, the individual has a genetic disorder, for example, one of the diseases described herein. In some embodiments, the individual is at risk of having a disease, for example, one of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by an insufficient amount of protein or insufficient protein activity. If the individual is at "increased risk" of having a disease or disorder caused by an insufficient amount of protein or insufficient protein activity, the method involves preventive or prophylactic treatment. For example, an individual may be at increased risk of having such a disease or disorder due to a family history of the disease. Typically, an individual at increased risk of having such a disease or disorder would benefit from prophylactic treatment (for example, by preventing or delaying the onset or progression of the disease or disorder). In embodiments, the fetus is treated in utero, for example, by administering the ASO composition directly or indirectly to the fetus (for example, via the mother).

[0103]

[0153] The preferred administration route for ASO of the present invention depends on the cell type to which ASO delivery is desired. It may be changed. Multiple tissues and organs are affected by Dravet syndrome; generalized epileptic febrile seizures plus type 2; familial febrile seizures 3A; familial hemiplegic migraine 3; autism; early infantile epileptic encephalopathy 13; sick sinus syndrome 1; Alzheimer's disease, or SUDEP, with the brain being the tissue most significantly affected. The ASO of the present invention is administered parenterally to the patient, for example, by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreous injection, or intravenous injection. It can be administered by injection.

[0104]

[0154] In some embodiments, the disease or condition is Na v 1.1 (SCN1A gene) It is triggered by mutations in the protein encoded by the offspring. In some cases, the mutation is Na v This is a loss-of-function mutation in 1.1. In some cases, Na v Loss-of-function mutations in 1.1 are wild-type Na v Compared to the functions of 1.1, Na v 1.1 contains one or more mutations that reduce or impair the function of (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some cases, Na v Loss-of-function mutations in 1.1 include one or more mutations that result in a disease phenotype. Exemplary loss-of-function mutations include, but are not limited to, R859C, T875M, V1353L, I1656M, R1657C, A1685V, M1841T, and R1916G.

[0105]

[0155] In other examples, the mutation is Na v This is a gain-of-function mutation in 1.1. In such cases, gain-of-function mutations result in wild-type Na v Compared to the functions of 1.1, Na v 1.1 comprises one or more mutations that prolong the activation of (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In such cases, Na vA gain-of-function mutation in 1.1 includes one or more mutations that result in a disease phenotype. Exemplary gain-of-function mutations include, but are not limited to, D188V, W1204R, R1648H, and D1866Y.

[0106]

[0156] In some embodiments, the disease or condition is encephalopathy. In some cases, the brain Symptoms include Na v It is triggered by loss-of-function mutations in 1.1.

[0157] In some embodiments, the encephalopathy is an epileptic encephalopathy. Encephalopathy includes, but is not limited to, Dravet syndrome (DS) (also known as severe myoclonic epilepsy of infants or SMEI); borderline severe myoclonic epilepsy of infants (SMEI) (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic apoptosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); early infant SCN1A encephalopathy; early infant epileptic encephalopathy (EIEE); or sick sinus syndrome1. In some embodiments, the disease or condition is an epileptic encephalopathy selected at random from Dravet syndrome (DS) (also known as severe myoclonic epilepsy of infants or SMEI); borderline severe myoclonic epilepsy of infants (SMEI) (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic apoptosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); and sick sinus syndrome1.

[0107]

[0158] In some cases, GEFS+ is generalized epileptic febrile seizures plus type 2.

[0159] In some cases, the febrile seizure is classified as familial febrile seizure 3A.

[0160] In some cases, SMEB is SMEB without generalized spikes (SMEB- This includes SW, SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more SMEI features (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0108]

[0161] In some embodiments, Na v Triggered by loss-of-function mutations in 1.1 The diseases or conditions that are treated include Dravet syndrome (DS) (also known as SMEI); in infants. Severe myoclonic epilepsy (SMEI) borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); infantile epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic apoptosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; pediatric alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; infantile SCN1A encephalopathy; infantile epileptic encephalopathy (EIEE); autism; or infantile malignant migratory partial seizures.

[0109]

[0162] In some embodiments, the disease or condition is Na v Feature acquisition in 1.1 It is triggered by type mutation. Na v 1.1 Examples of diseases or conditions associated with gain-of-function mutations include, but are not limited to, migraines. In some cases, Na v 1.1 Migraine is a disease or condition induced by gain-of-function mutations.

[0110]

[0163] In some cases, the migraine is familial hemiplegic migraine 3.

[0164] In some embodiments, the disease or condition is Na v 1.1 In predisposed epilepsy Na v 1.1 Predisposing epilepsy is caused by Na v Loss-of-function mutations in 1.1, or Na v It may include gain-of-function mutations as described in 1.1. In some cases, Na v 1.1 Predisposing epilepsy involves one or more inherited mutations. In other cases, Na v 1.1 Predisposing epilepsy involves one or more de novo mutations. In some cases, Na v 1.1 Predisposing epilepsy includes Dravet syndrome (DS) (also known as severe myoclonic epilepsy of infants or SMEI); borderline severe myoclonic epilepsy of infants (SMEI) (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic apoptosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; early infant SCN1A encephalopathy; early infant epileptic encephalopathy (EIEE); sudden unexpected death in epilepsy (SUDEP); or infant malignant migraine partial seizures. In some cases, Na v Na related to loss-of-function mutations in 1.1 v1.1 Examples of predisposing epilepsy include Dravet syndrome (DS) (also known as severe myoclonic epilepsy of infants or SMEI); borderline severe myoclonic epilepsy of infants (SMEI) (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic apoptosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; early infant SCN1A encephalopathy; early infant epileptic encephalopathy (EIEE); sudden unexpected death in epilepsy (SUDEP); and malignant migraine partial seizures of infants.

[0111]

[0165] In some embodiments, a disease or condition is a haploinsufficiency of the SCN1A gene. Related to: Exemplary diseases or conditions associated with SCN1A gene haploinsufficiency include, but are not limited to, Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infants (SMEI) borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic apoptosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; early infant SCN1A encephalopathy; early infant epileptic encephalopathy (EIEE); or infant malignant migratory partial seizures. In some cases, the disease or condition may be Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infants (SMEI) borderline (SMEB); febrile seizures (FS); or generalized epilepsy with febrile seizures plus (GEFS+). ); Early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic ataxia; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; early infant SCN1A encephalopathy; early infant epileptic encephalopathy (EIEE); or infant malignant migratory partial seizures.

[0112]

[0166] In some cases, the disease or condition is Dravet syndrome (DS).

[0167] Dravet syndrome (DS), also known as severe myoclonic epilepsy of infants (SMEI), is Dravet syndrome is an epileptic encephalopathy that manifests in the first year of life. It is an increasingly recognized epileptic encephalopathy, with its clinical diagnosis supported by the finding of sodium channel gene mutations in approximately 70–80% of patients. Mutations in ion channel genes play a major role in the pathogenesis of a range of epileptic syndromes, and consequently, some epilepsies are considered channel diseases. Voltage-gated sodium channels (VGSCs) play an essential role in neuronal excitability, and therefore it is not surprising that many mutations associated with DS have been identified in genes encoding VGSC subunits. The disease is described, for example, by Mulley et al., 2005, and in OMIM#607208 (Online Mendelian Inheritance in Man, Johns Hopkins University, 1966–2015), both of which are incorporated herein by reference.

[0113]

[0168] Between 70% and 80% of patients have a sodium channel α1 subunit gene. Individuals with SCN1A abnormalities have truncated mutations, accounting for approximately 40% of cases, and are strongly correlated with an earlier age of seizure onset. Sequence mutations are found in approximately 70% of cases, including truncated (40%) and missense mutations (40%), with the remainder being splice site changes. While most mutations are de novo, familial mutations occur in 5–10% of cases and are typically missense. The remaining SCN1A mutations include splice site and missense mutations, the majority of which are classified as sodium channel pore formation regions. Currently, more than 500 mutations are associated with DS and are randomly distributed in the gene (Mulley et al., Neurol. 2006, 67, pp. 1094–1095).

[0114]

[0169] The SCN1A gene is a sodium channel gene located on human chromosome 2q24. Located in the raster, it encodes the pore-forming α subunit known as Nav1.1 of the neuronal voltage-gated sodium channel. The SCN1A gene occupies approximately 100 kb of genomic DNA and contains 26 exons. The SCN1A protein consists of four domains, each with six transmembrane segments. Two splice variants have been identified, resulting in long and short isoforms, differing in the presence or absence of 11 amino acids in the cytoplasmic loop between domains 1 and 2 of exon 11 (Miller et al., 1993–2015, and Mulley et al., 2005, 25, pp. 535–542, incorporated herein by reference).

[0115]

[0170] Alternative splicing events in the SCN1A gene result in abnormal protein development. Therapeutic agents that can target alternative splicing events in the SCN1A gene, potentially resulting in unproductive mRNA transcripts, can modulate the expression levels of functional proteins and / or inhibit abnormal protein expression in DS patients. Such therapeutic agents can be used to treat conditions caused by SCN1A protein deficiency.

[0116]

[0171] Alternative splicing events that can result in unproductive mRNA transcripts One possibility is the addition of nonsense mutations to mRNA transcripts, which can induce a nonsense mutation-dependent mRNA degradation mechanism. This involves exon inclusion. The Disclosure provides compositions and methods for modulating alternative splicing of SCN1A to increase the production of protein-coding mature mRNA, and thus translated functional SCN1A protein. These compositions and methods include antisense oligomers (ASOs) that can induce exon skipping and promote constitutive splicing of SCN1A mRNA precursors. In various embodiments, functional SCN1A protein can be increased using the methods of the Disclosure to treat conditions caused by SCN1A protein deficiency.

[0117]

[0172] In some cases, the disease or condition is SMEB (Small-Symptom-Ecological Breakdown).

[0173] In some cases, the disease or condition is GEFS+.

[0174] In some cases, the disease or condition may be a febrile seizure (e.g., familial febrile seizures 3A). That is the case.

[0118]

[0175] In some cases, the disease or condition may be autism (autism spectrum disorder or A (Also known as SD)

[0176] In some cases, the disease or condition may be migraine (e.g., familial hemiplegic migraine 3). )

[0119]

[0177] In some cases, the disease or condition is Alzheimer's disease.

[0178] In some embodiments, the disease or condition is SCN2A encephalopathy.

[0179] In some embodiments, the disease or condition is SCN8A encephalopathy.

[0120]

[0180] In some embodiments, the disease or condition is an SCN5A arrhythmia.

[0181] In this embodiment, the antisense oligonucleotide crosses the blood-brain barrier. The target antisense oligonucleotide is administered by any method known in the art together with one or more agents capable of promoting the penetration of the target antisense oligonucleotide. For example, the delivery of the agent by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Patent No. 6,632,427, “Adenoviral-vector-mediated gene transfer into medullary motor neurons,” which is incorporated herein by reference. Direct delivery of the vector to the brain, e.g., the striatum, thalamus, hippocampus, or substantia nigra, is described, for example, in U.S. Patent No. 6,756,523, “Adenovirus vectors for This is described in "the transfer of foreign genes into cells of the central nervous system particularly in the brain."

[0121]

[0182] In this embodiment, the antisense oligonucleotide is used for a desired pharmaceutical or The antisense oligonucleotide is coupled or conjugated with a drug that provides pharmacodynamic properties. In embodiments, the antisense oligonucleotide is coupled with a substance known in the art, such as an antibody against a transferrin receptor, which facilitates penetration or transport across the blood-brain barrier. In embodiments, the antisense oligonucleotide is coupled with a viral vector to, for example, make the antisense compound more effective or increase its transport across the blood-brain barrier. In several embodiments, osmotic blood-brain barrier disruption is caused by sugars, such as mesoerythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dalcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, such as glutamine, lysine, and alpha- This is aided by infusion of ginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood-brain barrier penetration are, for example, U.S. Patent No. 9,193,969, “Compositions and methods for selective delivery of oligonucleotides,” each of which is incorporated herein by reference. "Molecules to specific neuron types," U.S. Patent No. 4,866,042, "Method for the delivery of genetic material across the blood brain It is described in "barrier," U.S. Patent No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Patent No. 6,936,589, "Parenteral delivery systems."

[0122]

[0183] In embodiments, the ASO of the present invention is incorporated herein by reference, for example. It is coupled with dopamine reuptake inhibitors (DRIs), selective serotonin reuptake inhibitors (SSRIs), norepinephrine reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs) using the method described in U.S. Patent No. 9,193,969.

[0123]

[0184] In the embodiment, the object treated using the method and composition is in a state of The above is evaluated using any method known and described in the art. Methods for identifying further ASOs that induce exon skipping

[0185] SCN1A NIE-containing mRNA precursor exon skipping induction AS Methods for identifying or determining ASOs are also within the scope of this disclosure. For example, a method may include a step of identifying or determining an ASO that induces pseudoexon skipping of an SCN1A NIE-containing mRNA precursor. ASOs that specifically hybridize to different nucleotides within a target region of the mRNA precursor may be screened to identify or determine ASOs that improve the rate and / or range of splicing of the target intron. In some embodiments, the ASO may block or interfere with the binding site of a splicing repressor / silencer. Any method known in the art may be used to identify (determine) an ASO that, when hybridized to a target region of an exon, results in a desired effect (e.g., pseudoexon skipping, protein or functional RNA production). These methods can also be used to identify ASOs that induce exon skipping of an intron by binding to a targeting region in an intron adjacent to the intron or non-inclusion exon. An example of a method that may be used is provided below.

[0124]

[0186] The ASO "walking" round of screening involves the mRNA precursor This may be carried out using ASOs designed to hybridize to the target region. For example, ASOs used for ASO walking can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the enclosing exon (e.g., a portion of the sequence of the exon located upstream of the target / enclosing exon) to approximately 100 nucleotides downstream of the 3' splice site of the target / enclosing exon, and / or from approximately 100 nucleotides upstream of the 5' splice site of the enclosing exon to approximately 100 nucleotides downstream of the 5' splice site of the target / enclosing exon (e.g., a portion of the sequence of the exon located downstream of the target / enclosing exon). For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 3' splice site of the target / enclosing exon. A second ASO may be designed to hybridize to the target / enclosing exon The ASO may be designed to specifically hybridize to nucleotides +11 to +25 relative to the 3' splice site. The ASO is designed to span the target region of the mRNA precursor. In embodiments, the ASO can be tiled more closely, for example, every 1, 2, 3, or 4 nucleotides. Furthermore, the ASO can be tiled from 100 nucleotides downstream of the 5' splice site to 100 nucleotides upstream of the 3' splice site. In some embodiments, the ASO can be tiled from approximately 1,160 nucleotides upstream of the 3' splice site to approximately 500 nucleotides downstream of the 5' splice site. In some embodiments, the ASO can be tiled from approximately 500 nucleotides upstream of the 3' splice site to approximately 1,920 nucleotides downstream of the 3' splice site.

[0125]

[0187] One or more ASOs, or a control ASO (hybridized into the target region) ASOs having scrambled sequences, which are sequences not expected to be included, are delivered to disease-associated cell lines expressing a target mRNA precursor (e.g., NIE-containing mRNA precursors as described herein), for example, by transfection. The exon-skipping effect of each ASO may be assessed by any method known in the art, for example, by reverse transcription (RT)-PCR using primers spanning splice junctions, as described in Example 4. A reduction or absence of longer RT-PCR products in ASO-treated cells compared to control ASO-treated cells, produced using primers spanning regions containing including exons (e.g., adjacent exons of NIE), indicates enhanced splicing of the target NIE. In some embodiments, exon-skipping efficiency (or splicing efficiency of splicing introns containing NIE), the ratio of spliced ​​mRNA precursors to unspliced ​​mRNA precursors, the rate of splicing, or the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target mRNA precursor can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for assessing and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, may be used.

[0126]

[0188] The second round of screening, called ASO "microwalking," involves mRN This may be carried out using an ASO designed to hybridize to the target region of the A precursor. The ASO used for ASO microwalking is tiled nucleotide by nucleotide to further refine the nucleotide acid sequence of the mRNA precursor, which results in exon skipping (or enhanced splicing of NIE) when hybridized with the ASO.

[0127]

[0189] The region defined by ASO, which facilitates the splicing of target introns, These are explored in more detail by ASO "microwalks" with ASOs arranged at 1nt step intervals, and by longer ASOs, typically 18–25nt.

[0128]

[0190] As described above regarding ASO gait, ASO microgait is a type of gait. This is carried out by delivering multiple ASOs, or a control ASO (an ASO having a scrambled sequence that is not expected to hybridize to the target region), to disease-associated cell lines expressing the target mRNA precursor, for example, by transfection. The splicing-inducing effect of each ASO may be assessed by any method known in the art, for example by reverse transcription (RT)-PCR using primers spanning the NIE, as described herein (see, for example, Example 4). A reduction or absence of longer RT-PCR products compared to those in cells indicates enhanced exon skipping (or splicing of target introns containing NIEs). In some embodiments, exon skipping efficiency (or splicing efficiency of splicing introns containing NIEs), the ratio of spliced ​​mRNA precursors to unspliced ​​mRNA precursors, the rate of splicing, or the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target mRNA precursor can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for assessing and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, may be used.

[0129]

[0191] When hybridizing to the mRNA precursor region, exon skipping occurs. ASOs that result in enhanced splicing of introns containing NIEs and increased protein production may be tested in vivo using animal models, e.g., transgenic mouse models with full-length human genes knocked in, or humanized mouse models of the disease. The preferred route of administration of ASOs may vary depending on the disease and / or the cell type to which ASO delivery is desired. ASOs may be administered, for example, by intrathecal, intraventricular, intraperitoneal, intramuscular, subcutaneous, intravitreous, or intravenous injection. After administration, the cells, tissues, and / or organs of the model animals may be assessed to determine the effect of ASO treatment by evaluating splicing (efficiency, rate, and range) and protein production, for example, by methods known in the art and described herein. Animal models may also serve as any phenotypic or behavioral indicators of disease or disease severity.

[0130]

[0192] As described in various examples herein, the expression in the human SCN1A gene Exon 20x is equivalent to exon 21x in the mouse SCN1A gene.

[0193] In the presence of an NMD inhibitor, for example, cycloheximide, NMD-inducing exo Methods for identifying or validating the 'n' are also within the scope of this disclosure. Exemplary methods are provided in Figure 3 and Example 2.

[0131]

[0194] Specific Embodiments

[0195] Embodiment 1. mR containing a nonsense mutation-dependent RNA degradation mechanism-inducing exon. A method for regulating the expression of SCN1A protein in cells having mRNA encoding SCN1A protein, which is NA (NMD exon mRNA), comprising the step of contacting a therapeutic agent with the cells, whereby the therapeutic agent regulates the splicing of the NMD exon from the NMD exon mRNA encoding SCN1A protein, thereby regulating the level of the processed mRNA encoding SCN1A protein and regulating the expression of SCN1A protein in the cells.

[0132]

[0196] Embodiment 2. A method for treating a disease or condition in a subject in need thereof by regulating the expression of SCN1A protein in the subject's cells, comprising contacting the subject's cells with a therapeutic agent that regulates the splicing of the NMD exon from the mRNA in cells containing a nonsense mutation-dependent mRNA decay mechanism-inducing exon (NMD exon) and encoding SCN1A, thereby regulating the level of the processed mRNA encoding SCN1A protein and regulating the expression of SCN1A protein in the subject's cells.

[0133]

[0133]

[0197] Embodiment 3. The method according to embodiment 1 or 2, wherein the therapeutic agent is (a) binding to a targeting portion of the NMD exon mRNA encoding SCN1A, (b) regulating the binding of a factor involved in the splicing of the NMD exon mRNA, or (c) a combination of (a) and (b)

[0134]

[0198] Embodiment 4. The method according to embodiment 3, wherein the therapeutic agent interferes with the binding of a factor involved in the splicing of the NMD exon from the region of the targeting portion.

[0199] Embodiment 5. The method according to embodiment 3 or 4, wherein the targeting portion is proximal to the NMD exon.

[0135]

[0200] Embodiment 6. The targeting portion is the 5' end of the NMD exon, up to approximately 1500 The method according to any one of Embodiments 3 to 5, located upstream of a nucleotide, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides.

[0136]

[0201] Embodiment 7. The targeting portion is at least about 15 of the 5' end of the NMD exon. The method according to any one of Embodiments 3 to 6, located upstream of 00 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides, approximately 40 nucleotides, approximately 30 nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, approximately 2 nucleotides, and approximately 1 nucleotide.

[0137]

[0202] Embodiment 8. The targeting portion is the 3' end of the NMD exon, up to approximately 1500 The method according to any one of Embodiments 3 to 5, wherein the nucleotides are located downstream of approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides.

[0138]

[0203] Embodiment 9. The targeting portion is at least about 15 of the 3' end of the NMD exon. The method according to any one of Embodiments 3 to 5 or 8, located downstream of 00 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides, approximately 40 nucleotides, approximately 30 nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, approximately 2 nucleotides, and approximately 1 nucleotide.

[0139]

[0204] Embodiment 10. The targeting portion is an NMD exon mRN encoding SCN1A. The method according to any one of embodiments 3 to 9, wherein the intron region is located between two canonical exon regions of A, and the intron region contains an NMD exon.

[0140]

[0205] Embodiment 11. The targeting portion at least partially overlaps with the NMD exons. The method according to any one of embodiments 3 to 10.

[0206] Embodiment 12. The targeting portion is an intron upstream of the NMD exon and at least The method according to any one of embodiments 3 to 11, which also partially overlaps.

[0141]

[0207] Embodiment 13. The targeting portion is a 5'NMD exon-intron junction or The method according to any one of embodiments 3 to 12, comprising a 3'NMD exon-intron junction.

[0142]

[0208] Embodiment 14. Embodiments 3 to 13, wherein the targeting portion is located within an NMD exon. Any one of the following methods.

[0209] Embodiment 15. The targeting portion is approximately 5, 6, 7, 8, 9, 10 of the NMD exons. The method according to any one of embodiments 3 to 14, comprising 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides.

[0143]

[0210] Embodiment 16. The NMD exon mRNA encoding SCN1A is sequence number The method according to any one of Embodiments 1 to 15, comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to any one of 2 or 7 to 10.

[0144]

[0211] Embodiment 17. The NMD exon mRNA encoding SCN1A is sequence number The method according to any one of Embodiments 1 to 16, encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to 1 or 3-6.

[0145]

[0212] Embodiment 18. The targeted region is the genomic region GRCh37 / hg19:chr2 The method according to any one of Embodiments 3 to 17, wherein the nucleotides are located upstream of 166,863,803, at a maximum of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides.

[0146]

[0213] Embodiment 19. The targeted region is the genomic region GRCh37 / hg19:chr2 The method according to any one of Embodiments 3 to 18, which is upstream of 166,863,803 by about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, and about 1 nucleotide.

[0147]

[0214] Embodiment 20. The targeted portion is the genomic site GRCh37 / hg19:chr2 The method according to any one of Embodiments 3 to 17, which is downstream of 166,863,740 by up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides.

[0148]

[0215] Embodiment 21. The targeted portion is the genomic site GRCh37 / hg19:chr2 The method according to any one of Embodiments 3 to 17 or 20, which is downstream of 166,863,740 by about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about ......400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, and about 1 nucleotide.

[0149]

[0216] Embodiment 22. The targeted portion of the NMD exon mRNA encoding SCN1A The method according to any one of Embodiments 3 to 21, wherein the sequence includes a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to a region containing at least eight consecutive nucleic acids of SEQ ID NOs. 2 or 7-10.

[0150]

[0217] Embodiment 23. The therapeutic agent is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 22, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 21-67, 210-256, or 304-379.

[0151]

[0218] Embodiment 24. Targeted portion of NMD exon mRNA encoding SCN1A The method according to any one of Embodiments 3 to 21, wherein the nonsense mutation-dependent RNA degradation mechanism induction exon 20x of SCN1A is present.

[0152]

[0219] Embodiment 25. The therapeutic agent is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 24, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 42-50 or 231-239.

[0153]

[0220] Embodiment 26. Targeted portion of NMD exon mRNA encoding SCN1A The method according to any one of Embodiments 3 to 21, wherein the exon 20x is located upstream or downstream of the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A.

[0154]

[0221] Embodiment 27. The therapeutic agent is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 26, comprising sequences that are at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 21-38, 53-67, 210-227, or 242-256.

[0155]

[0222] Embodiment 28. The targeting portion of the NMD exon mRNA is the exo of SCN1A. The method according to any one of embodiments 3 to 21, including an exon-intron junction of 20x.

[0156]

[0223] Embodiment 29. The therapeutic agent is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 28, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 39-41, 51, 52, 228-230, 240, or 241.

[0157]

[0224] Embodiment 30. The therapeutic agent is a processing agent that encodes the SCN1A protein. The method according to any one of embodiments 1 to 29, which facilitates the elimination of NMD exons from the mRNA.

[0158]

[0225] Embodiment 31. In cells that have come into contact with the therapeutic agent, the SCN1A protein is controlled The elimination of NMD exons from the processed mRNA encoding the SCN1A protein in control cells was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, and 1 The method according to Embodiment 30, which increases by 0.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0159]

[0226] Embodiment 32. The therapeutic agent encodes the SCN1A protein in cells. The method according to embodiment 30 or 31, which increases the level of processed mRNA.

[0160]

[0227] Embodiment 33. In cells that have come into contact with the therapeutic agent, the SCN1A protein is controlled The amount of processed mRNA encoding the SCN1A protein in control cells was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 7 times, and approximately 1.1 to 9 times, compared to the total amount of processed mRNA encoding the SCN1A protein in control cells. The method according to any one of embodiments 30 to 32, which increases by 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0161]

[0228] Embodiment 34. The therapeutic agent increases the expression of the SCN1A protein in cells. The method according to any one of embodiments 30 to 33.

[0229] Embodiment 35. The amount of SCN1A produced in cells that have come into contact with the therapeutic agent. Compared to the total amount of SCN1A produced in control cells, the amounts were approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 7 times, 2 to 8 times, 2 to 9 times, and 3 to The method according to any one of embodiments 30 to 34, which increases by 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0162]

[0230] Embodiment 36. A disease or condition caused by a loss-of-function mutation in Nav1.1 The method according to any one of embodiments 2 to 35, which is triggered by the above.

[0231] Embodiment 37. The disease or condition is related to the haploinsufficiency of the SCN1A gene, The method according to any one of Embodiments 2 to 36, wherein the subject has a first allele encoding a functional SCN1A, and a second allele in which SCN1A is not produced or is produced at a reduced level, or a second allele encoding a non-functional SCN1A or a partially functional SCN1A.

[0163]

[0232] Embodiment 38. Any of Embodiments 2 to 37, wherein the disease or condition is encephalopathy. One method.

[0233] Embodiment 39. The method according to Embodiment 38, wherein the encephalopathy is epileptic encephalopathy.

[0164]

[0234] Embodiment 40. The disease or condition is Dravet syndrome (DS); severe myoclosis of infants. Knee epilepsy (SMEI), borderline epilepsy (SMEB); febrile seizures (FS); generalized epilepsy The method according to any one of Embodiments 2 to 37, wherein the condition is: seizures plus (GEFS+); early infantile epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic astosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; pediatric alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; autism; or infantile malignant migratory partial seizures.

[0165]

[0235] Embodiment 41. An embodiment in which GEFS+ is generalized epileptic febrile seizures plus type 2. The method described in condition 40.

[0236] Embodiment 42. The embodiment described in Embodiment 40, wherein the febrile seizure is familial febrile seizure 3A. method.

[0166]

[0237] Embodiment 43. SMEB is SMEB without generalized spikes (SMEB-SW The method according to Embodiment 40, wherein the condition is SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more SMEI features (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0167]

[0238] Embodiment 44. The therapeutic agent is a processing agent that encodes the SCN1A protein. The method according to any one of embodiments 1 to 43, which promotes the elimination of NMD exons from the mRNA and increases the expression of SCN1A in cells.

[0168]

[0239] Embodiment 45. The therapeutic agent is an antisense oligomer (ASO), and the ASO is The method according to any one of Embodiments 1 to 44, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs. 22-24, 26, 27, 29-35, 37-62, 64-67, or 304-379.

[0169]

[0240] Embodiment 46. The therapeutic agent is a processing agent that encodes the SCN1A protein. The method according to any one of embodiments 1 to 29, which inhibits the elimination of NMD exons from the mRNA.

[0170]

[0241] Embodiment 47. In cells that have come into contact with the therapeutic agent, the SCN1A protein is controlled The elimination of NMD exons from the processed mRNA encoding the SCN1A protein in control cells was approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, 1 / 1.1 to 1 / 9, 1 / 2 to 1 / 5, and 1 / 2 to 6. The method according to Embodiment 46, which reduces to 1 / 2 to 1 / 7, 1 / 2 to 1 / 8, 1 / 2 to 1 / 9, 1 / 3 to 1 / 6, 1 / 3 to 1 / 7, 1 / 3 to 1 / 8, 1 / 3 to 1 / 9, 1 / 4 to 1 / 7, 1 / 4 to 1 / 8, 1 / 4 to 1 / 9, at least 1 / 1.1, at least 1 / 1.5, at least 1 / 2, at least 1 / 2.5, at least 1 / 3, at least 1 / 3.5, at least 1 / 4, at least 1 / 5, or at least 1 / 10.

[0171]

[0242] Embodiment 48. The therapeutic agent encodes the SCN1A protein in cells. The method according to embodiment 46 or 47, which reduces the level of processed mRNA.

[0172]

[0243] Embodiment 49. In cells that have come into contact with the therapeutic agent, the SCN1A protein is controlled The amount of processed mRNA encoding the SCN1A protein in control cells was approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.1 to 1 / 5, 1 / 1.1 to 1 / 6, 1 / 1.1 to 1 / 7, 1 / 1.1 to 1 / 8, 1 / 1.1 to 1 / 9, 1 / 2 to 1 / 5, 1 / 2 to 1 / 6, and 1 / 2 to 1 / 7. The method according to any one of embodiments 46 to 48, which reduces to about 1 / 2 to about 1 / 8, about 1 / 2 to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1.1, at least about 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 5, or at least about 1 / 10.

[0173]

[0244] Embodiment 50. The therapeutic agent reduces the expression of the SCN1A protein in cells. The method according to any one of embodiments 46 to 49.

[0245] Embodiment 51. The amount of SCN1A produced in cells that have come into contact with the therapeutic agent. Compared to the total amount of SCN1A produced in control cells, approximately 1 / 1.1 to 1 / 10, approximately 1 / 1.5 to 1 / 10, approximately 1 / 2 to 1 / 10, approximately 1 / 3 to 1 / 10, approximately 1 / 4 to 1 / 10, approximately 1 / 1.1 to 1 / 5, approximately 1 / 1.1 to 1 / 6, approximately 1 / 1.1 to 1 / 7, approximately 1 / 1.1 to 1 / 8, approximately 1 / 1.1 to 1 / 9, approximately 1 / 2 to 1 / 5, approximately 1 / 2 to 1 / 6, approximately 1 / 2 to 1 / 7, approximately 1 / 2 to 1 / 8, approximately 1 / 2 The method according to any one of embodiments 46 to 50, which reduces to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1 / 1.1, at least about 1 / 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 5, or at least about 1 / 10.

[0174]

[0246] Embodiment 52. A disease or condition caused by a gain-of-function mutation in Nav1.1 The method according to any one of embodiments 2 to 29 or 46 to 49, which is induced as follows.

[0175]

[0247] Embodiment 53. The target is an allele produced at a level in which SCN1A is increased, The method according to Embodiment 52, wherein the allele has an allele encoding a mutant SCN1A that induces increased activity of Nav1.1 in cells.

[0176]

[0248] Embodiment 54. The disease or condition described in Embodiment 52 or 53, wherein the disease or condition is migraine. Method of loading.

[0249] Embodiment 55. The migraine is familial hemiplegic migraine 3, as described in Embodiment 54. The method.

[0177]

[0250] Embodiment 56. The disease or condition is Nav1.1 diathesis epilepsy, The method described in any one of states 2 to 49.

[0251] Embodiment 57. The therapeutic agent is a processing agent that encodes the SCN1A protein. The method according to any one of embodiments 46 to 56, which inhibits the elimination of NMD exons from the mRNA and reduces the expression of SCN1A in cells.

[0178]

[0252] Embodiment 58. The therapeutic agent is an antisense oligomer (ASO), and the ASO is The method according to any one of embodiments 46 to 57, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 21, 25, 28, 36, or 63.

[0179]

[0253] Embodiment 59. The therapeutic agent is an antisense oligomer (ASO), and antisense The method according to any one of Embodiments 1 to 58, wherein the lance oligomer includes a skeletal modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0180]

[0254] Embodiment 60. The therapeutic agent is an antisense oligomer (ASO), and antisense The method according to any one of Embodiments 1 to 59, wherein the lance oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0181]

[0255] Embodiment 61. The therapeutic agent is an antisense oligomer (ASO), and antisense The method according to any one of Embodiments 1 to 60, wherein the lance oligomer comprises at least one modified sugar moiety.

[0182]

[0256] Embodiment 62. The method according to Embodiment 61, wherein each sugar portion is a modified sugar portion.

[0257] Embodiment 63. The therapeutic agent is an antisense oligomer (ASO), and antisense Nucleic acid oligomers are 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 A method according to any one of Embodiments 1 to 62, comprising up to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or up to 12 to 15 nucleic acid bases.

[0183]

[0258] Embodiment 64. The therapeutic agent is an antisense oligomer (ASO), and antisense The method according to any one of Embodiments 3 to 63, wherein the lance oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeting region of the protein-coding NMD exon mRNA.

[0184]

[0259] Embodiment 65. Assessment of SCN1A mRNA or protein expression. A method according to any one of embodiments 1 to 64, further comprising the step of [doing something].

[0260] Embodiment 66. Described in any one of Embodiments 2 to 65, wherein the subject is a human. The method.

[0185]

[0261] Embodiment 67. Any one of Embodiments 2 to 65, wherein the subject is a non-human animal. Methods used.

[0262] Embodiment 68. Embodiments 2 to 65, wherein the subject is a fetus, embryo, or child. Either one of the methods.

[0186]

[0263] Embodiment 69. Any one of Embodiments 1 to 68, wherein the cells are ex vivo. Methods used.

[0264] Embodiment 70. The therapeutic agent is administered via intrathecal injection, intraventricular injection, intraperitoneal injection, or intramuscular injection. The method according to any one of Embodiments 2 to 69, administered by internal injection, subcutaneous injection, intravitreous injection, or intravenous injection.

[0187]

[0265] Embodiment 71. An embodiment further comprising the step of administering a second therapeutic agent to a target. The method described in any one of states 2 to 65.

[0266] Embodiment 72. The method according to Embodiment 71, wherein the second therapeutic agent is a low molecular weight agent.

[0188]

[0267] Embodiment 73. The method according to Embodiment 71, wherein the second therapeutic agent is an ASO.

[0268] Embodiment 74. ASO is less than any one of sequence numbers 115 to 161. The method according to Embodiment 73, comprising sequences that are at least 80%, 85%, 90%, 95%, 97%, or 100% complementary.

[0189]

[0269] Embodiment 75. The second therapeutic agent corrects intron retention, as described in Embodiment 71. Method of loading.

[0270] Embodiment 76. The disease or condition is Alzheimer's disease, SCN2A encephalopathy, SC The method according to any one of embodiments 2 to 65, wherein the condition is N8A encephalopathy or SCN5A arrhythmia.

[0190]

[0271] Embodiment 77. The disease or condition is Alzheimer's disease, SCN2A encephalopathy, SC The method according to embodiment 30, 32, or 34, wherein the condition is N8A encephalopathy or SCN5A arrhythmia.

[0191]

[0272] Embodiment 78. Dravet syndrome (DS) in general, in subjects requiring it. A method for treating epileptic febrile seizures plus type 2; familial febrile seizures 3A; familial hemiplegic migraine 3; autism; early infantile epileptic encephalopathy 13; sick sinus syndrome 1; Alzheimer's disease, or sudden unexpected death (SUDEP) in epilepsy by increasing the expression of a target protein or functional RNA by cells of interest, wherein the cells have mRNA containing a nonsense mutation-dependent RNA degradation mechanism-inducing exon (NMD exon mRNA), the NMD exon mRNA encoding a target protein or functional RNA, and the method comprises the step of contacting the cells of interest with a therapeutic agent that binds to a targeting portion of the NMD exon mRNA encoding the target protein or functional RNA, thereby eliminating the nonsense mutation-dependent RNA degradation mechanism-inducing exon from the NMD exon mRNA encoding the target protein or functional RNA, thereby increasing the level of processed mRNA encoding the target protein or functional RNA and increasing the expression of the target protein or functional RNA in the cells of interest.

[0192]

[0273] Embodiment 79. The method described in Embodiment 78, wherein the target protein is SCN1A. Law.

[0274] Embodiment 80. m containing an exon that induces a nonsense mutation-dependent RNA degradation mechanism. A method for increasing the expression of the SCN1A protein in cells having RNA (NMD exon mRNA) that encodes the SCN1A protein, comprising the step of contacting the cells with a drug that binds to a targeting region of the NMD exon mRNA encoding the SCN1A protein, thereby eliminating the nonsense mutation-dependent RNA degradation mechanism-inducing exon from the NMD exon mRNA encoding the SCN1A protein, thereby increasing the level of processed mRNA encoding the SCN1A protein and increasing the expression of the SCN1A protein in the cells.

[0193]

[0275] Embodiment 81. A disease or condition in the subject requiring it, in the cells of the subject. A method for treating a disease or condition by increasing the expression of the SCN1A protein, comprising the step of contacting the target cells with a therapeutic agent that binds to a targeting portion of the nonsense mutation-dependent RNA degradation mechanism-inducing exon mRNA encoding the SCN1A protein or functional SCN1A RNA, thereby eliminating the nonsense mutation-dependent RNA degradation mechanism-inducing exon from the NMD exon mRNA encoding the SCN1A protein or functional SCN1A RNA, thereby increasing the level of processed mRNA encoding the SCN1A protein or functional SCN1A RNA, thereby increasing the expression of the SCN1A protein or functional SCN1A RNA in control cells, wherein the disease or condition is related to mutations in genes other than the SCN1A gene, abnormal expression of proteins encoded by genes other than the SCN1A gene, or abnormal expression of RNA encoded by genes other than the SCN1A gene.

[0194]

[0276] Embodiment 82. The symptoms of the disease or condition are present in approximately half, one-third, one-quarter, The method according to Embodiment 81, which reduces the amount to one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, one-tenth, or less.

[0195]

[0277] Embodiment 83. Symptoms of a disease or condition include increased expression of the SCN1A protein. The method according to embodiment 81 or 82, which also reduces by approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.

[0196]

[0278] Embodiment 84. The progression of a disease or condition leads to an increase in the expression of the SCN1A protein. The method according to any one of embodiments 81 to 83, which also reduces the amount to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, or less.

[0197]

[0279] Embodiment 85. The progression of a disease or condition leads to an increase in the expression of the SCN1A protein. The method according to any one of embodiments 81 to 84, which also reduces by approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.

[0198]

[0280] Embodiment 86. Expression of SCN1A protein or functional SCN1A RNA The method according to any one of embodiments 81 to 85, wherein increasing compensates for mutations in genes other than the SCN1A gene, abnormal expression of proteins encoded by genes other than the SCN1A gene, or abnormal expression of RNA encoded by genes other than the SCN1A gene.

[0199]

[0281] Embodiment 87. The disease or condition is early infantile epileptic encephalopathy (encephalopathy) The method according to any one of embodiments 81 to 86, wherein ophathy)13.

[0282] Embodiment 88. Embodiment 81, wherein the subject has a mutation in the SCN8A gene. The method described in any one of 87.

[0200]

[0283] Embodiment 89. Embodiments 81 to 8, wherein the disease or condition is sick sinus syndrome 1. The method described in any one of the six.

[0284] Embodiment 90. Embodiment 81, in which the subject has a mutation in the SCN5A gene. The method described in either 86 or 88.

[0201]

[0285] Embodiment 91. From Embodiment 81, wherein the disease or condition is Alzheimer's disease. The method described in any one of the 86.

[0286] Embodiment 92. A method for treating a disease or condition in an object that requires it. The method comprises the step of administering a composition containing an antisense oligomer to a subject, wherein the antisense oligomer is present in an amount of at least 80% to 85% relative to the intron 20 of SCN1A. A method comprising a sequence of at least eight consecutive nucleotides that are 90%, 95%, 97%, or 100% complementary.

[0202]

[0287] Embodiment 93. A method for treating a disease or condition in an object that requires it. A method comprising the step of administering to a subject a composition comprising an antisense oligomer, wherein the antisense oligomer comprises a sequence of at least eight consecutive nucleotides that is at least 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs. 7 to 10.

[0203]

[0288] Embodiment 94. A nonsense mutation-dependent RNA degradation mechanism-inducing exon targets the target tongue. The method according to any one of embodiments 78 to 93, wherein the mRNA is excised from an NMD exon mRNA encoding a protein or functional RNA.

[0204]

[0289] Embodiment 95. The target protein induces a nonsense mutation-dependent RNA degradation mechanism. The method according to any one of embodiments 78 to 94, which does not include an amino acid sequence encoded by xon.

[0205]

[0290] Embodiment 96. Embodiment 78 or later, in which the target protein is a full-length target protein. The method described in any one of the 95.

[0291] Embodiment 97. The drug is complementary to the targeting region of the NMD exon mRNA. The method according to any one of embodiments 78 to 96, wherein the antisense oligomer (ASO) is an antisense oligomer (ASO).

[0206]

[0292] Embodiment 98. The embodiments of Embodiments 78 to 97, wherein mRNA is an mRNA precursor. Either one of the methods.

[0293] Embodiment 99. The contact step is a step in which the therapeutic agent is brought into contact with mRNA. The method according to any one of embodiments 78 to 98, comprising the mRNA located in the nucleus of a cell.

[0207]

[0294] Embodiment 100. A target protein or functional RNA is a target in a target The method according to any one of embodiments 78 to 99, for correcting a protein or functional RNA deficiency.

[0208]

[0295] Embodiment 101. Cells are affected by the amount or activity of the SCN1A protein deficiency. The method according to any one of embodiments 78 to 100, wherein the object is in or originates from having a state caused by the above.

[0209]

[0296] Embodiment 102. The amount of deficiency of the target protein corresponds to haploinsufficiency of the target protein. The method according to any one of embodiments 78 to 101, wherein the target has a first allele encoding a functional target protein and a second allele that does not produce the target protein or produces it at a reduced level, or a second allele encoding a non-functional or partially functional target protein, and the antisense oligomer binds to the targeting portion of the NMD exon mRNA transcribed from the first allele.

[0210]

[0297] Embodiment 103. The subject is a result of a deficiency in the quantity or function of the target protein. It has a condition caused by a disorder that occurs as a result, (a) (i) The target protein is produced at a reduced level compared to production from the wild-type allele. (ii) The target protein is produced in a form with reduced function compared to an equivalent wild-type protein, or (iii) The target protein is not produced. The first variant allele, and (b) (i) The target protein is produced at a reduced level compared to production from the wild-type allele. (ii) The target protein is produced in a form with reduced function compared to an equivalent wild-type protein, or (iii) The target protein is not produced. Second mutant allele The method according to any one of embodiments 78 to 101, wherein the subject has a first mutant allele (a)(iii), the second mutant allele is (b)(i) or (b)(ii), and if the subject has a second mutant allele (b)(iii), the first mutant allele is (a)(i) or (a)(ii), and the NMD exon mRNA is transcribed from the first mutant allele which is (a)(i) or (a)(ii), and / or the second allele which is (b)(i) or (b)(ii).

[0211]

[0298] Embodiment 104. Target protein is reduced compared to an equivalent wild-type protein. The method according to Embodiment 103, which is produced in a form having the function described above.

[0299] Embodiment 105. The target protein is complete compared to an equivalent wild-type protein. The method according to Embodiment 103, which is produced in a functional form.

[0212]

[0300] Embodiment 106. The targeting region of the NMD exon mRNA is affected by a nonsense mutation. The method according to any one of embodiments 78 to 105, located within an exon that induces the RNA degradation mechanism.

[0213]

[0301] Embodiment 107. The targeting portion of the NMD exon mRNA is affected by a nonsense mutation. The method according to any one of embodiments 78 to 105, wherein the exon is either upstream or downstream of the RNA degradation mechanism-inducing exon.

[0214]

[0302] Embodiment 108. NMD exon mRNA is sequence numbers 2, 7-10, 12, The method according to any one of embodiments 78 to 107, comprising sequences having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to any one of 17 to 20.

[0215]

[0303] Embodiment 109. NMD exon mRNA is sequence numbers 1, 3-6, 11, The method according to any one of embodiments 78 to 107, encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to 13-16.

[0216]

[0304] Embodiment 110. The targeting portion of NMD exon mRNA is sequence numbers 2, 7~ The method according to any one of embodiments 78 to 107, comprising a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to a region comprising at least eight consecutive nucleic acids 10, 12, and 17-20.

[0217]

[0305] Embodiment 111. The drug is an antisense oligomer (ASO), and the ASO is The method according to any one of embodiments 78 to 110, comprising an array that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 21 to 114.

[0218]

[0306] Embodiment 112. The targeting portion of the NMD exon mRNA is the SCN1A The sense mutation-dependent RNA degradation mechanism is located within the induction exon 20x of embodiments 78 to 105. Any one of the following methods.

[0219]

[0307] Embodiment 113. The drug is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 112, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 42-50 or 231-239.

[0220]

[0308] Embodiment 114. The targeting portion of the NMD exon mRNA is the SCN1A The method according to any one of embodiments 78 to 105, wherein the exon 20x is located upstream or downstream of the sense mutation-dependent RNA degradation mechanism induction exon.

[0221]

[0309] Embodiment 115. The drug is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 114, comprising sequences that are at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 21-38, 53-67, 210-227, or 242-256.

[0222]

[0310] Embodiment 116. The targeting portion of the NMD exon mRNA is the exon of SCN1A. The method according to any one of embodiments 78 to 105, including an exon-intron junction of Son 20x.

[0223]

[0311] Embodiment 117. The drug is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 116, comprising sequences that are at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 39-41, 51, 52, 228-230, 240, or 241.

[0224]

[0312] Embodiment 118. The targeting portion of the NMD exon mRNA is the nan of Scn1a. The method according to any one of embodiments 78 to 105, located within sense mutation-dependent RNA degradation mechanism induction exon 21x.

[0225]

[0313] Embodiment 119. The drug is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 118, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 89 to 97.

[0226]

[0314] Embodiment 120. The targeting portion of the NMD exon mRNA is the nan of Scn1a. The method according to any one of embodiments 78 to 105, wherein the exon 21x is either upstream or downstream of the sense mutation-dependent RNA degradation mechanism induction exon 21x.

[0227]

[0315] Embodiment 121. The drug is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 120, comprising sequences that are at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 68-85 and 100-114.

[0228]

[0316] Embodiment 122. The targeting portion of the NMD exon mRNA is the exon of Scn1a. The method according to any one of embodiments 78 to 105, including an exon-intron junction of Son 21x.

[0229]

[0317] Embodiment 123. The drug is an antisense oligomer (ASO), and the ASO is The method according to Embodiment 122, comprising sequences that are at least approximately 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 86-88 and 98-99.

[0230]

[0318] Embodiment 124. The target protein produced is a full-length protein or wild-type protein. The method according to any one of embodiments 78 to 123, wherein the protein is a protein.

[0319] Embodiment 125. Produced in cells in contact with antisense oligomers. The total amount of processed mRNA encoding the target protein or functional RNA produced in control cells was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, and 2 times greater than the total amount of processed mRNA encoding the target protein or functional RNA produced in control cells. The method according to any one of embodiments 78 to 124, increasing by approximately 6 times, approximately 2 to approximately 7 times, approximately 2 to approximately 8 times, approximately 2 to approximately 9 times, approximately 3 to approximately 6 times, approximately 3 to approximately 7 times, approximately 3 to approximately 8 times, approximately 3 to approximately 9 times, approximately 4 to approximately 7 times, approximately 4 to approximately 8 times, approximately 4 to approximately 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

[0231]

[0320] Embodiment 126. Produced by cells in contact with antisense oligomers The total amount of target protein produced by the control cells is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 7 times, 2 to 8 times, and 2 to 9 times compared to the total amount of target protein produced by the control cells. The method according to any one of embodiments 78 to 124, which increases by a factor of 1 / 2, approximately 3 to approximately 6 times, approximately 3 to approximately 7 times, approximately 3 to approximately 8 times, approximately 3 to approximately 9 times, approximately 4 to approximately 7 times, approximately 4 to approximately 8 times, approximately 4 to approximately 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

[0232]

[0321] Embodiment 127. The drug is an antisense oligomer (ASO), and the antisense The method according to any one of embodiments 78 to 126, wherein the lance oligomer includes a skeletal modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0233]

[0322] Embodiment 128. The drug is an antisense oligomer (ASO), and the antisense The method according to any one of Embodiments 78 to 127, wherein the lance oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0234]

[0323] Embodiment 129. The drug is an antisense oligomer (ASO), and the antisense The method according to any one of embodiments 78 to 128, wherein the lance oligomer comprises at least one modified sugar moiety.

[0235]

[0324] Embodiment 130. The method according to Embodiment 129, wherein each sugar portion is a modified sugar portion. .

[0325] Embodiment 131. The drug is an antisense oligomer (ASO), and the antisense Nucleic acid oligomers are 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, and 11 to 25 nucleic acid bases. The method according to any one of embodiments 78 to 130, comprising 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or 12 to 15 nucleic acid bases.

[0236]

[0326] Embodiment 132. The drug is an antisense oligomer (ASO), and the antisense The method according to any one of embodiments 78 to 131, wherein the lance oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeting region of the protein-coding NMD exon mRNA.

[0237]

[0327] Embodiment 133. Assessment of SCN1A mRNA or protein expression. The method according to any one of embodiments 78 to 132, further comprising the step of doing the following.

[0328] Embodiment 134. Dravet syndrome; generalized epileptic febrile seizures plus type 2; familial fever The method according to any one of Embodiments 1 to 133, wherein a seizure 3A; familial hemiplegic migraine 3; autism; early infantile epileptic encephalopathy 13; sick sinus syndrome 1; Alzheimer's disease; or sudden unexpected death (SUDEP) in epilepsy is treated, and an antisense oligomer binds to a targeting portion of SCN1A NMD exon mRNA, the targeting portion being within a sequence selected from SEQ ID NOs: 7-10 and 17-20.

[0238]

[0329] Embodiment 135. Any one of Embodiments 78 to 134, wherein the subject is a human. Methods used.

[0330] Embodiment 136. Any of Embodiments 78 to 135, wherein the subject is a non-human animal. One method.

[0239]

[0331] Embodiment 137. Embodiments 78 to 13, in which the subject is a fetus, embryo, or child. The method described in any one of the six.

[0332] Embodiment 138. Any of Embodiments 78 to 137, wherein the cells are ex vivo. One method.

[0240]

[0333] Embodiment 139. The therapeutic agent is administered via intrathecal injection, intraventricular injection, intraperitoneal injection, or intramuscular injection. The method according to any one of embodiments 78 to 138, administered by intramuscular injection, subcutaneous injection, intravitreal injection, or intravenous injection.

[0241]

[0334] Embodiment 140. An embodiment further comprising the step of administering a second therapeutic agent to a target. The method described in any of forms 78 to 139.

[0335] Embodiment 141. The method according to Embodiment 140, wherein the second therapeutic agent is a low molecular weight agent. .

[0242]

[0336] Embodiment 142. The method according to Embodiment 140, wherein the second therapeutic agent is ASO. .

[0337] Embodiment 143. The ASO corresponds to any one of sequence numbers 115 to 161. The method according to Embodiment 142, comprising sequences that are at least about 80%, 85%, 90%, 95%, 97%, or 100% identical.

[0243]

[0338] Embodiment 144. Embodiment 140 in which the second therapeutic agent corrects intron retention. The method described in any one of 142.

[0339] Embodiment 145. Used in the method described in any one of Embodiments 78 to 144 Antisense oligomers used in various applications.

[0244]

[0340] Embodiment 146. At least approximately for any one of Sequence IDs 21 to 114 Antisense oligomers containing sequences having 80%, 85%, 90%, 95%, 97%, or 100% sequence identity.

[0245]

[0341] Embodiment 147. The antisense oligomer of Embodiment 145 or 146, A pharmaceutical composition containing excipients.

[0342] Embodiment 148. A method for treating an object that requires it, wherein Embodiment 1 A method comprising the step of administering 47 pharmaceutical compositions, wherein the administration step is by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreous injection, or intravenous injection.

[0246]

[0343] Embodiment 149. Increasing the expression of a target protein or functional RNA by cells. A composition comprising a therapeutic agent for use in a method of treating a disease or condition associated with a deficiency protein or deficiency functional RNA in a subject requiring such treatment, wherein the deficiency protein or deficiency functional RNA is deficient in the subject in terms of quantity or activity, and the target protein is (a) Deficient protein, or (b) Compensatory proteins that functionally increase or replace the deficient protein in the target. Therefore, functional RNA is (c) Deficient RNA, or (d) Compensatory functional RNA that functionally increases or replaces the deficient functional RNA in the target. And,

[0344] The therapeutic agent encodes a target protein or functional RNA, specifically NMD exon m A composition that enhances the elimination of nonsense mutation-dependent RNA degradation mechanism-inducing exons from RNA, thereby increasing the production or activity of a target protein or functional RNA in a subject.

[0247]

[0345] Embodiment 150. A method for treating a disease or condition in a subject that requires it. A composition comprising a therapeutic agent for use in a law, wherein the method comprises the step of regulating the expression of the SCN1A protein by cells of interest, wherein the cells have mRNA encoding the SCN1A protein, which is mRNA containing a nonsense mutation-dependent RNA degradation mechanism-inducing exon (NMD exon mRNA), and the method comprises the step of contacting the cells with the therapeutic agent, thereby regulating the elimination of the nonsense mutation-dependent RNA degradation mechanism-inducing exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of processed mRNA encoding the SCN1A protein, and regulating the expression of the SCN1A protein in cells of interest.

[0248]

[0346] Embodiment 151. The disease or condition is Dravet syndrome (DS); severe infantile myocclusion A composition according to Embodiment 150, selected from the group consisting of Ronnie's epilepsy (SMEI) borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infantile epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic ataxia; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; pediatric alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; autism; or familial hemiplegic migraine3; and Alzheimer's disease.

[0249]

[0347] Embodiment 152. SCN1A protein and NMD exon mRNA are SC The composition according to embodiment 150 or 151, encoded by the N1A gene.

[0348] Embodiment 153. Nonsense mutation-dependent RNA degradation mechanism-inducing exons, SCN A composition according to any one of embodiments 149 to 152, which is excised from NMD exon mRNA encoding the 1A protein.

[0250]

[0349] Embodiment 154. The SCN1A protein is a nonsense mutation-dependent RNA degradation machine. A composition according to any one of embodiments 149 to 153, which does not contain an amino acid sequence encoded by a structural induction exon.

[0251]

[0350] Embodiment 155. The SCN1A protein is a full-length SCN1A protein. The composition according to any one of embodiments 149 to 154.

[0351] Embodiment 156. The therapeutic agent interacts with the targeting region of the NMD exon mRNA. A composition according to any one of embodiments 149 to 155, wherein the composition is a complementary antisense oligomer (ASO).

[0252]

[0352] Embodiment 157. The therapeutic agent is an antisense oligomer (ASO), and anti The composition according to any one of embodiments 149 to 156, wherein the sense oligomer targets a portion of NMD exon mRNA located within a nonsense mutation-dependent RNA degradation mechanism-inducing exon.

[0253]

[0353] Embodiment 158. The therapeutic agent is an antisense oligomer (ASO), and anti The composition according to any one of embodiments 149 to 156, wherein the sense oligomer targets a portion of NMD exon mRNA located upstream or downstream of a nonsense mutation-dependent RNA degradation mechanism-inducing exon.

[0254]

[0354] Embodiment 159. Embodiments 149 to 1, wherein the target protein is SCN1A. The composition described in any one of 58.

[0355] Embodiment 160. NMD exon mRNA is sequence numbers 2, 7-10, 12, The composition according to Embodiment 159, comprising sequences having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to any one of 17 to 20.

[0255]

[0356] Embodiment 161. NMD exon mRNA is sequence numbers 1, 3-6, 11, The composition according to Embodiment 159, encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to 13-16.

[0256]

[0357] Embodiment 162. The targeting portion of NMD exon mRNA is sequence numbers 2, 7~ The composition according to Embodiment 159, comprising a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to a region comprising at least eight consecutive nucleic acids numbered 10, 12, and 17-20.

[0257]

[0358] Embodiment 163. The targeting portion of the NMD exon mRNA is (i) nonsense A composition according to any one of Embodiments 159 to 162, comprising (ii) located within mutation-dependent RNA degradation mechanism-inducing exon 20x, either upstream or downstream of nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x, or (iii) an exon-intron junction of nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x.

[0258]

[0359] Embodiment 164. The therapeutic agent is an antisense oligomer (ASO), and ASO Embodiments 159 to 163 include sequences that are at least approximately 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of sequence numbers 21 to 114. The composition described in any one of the following.

[0259]

[0360] Embodiment 165. A disease or condition in which a loss-of-function mutation occurs in Nav1.1. The composition described in any one of embodiments 149 to 164 is thus induced.

[0361] Embodiment 166. The disease or condition is related to the haploinsufficiency of the SCN1A gene. The composition according to any one of Embodiments 149 to 165, wherein the subject comprises a first allele encoding a functional SCN1A, and a second allele in which SCN1A is not produced or is produced at a reduced level, or a second allele encoding a non-functional SCN1A or a partially functional SCN1A.

[0260]

[0362] Embodiment 167. A disease or condition, optionally resulting in loss of function in Nav1.1. A composition according to any one of embodiments 149 to 166, which is an encephalopathy induced by a dysmorphic mutation.

[0261]

[0363] Embodiment 168. The composition according to Embodiment 167, wherein the encephalopathy is epileptic encephalopathy. thing.

[0364] Embodiment 169. The disease or condition is Dravet syndrome (DS); severe infantile myocclusion The composition according to Embodiment 165 or 166, wherein Ronnie's epilepsy (SMEI) borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic ataxia; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; pediatric alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; autism; or infant malignant migratory partial seizures.

[0262]

[0365] Embodiment 170. GEFS+ is generalized epileptic febrile seizures plus type 2. The composition described in Form 168.

[0366] Embodiment 171. The febrile seizure is familial febrile seizure 3A, as described in Embodiment 168. The composition of the material.

[0263]

[0367] Embodiment 172. SMEB is SMEB without generalized spikes (SMEB-S The composition according to Embodiment 168, wherein W) SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more SMEI features (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0264]

[0368] Embodiment 173. The therapeutic agent is a processing agent that encodes the SCN1A protein. A composition according to any one of embodiments 165 to 172, which promotes the elimination of NMD exons from mRNA and increases the expression of SCN1A in cells.

[0265]

[0369] Embodiment 174. The therapeutic agent is an antisense oligomer (ASO), and ASO The composition according to any one of embodiments 165 to 173, wherein the sequence is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 22-24, 26, 27, 29-35, 37-62, or 64-67.

[0266]

[0370] Embodiment 175. A disease or condition in which a gain-of-function mutation occurs in Nav1.1. The composition described in any one of embodiments 149 to 164 is thus induced.

[0371] Embodiment 176. The target is an allergen produced at a level in which SCN1A is increased. Or the composition according to any one of embodiments 149 to 164 or 175, having an allele encoding a mutant SCN1A that induces increased activity of Nav1.1 in cells thing.

[0267]

[0372] Embodiment 177. Embodiments 149 to 164, in which the disease or condition is migraine. The composition described in any one of 175 or 176.

[0373] Embodiment 178. The migraine is familial hemiplegic migraine 3, as in Embodiment 177. The composition described.

[0268]

[0374] Embodiment 179. The disease or condition is Nav1.1 diathesis epilepsy, A composition according to any one of forms 149 to 164, 175, or 176.

[0375] Embodiment 180. The therapeutic agent is a processing agent that encodes the SCN1A protein. A composition according to any one of embodiments 149 to 164 or 175 to 179, which inhibits the elimination of NMD exons from the mRNA and reduces the expression of SCN1A in cells.

[0269]

[0376] Embodiment 181. The therapeutic agent is an antisense oligomer (ASO), and ASO The composition according to any one of embodiments 149 to 164 or 175 to 180, comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 21, 25, 28, 36, or 63.

[0270]

[0377] Embodiment 182. Processes encoding a target protein or functional RNA. The composition according to any one of Embodiments 149 to 181, wherein the mRNA that is extracted is full-length mature mRNA or wild-type mature mRNA.

[0271]

[0378] Embodiment 183. The target protein produced is a full-length protein or wild-type protein. A composition according to any one of embodiments 149 to 182, wherein the composition is a protein.

[0379] Embodiment 184. The therapeutic agent is an antisense oligomer (ASO), and anti The composition according to any one of Embodiments 149 to 183, wherein the sense oligomer includes a skeletal modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0272]

[0380] Embodiment 185. The therapeutic agent is an antisense oligomer (ASO), and the A The composition according to any one of embodiments 149 to 184, wherein the antisense oligomer is an antisense oligonucleotide.

[0273]

[0381] Embodiment 186. The therapeutic agent is an antisense oligomer (ASO), and anti The composition according to any one of Embodiments 149 to 185, wherein the sense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0274]

[0382] Embodiment 187. The therapeutic agent is an antisense oligomer (ASO), and anti The composition according to any one of embodiments 149 to 186, wherein the sense oligomer comprises at least one modified sugar moiety.

[0275]

[0383] Embodiment 188. The composition according to Embodiment 187, wherein each sugar portion is a modified sugar portion. thing.

[0384] Embodiment 189. The therapeutic agent is an antisense oligomer (ASO), and anti Sense oligomers are 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 A composition according to any one of embodiments 149 to 188, comprising a nucleic acid base, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or 12 to 15 nucleic acid bases.

[0276]

[0385] Embodiment 190. A composition comprising an antisense oligomer, wherein the antisense A composition comprising a sequence of at least eight consecutive nucleotides, wherein the oligomer is at least 80%, 85%, 90%, 95%, 97%, or 100% complementary to intron 20 of SCN1A.

[0277]

[0386] Embodiment 191. A composition comprising an antisense oligomer, wherein the antisense oligomer is present. A composition comprising a sequence of at least eight consecutive nucleotides, wherein the oligomer is at least 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 7-10.

[0278]

[0387] Embodiment 192. Treatment of any of the compositions described in Embodiments 149 to 191. A pharmaceutical composition comprising an agent and an excipient.

[0388] Embodiment 193. A method for treating an object that requires it, wherein Embodiment 1 A method comprising the step of administering 92 pharmaceutical compositions, wherein the administration step is by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreous injection, or intravenous injection.

[0279]

[0389] Embodiment 194. Hybridizing the target sequence of the SCN1A mRNA transcript A pharmaceutical composition comprising an antisense oligomer and a pharmaceutically acceptable excipient, wherein the SCN1A mRNA transcript contains a nonsense mutation-dependent RNA degradation mechanism-inducing exon, and the antisense oligomer induces the exclusion of the nonsense mutation-dependent RNA degradation mechanism-inducing exon from the SCN1A mRNA transcript.

[0280]

[0390] Embodiment 195. The SCN1A mRNA transcript is the SCN1A NMD exon The pharmaceutical composition according to Embodiment 194, which is an mRNA transcript.

[0391] Embodiment 196. Targeting portion of SCN1A NMD exon mRNA transcript The pharmaceutical composition according to Embodiment 194 or 195, wherein (i) is located within the nonsense mutation-dependent RNA degradation mechanism inducing exon 20x, (ii) is located upstream or downstream of the nonsense mutation-dependent RNA degradation mechanism inducing exon 20x, or (iii) includes an exon-intron junction of the nonsense mutation-dependent RNA degradation mechanism inducing exon 20x.

[0281]

[0392] Embodiment 197. The SCN1A NMD exon mRNA transcript is SEQ ID NO: 1 The pharmaceutical composition according to Embodiment 194 or 196, encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of 3-6, 11, and 13-16.

[0282]

[0393] Embodiment 198. The SCN1A NMD exon mRNA transcript is SEQ ID NO: 2 The pharmaceutical composition according to Embodiment 194 or 196, comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any one of 7-10, 12, and 17-20.

[0283]

[0394] Embodiment 199. The antisense oligomer is phosphorothioate-linked or A pharmaceutical composition according to Embodiment 194, comprising a skeletal modification including phosphorodiamidate linkage.

[0395] Embodiment 200. Antisense oligomer is an antisense oligonucleotide. The pharmaceutical composition according to Embodiment 194.

[0284]

[0396] Embodiment 201. The antisense oligomer is phosphorodiamidate morphol. The pharmaceutical composition according to Embodiment 194, comprising a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0285]

[0397] Embodiment 202. The antisense oligomer comprises at least one modified sugar moiety The pharmaceutical composition described in Embodiment 194.

[0398] Embodiment 203. The antisense oligomer comprises 8 to 50 nucleic acid bases, 8 to 4 0 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases A pharmaceutical composition according to embodiment 194, comprising 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or up to 12 to 15 nucleic acid bases.

[0286]

[0399] Embodiment 204. The antisense oligomer is SCN1A NMD exon m The pharmaceutical composition according to Embodiment 194 or 195, which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeted portion of the RNA transcript.

[0287]

[0400] Embodiment 205. Targeting portion of SCN1A NMD exon mRNA transcript The pharmaceutical composition according to Embodiment 194 or 195, wherein the sequence is located within a sequence selected from Sequence IDs 2, 7-10, 12, and 17-20.

[0288]

[0401] Embodiment 206. The antisense oligomer is any of SEQ ID NOs. 21 to 114. The pharmaceutical composition according to Embodiment 194, comprising a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to one of them.

[0289]

[0402] Embodiment 207. An antisense oligomer is selected from SEQ ID NOs: 21-114. A pharmaceutical composition according to Embodiment 194, comprising the nucleotide sequence.

[0403] Embodiment 208. Intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection A pharmaceutical composition according to any one of embodiments 194 to 207, formulated for injection, intravitreal injection, or intravenous injection.

[0290]

[0404] Embodiment 209. Inducing the processing of a deficient SCN1A mRNA transcript, A method for producing a fully processed SCN1A mRNA transcript encoding a functional form of the SCN1A protein by facilitating the removal of nonsense mutation-dependent RNA degradation mechanism-inducing exons,

[0405] (a) A step of bringing the antisense oligomer into contact with the target cells,

[0406] (b) Hybridize antisense oligomers into deficient SCN1A mRNA transcripts The soybean step involves sowing a deficient SCN1A mRNA transcript that can encode a functional form of the SCN1A protein and contains at least one nonsense mutation-dependent RNA molecule. Steps including the solution mechanism-inducing exon,

[0407] (c) At least one nonsense mutation in the deficient SCN1A mRNA transcript. The steps include removing the RNA degradation mechanism-dependent inducing exon to produce a fully processed SCN1A mRNA transcript encoding the functional form of the SCN1A protein, and

[0408] (d) From fully processed SCN1A mRNA transcript Steps to translate the functional form of a protein A method that includes this.

[0291]

[0409] Embodiment 210. The amount or activity of the SCN1A protein is deficient. A method for treating a subject having a condition to be induced, comprising the step of administering to the subject an antisense oligomer containing a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 24-114.

[0292]

[0410] Embodiment 211. Gene expression of target protein or functional RNA by cells. A method for screening drugs that increase, wherein cells have mRNA containing nonsense mutation-dependent RNA degradation mechanism-inducing exons (NMD exon mRNA), and the NMD exon mRNA encodes a target protein or functional RNA, and the method (a) A step of contacting the first cell with a test drug that targets NMD exon mRNA, (b) A step of bringing the control drug into contact with the second cell, (c) A step of determining a first level in a first cell, wherein the first level is the level of (i) an RNA transcript encoded by NMD exon mRNA that does not contain RNA degradation mechanism-inducing exons, or (ii) a protein encoded by NMD exon mRNA that does not contain an amino acid sequence encoded by RNA degradation mechanism-inducing exons. (d) A step of determining a second level in a second cell, wherein the second level is the level of (i) an RNA transcript encoded by NMD exon mRNA that does not contain RNA degradation mechanism-inducing exons, or (ii) a protein encoded by NMD exon mRNA that does not contain an amino acid sequence encoded by RNA degradation mechanism-inducing exons. (Level 1 is higher than Level 2), and (e) Step of selecting the test reagent Methods that include...

[0293]

[0411] Embodiment 212. Gene expression of target protein or functional RNA by cells. A method for screening drugs that increase, wherein cells have mRNA containing nonsense mutation-dependent RNA degradation mechanism-inducing exons (NMD exon mRNA), and the NMD exon mRNA encodes a target protein or functional RNA, and the method (a) A step of contacting the first cell with a test drug that targets NMD exon mRNA, (b) A step of bringing the control drug into contact with the second cell, (c) A step of determining a first level in a first cell, wherein the first level is the level of (i) an RNA transcript encoded by NMD exon mRNA containing an RNA degradation mechanism-inducing exon, or (ii) a protein encoded by NMD exon mRNA containing an amino acid sequence encoded by an RNA degradation mechanism-inducing exon. (d) A step of determining a second level in a second cell, wherein the second level is encod...

Claims

1. A method for regulating the expression of the SCN1A protein in cells having mRNA encoding the SCN1A protein, comprising the step of contacting a cell with a therapeutic agent, wherein the therapeutic agent modulates the splicing of NMD exons from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of processed mRNA encoding the SCN1A protein and regulating the expression of the SCN1A protein in the cell.

2. A method for treating a disease or condition in a subject requiring treatment of the disease or condition by regulating the expression of the SCN1A protein in cells of the subject, comprising the step of contacting the cells of the subject with a therapeutic agent containing a nonsense mutation-dependent mRNA degradation mechanism-inducing exon (NMD exon) that modulates the splicing of NMD exons from mRNA in the cells encoding SCN1A, thereby regulating the level of processed mRNA encoding the SCN1A protein and regulating the expression of the SCN1A protein in cells of the subject.

3. The treatment drug, (a) Binds to the target region of the NMD exon mRNA encoding SCN1A, (b) Regulate the binding of factors involved in splicing NMD exon mRNA, or (c) Combination of (a) and (b) The method according to claim 1 or 2.

4. The method according to claim 3, wherein the therapeutic agent interferes with the binding of factors involved in the splicing of NMD exons from the region of the targeted area.

5. The method according to claim 3, wherein the targeting portion is located proximal to the NMD exon.

6. The method according to claim 5, wherein the targeting region is located at a maximum of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides upstream of the 5' end of the NMD exon.

7. The method according to claim 5, wherein the targeting region is located upstream of the 5' end of the NMD exon by at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, and about 1 nucleotide.

8. The method according to claim 5, wherein the targeting region is located at a maximum of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides downstream of the 3' end of the NMD exon.

9. The method according to claim 5, wherein the targeting portion is located at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, and about 1 nucleotide downstream of the 3' end of the NMD exon.

10. The method according to claim 3, wherein the targeting portion is located in an intron region between two canonical exon regions of an NMD exon mRNA encoding SCN1A, and the intron region contains an NMD exon.

11. The method according to claim 3, wherein the targeted portion at least partially overlaps with the NMD exon.

12. The method according to claim 3, wherein the targeted portion at least partially overlaps with an intron upstream of the NMD exon.

13. The method according to claim 3, wherein the targeted portion includes a 5'NMD exon-intron junction or a 3'NMD exon-intron junction.

14. The method according to claim 3, wherein the targeting portion is located within an NMD exon.

15. The method according to claim 3, wherein the targeted portion comprises approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of an NMD exon.

16. The method according to claim 1 or 2, wherein the NMD exon mRNA encoding SCN1A comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any one of sequence numbers 2 or 7-10.

17. The method according to claim 1 or 2, wherein the NMD exon mRNA encoding SCN1A is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with SEQ ID NOs: 1 or 3-6.

18. The method according to claim 5, wherein the targeting region is located at a maximum of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides upstream of the genomic region GRCh37 / hg19:chr2:166,863,803.

19. The method according to claim 5, wherein the targeting region is located approximately 1,000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, 40 nucleotides, 30 nucleotides, 20 nucleotides, 10 nucleotides, 5 nucleotides, 4 nucleotides, 2 nucleotides, and 1 nucleotide upstream of the genomic site GRCh37 / hg19:chr2:166,863,803.

20. The method according to claim 5, wherein the targeting region is located at a maximum of approximately 1,500 nucleotides, approximately 1,000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides downstream of genomic site GRCh37 / hg19:chr2:166,863,740.

21. The method according to claim 5, wherein the targeting region is located approximately 1,000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides, approximately 40 nucleotides, approximately 30 nucleotides, approximately 20 nucleotides, approximately 10 nucleotides, approximately 5 nucleotides, approximately 4 nucleotides, approximately 2 nucleotides, and approximately 1 nucleotide downstream of genomic site GRCh37 / hg19:chr2:166,863,740.

22. The method according to claim 3, wherein the targeting portion of the NMD exon mRNA encoding SCN1A comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to a region comprising at least eight consecutive nucleic acids of sequence number 2 or 7-10.

23. The method according to claim 1 or 2, wherein the therapeutic agent is an antisense oligomer (ASO), and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 21-67, 210-256, or 304-379.

24. The method according to claim 3, wherein the targeting portion of the NMD exon mRNA encoding SCN1A is located within the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A.

25. The method according to claim 24, wherein the therapeutic agent is an antisense oligomer (ASO), and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 42-50 or 231-239.

26. The method according to claim 3, wherein the targeting region of the NMD exon mRNA encoding SCN1A is located upstream or downstream of the nonsense mutation-dependent RNA degradation mechanism-inducing exon 20x of SCN1A.

27. The method according to claim 26, wherein the therapeutic agent is an antisense oligomer (ASO), and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 21-38, 53-67, 210-227, or 242-256.

28. The method according to claim 3, wherein the targeting portion of the NMD exon mRNA includes the exon-intron junction of exon 20x of SCN1A.

29. The method according to claim 28, wherein the therapeutic agent is an antisense oligomer (ASO), and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs. 39-41, 51, 52, 228-230, 240, or 241.

30. The method according to claim 1 or 2, wherein the therapeutic agent promotes the elimination of NMD exons from processed mRNA encoding the SCN1A protein.

31. In cells exposed to the therapeutic agent, the elimination of NMD exons from processed mRNA encoding the SCN1A protein was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, and 1.1 times greater compared to the elimination of NMD exons from processed mRNA encoding the SCN1A protein in control cells. The method according to claim 30, which increases by approximately 9 times, approximately 2 to approximately 5 times, approximately 2 to approximately 6 times, approximately 2 to approximately 7 times, approximately 2 to approximately 8 times, approximately 2 to approximately 9 times, approximately 3 to approximately 6 times, approximately 3 to approximately 7 times, approximately 3 to approximately 8 times, approximately 3 to approximately 9 times, approximately 4 to approximately 7 times, approximately 4 to approximately 8 times, approximately 4 to approximately 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

32. The method according to claim 30, wherein the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in cells.

33. The amount of processed mRNA encoding the SCN1A protein in cells exposed to the therapeutic agent was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, and 2 to 7 times compared to the total amount of processed mRNA encoding the SCN1A protein in control cells. The method according to claim 30, which increases by 5 times, approximately 2 to approximately 6 times, approximately 2 to approximately 7 times, approximately 2 to approximately 8 times, approximately 2 to approximately 9 times, approximately 3 to approximately 6 times, approximately 3 to approximately 7 times, approximately 3 to approximately 8 times, approximately 3 to approximately 9 times, approximately 4 to approximately 7 times, approximately 4 to approximately 8 times, approximately 4 to approximately 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

34. The method according to claim 30, wherein the therapeutic agent increases the expression of the SCN1A protein in cells.

35. The amount of SCN1A produced in cells exposed to the therapeutic agent was approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 7 times, and approximately The method according to claim 30, which increases by 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

36. If the disease or condition is Na ｖ The method according to claim 2, which is induced by a loss-of-function mutation in 1.

1.

37. The disease or condition is associated with haploinsufficiency of the SCN1A gene, and the subject is a first allele encoding functional SCN1A, and a second allele in which SCN1A is not produced or is produced at reduced levels, or non-functional SCN1A or partially functional SCN1A. The method according to claim 36, having a second allele that codes for A.

38. The method according to claim 36, wherein the disease or condition is encephalopathy.

39. The method according to claim 38, wherein the encephalopathy is epileptic encephalopathy.

40. The method according to claim 36, wherein the disease or condition is Dravet syndrome (DS); severe myoclonic epilepsy of infants (SMEI) borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); early infant epileptic encephalopathy13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic astosis; Lennox-Gastaut syndrome; West syndrome; idiopathic spasm; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome1; autism; or malignant partial seizures of infants.

41. The method according to claim 40, wherein GEFS+ is generalized epileptic febrile seizures plus type 2.

42. The method according to claim 40, wherein the febrile seizure is familial febrile seizure 3A.

43. The method according to claim 40, wherein SMEB is SMEB without generalized spike waves (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more SMEI features (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

44. The method according to claim 36, wherein the therapeutic agent promotes the elimination of NMD exons from processed mRNA encoding the SCN1A protein, thereby increasing the expression of SCN1A in cells.

45. The method according to claim 36, wherein the therapeutic agent is an antisense oligomer (ASO), and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs. 22-24, 26, 27, 29-35, 37-62, 64-67, or 304-379.

46. The method according to claim 1 or 2, wherein the therapeutic agent inhibits the elimination of NMD exons from processed mRNA encoding the SCN1A protein.

47. In cells exposed to the therapeutic agent, the elimination of NMD exons from processed mRNA encoding the SCN1A protein was approximately 1 / 1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1 to 1 / 5, 1 / 1 to 1 / 6, 1 / 1 to 1 / 7, 1 / 1 to 1 / 8, 1 / 1 to 1 / 9, and 1 / 2 to 5. The method according to claim 46, which reduces to 1 / 2, about 1 / 2 to about 1 / 6, about 1 / 2 to about 1 / 7, about 1 / 2 to about 1 / 8, about 1 / 2 to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, at least about 1.1, at least about 1.5, at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 5, or at least about 1 / 10.

48. The method according to claim 46, wherein the therapeutic agent reduces the level of processed mRNA encoding the SCN1A protein in cells.

49. The amount of processed mRNA encoding the SCN1A protein in cells exposed to the therapeutic agent was approximately 1 / 1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1 to 1 / 5, 1 / 1 to 1 / 6, 1 / 1 to 1 / 7, 1 / 1 to 1 / 8, 1 / 1 to 1 / 9, 1 / 2 to 1 / 5, or 1 / 2. The method according to claim 46, wherein the amount is reduced to approximately 1 / 6, from approximately 1 / 2 to approximately 1 / 7, from approximately 1 / 2 to approximately 1 / 8, from approximately 1 / 2 to approximately 1 / 9, from approximately 1 / 3 to approximately 1 / 6, from approximately 1 / 3 to approximately 1 / 7, from approximately 1 / 3 to approximately 1 / 8, from approximately 1 / 3 to approximately 1 / 9, from approximately 1 / 4 to approximately 1 / 7, from approximately 1 / 4 to approximately 1 / 8, from approximately 1 / 4 to approximately 1 / 9, at least approximately 1.1, at least approximately 1.5, at least approximately 1 / 2, at least approximately 1 / 2.5, at least approximately 1 / 3, at least approximately 1 / 3.5, at least approximately 1 / 4, at least approximately 1 / 5, or at least approximately 1 / 10.

50. The method according to claim 46, wherein the therapeutic agent reduces the expression of the SCN1A protein in cells.

51. The amount of SCN1A produced in cells exposed to the therapeutic agent was approximately 1 / 1.1 to 1 / 10, 1 / 1.5 to 1 / 10, 1 / 2 to 1 / 10, 1 / 3 to 1 / 10, 1 / 4 to 1 / 10, 1 / 1.5 to 1 / 5, 1 / 1.5 to 1 / 6, 1 / 1.5 to 1 / 7, 1 / 1.5 to 1 / 8, 1 / 1.5 to 1 / 9, 1 / 2 to 1 / 5, 1 / 2 to 1 / 6, 1 / 2 to 1 / 7. The method according to claim 46, wherein the amount is reduced to approximately 1 / 2 to approximately 1 / 8, approximately 1 / 2 to approximately 1 / 9, approximately 1 / 3 to approximately 1 / 6, approximately 1 / 3 to approximately 1 / 7, approximately 1 / 3 to approximately 1 / 8, approximately 1 / 3 to approximately 1 / 9, approximately 1 / 4 to approximately 1 / 7, approximately 1 / 4 to approximately 1 / 8, approximately 1 / 4 to approximately 1 / 9, at least approximately 1.1, at least approximately 1.5, at least approximately 1 / 2, at least approximately 1 / 2.5, at least approximately 1 / 3, at least approximately 1 / 3.5, at least approximately 1 / 4, at least approximately 1 / 5, or at least approximately 1 / 10.

52. If the disease or condition is Na ｖ The method according to claim 2, which is induced by a gain-of-function mutation in 1.

1.

53. The target is an allele produced at a level where SCN1A is increased, or Na in cells. ｖ The method according to claim 52, comprising an allele encoding a mutant SCN1A that induces increased activity of 1.

1.

54. The method according to claim 52, wherein the disease or condition is migraine.

55. The method according to claim 54, wherein the migraine is familial hemiplegic migraine 3.

56. Disease or condition is Na ｖ 1.1 The method according to claim 2, wherein the epilepsy is predisposed.

57. Claim 52, the therapeutic agent inhibits the elimination of NMD exons from processed mRNA encoding the SCN1A protein, thereby reducing the expression of SCN1A in cells. Method of description.

58. The method according to claim 52, wherein the therapeutic agent is an antisense oligomer (ASO), and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs: 21, 25, 28, 36, or 63.

59. The method according to claim 1 or 2, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer includes a skeletal modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

60. The method according to claim 1 or 2, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

61. The method according to claim 1 or 2, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

62. The method according to claim 61, wherein each sugar portion is a modified sugar portion.

63. The therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is composed of 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, and 10 to 30 The method according to claim 1 or 2, comprising nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or 12 to 15 nucleic acid bases.

64. The method according to claim 3, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeting portion of the protein-coding NMD exon mRNA.

65. The method according to claim 1, further comprising the step of assessing SCN1A mRNA or protein expression.

66. The method according to claim 2, wherein the subject is a human.

67. The method according to claim 2, wherein the subject is a non-human animal.

68. The method according to claim 2, wherein the subject is a fetus, embryo, or child.

69. The method according to claim 1 or 2, wherein the cells are ex vivo.

70. The method according to claim 2, wherein the therapeutic agent is administered by target intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreous injection, or intravenous injection.

71. The method according to claim 2, further comprising the step of administering a second therapeutic agent to a target.

72. The method according to claim 71, wherein the second therapeutic agent is a low molecular weight agent.

73. The method according to claim 71, wherein the second therapeutic agent is ASO.

74. The method according to claim 73, wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of sequence numbers 115 to 161.

75. The method according to claim 71, wherein the second therapeutic agent corrects intron retention.

76. The method according to claim 2, wherein the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy, or SCN5A arrhythmia.

77. The method according to claim 30, 32, or 34, wherein the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy, or SCN5A arrhythmia.

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