Antisense oligomers for treating conditions and diseases

Abstract

To provide therapeutic agents capable of treating conditions caused by SCN1A, SCN8A or SCN5A protein deficiency and uses thereof.SOLUTION: Alternative splicing events in SCN1A gene can lead to non-productive mRNA transcripts which in turn can lead to aberrant protein expression, and therapeutic agents which can target the alternative splicing events in SCN1A gene can modulate the expression level of functional proteins in Dravet Syndrome patients and / or inhibit aberrant protein expression. Such therapeutic agents can be used to treat a condition caused by SCN1A, SCN8A or SCN5A protein deficiency.SELECTED DRAWING: Figure 1AB

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 811,511 (filed February 27, 2019), the entire contents of which are incorporated herein by reference. [Background technology]

[0002]

[0002] Nervous system disorders are characterized by dysfunction of ion channels that mediate neuronal excitability, neuronal interactions, and general brain function. Mutations in the SCN1A gene, part of the SCN1A-SCN2A-SCN3A gene cluster that encodes the alpha pore-forming subunit of neuronal voltage-gated sodium channels, have been linked to the development of diseases under the nosology of diseases and conditions, such as Dravet syndrome (DS) (Miller, et al., 1993-2015, GeneReviews, Eds. Pagon RA, et al. Seattle, WA: University of Washington, Seattle, Bookshelf ID: NBK1318, and Mulley, et al., 2005, Hum. Mutat. 25: 535-542). Summary of the Invention

[0003]

[0003] Disclosed in one aspect of the present invention is a method for regulating the expression of SCN1A protein in a cell having an mRNA (NMD exon mRNA) containing a nonsense mutation-dependent RNA degradation-inducing exon and encoding the SCN1A protein, the method comprising contacting the cell with a therapeutic agent, whereby the therapeutic agent regulates the splicing of the NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of the processed mRNA encoding the SCN1A protein, and regulating the expression of the SCN1A protein in the cell, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is from about 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to about 100 nucleotides upstream from the 5' end of the NIE; or from about 100 nucleotides downstream from the 3' end of the NIE to about 1000 nucleotides downstream from the 3' end of the NIE. In some aspects, the therapeutic agent interferes with the binding of a factor involved in the splicing of the NMD exon from the region of the targeted portion. In some aspects, the targeted portion is at most 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 upstream from the 5' end of the NIE. In some aspects, the targeted portion is at least 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 upstream from the 5' end of the NIE.In some embodiments, the targeting moiety is located up to 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 downstream of the 3' end of the NIE. In some embodiments, the targeting moiety is at least 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 downstream of the 3' end of the NIE. It is downstream of the nucleotide, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide. In some embodiments, the therapeutic agent is an antisense oligomer (ASO). In some embodiments, 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: 12 - 731. In some embodiments, the therapeutic agent promotes the elimination of NMD exons from the processed mRNA encoding the SCN1A protein. In some embodiments, the elimination of NMD exons from the processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is about 1.1 - about 10 times, about 1.5 - about 10 times, about 2 - about 10 times, about 3 - about 10 times, about 4 - about 10 times, about 1.1 - about 5 times, about 1.1 - about 6 times, about 1.1 - about 7 times, about 1.1 - about 8 times, about 1.1 - about 9 times, about 2 - about 5 times, about 2 - about 6 times, about 2 - about 7 times, about 2 - about 8 times, about 2 - about 9 times, about 3 - about 6 times, about 3 - about 7 times, about 3 - about 8 times, about 3 - about 9 times, about 4 - about 7 times, about 4 - about 8 times, about 4 - 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 increased compared to the elimination of NMD exons from the processed mRNA encoding the SCN1A protein in control cells. In some embodiments, the therapeutic agent increases the level of the processed mRNA encoding the SCN1A protein in the cell.In some embodiments, the amount of processed mRNA encoding the SCN1A protein in cells contacted with a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increased compared to the total amount of processed mRNA encoding the SCN1A protein in control cells.

[0004]

[0004] Disclosed in certain aspects of the present invention is a method of treating a subject in need of treatment for a disease or disorder by modulating the expression of SCN1A protein in the cells of the subject, the method comprising contacting the cells of the subject with a therapeutic agent that modulates the splicing of a nonsense mutation-dependent mRNA decay-inducing exon (NMD exon) from an mRNA encoding SCN1A that contains an NMD exon in the cell, thereby modulating the level of processed mRNA encoding SCN1A protein and modulating the expression of SCN1A protein in the cells of the subject; wherein the therapeutic agent binds to a targeting portion of the NMD exon mRNA encoding SCN1A, and the targeting portion is from about 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to about 100 nucleotides upstream from the 5' end of the NIE; or from about 100 nucleotides downstream from the 3' end of the NIE to about 1000 nucleotides downstream from the 3' end of the NIE. In some aspects, the therapeutic agent interferes with the binding of factors involved in the splicing of the NMD exon from the region of the targeting portion. In some aspects, the targeting portion is at most 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 upstream from the 5' end of the NIE. In some aspects, the targeting portion is at least 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 upstream from the 5' end of the NIE. The ootide is located approximately 2 nucleotides, approximately 1 nucleotide upstream. In some embodiments, the targeting moiety is at most 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 downstream of the 3' end of the NIE. In some embodiments, the targeting moiety is at least 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 downstream of the 3' end of the NIE. In some embodiments, the therapeutic agent is an antisense oligomer (ASO). In some embodiments, 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: 12-731. In some embodiments, the therapeutic agent promotes the exclusion 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 a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increased compared to the elimination of NMD exons from processed mRNA encoding the SCN1A protein in control cells. In some embodiments, the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in the cells. In some embodiments, the amount of processed mRNA encoding the SCN1A protein in cells contacted with a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increased compared to the total amount of processed mRNA encoding the SCN1A protein in control cells. In some embodiments, the disease or condition is Na. vInduced by loss-of-function mutations in 1.1. In some embodiments, the disease or disorder is associated with haploinsufficiency of the SCN1A gene, where the subject has a first allele encoding functional SCN1A and a second allele that does not produce SCN1A or produces it at reduced levels, or a second allele encoding non-functional SCN1A or partially functional SCN1A. In some embodiments, the disease or disorder is encephalopathy. In some embodiments, the encephalopathy is epileptic encephalopathy. In some embodiments, the disease or disorder is Dravet syndrome (DS); severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); porencephaly syndrome 1; autism; or malignant migrating partial seizures in infancy. In some embodiments, GEFS+ is generalized epilepsy with febrile seizures plus type 2. In some embodiments, the febrile seizure is familial febrile seizure 3A. In some embodiments, SMEB is SMEB without generalized spike-and-wave (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O), or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0005]

[0005] Disclosed in certain embodiments of the present invention is a method of regulating the expression of SCN1A protein in a cell having mRNA encoding SCN1A protein, which is mRNA containing a nonsense mutation-dependent RNA degradation-inducing exon (NMD exon mRNA), the method comprising contacting the cell with a therapeutic agent, whereby the therapeutic agent regulates the splicing of the 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, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE.

[0006]

[0006] Disclosed in certain embodiments of the present invention is a method of treating a subject in need of treating a disease or condition by regulating the expression of SCN1A protein in the cells of the subject, the method comprising contacting the cells of the subject with a therapeutic agent that regulates the splicing of a nonsense mutation-dependent mRNA degradation-inducing exon (NMD exon) from mRNA 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 cells of the subject, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE.

[0007]

[0007] Disclosed in certain embodiments of the present invention 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: 12-731.

[0008]

[0008] In one aspect of the present invention, an antisense oligomer (ASO) consisting of a sequence selected from SEQ ID NOs: 12 to 731 is disclosed.

[0009] In one aspect of the present invention, a method for treating a subject in need of treating a disease or condition by modulating the expression of SCN1A protein in the cells of the subject is disclosed, the method comprising an ASO comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 12 to 731; or contacting an ASO consisting of a sequence selected from SEQ ID NOs: 12 to 731 with the cells of the subject.

[0009]

[0010] In one aspect of the present invention, a kit comprising an ASO comprising a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 12 to 731; or an ASO consisting of a sequence selected from SEQ ID NOs: 12 to 731 is disclosed.

[0010] Incorporation by reference

[0011] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0011]

[0012] The novel features of the invention are particularly pointed out 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 illustrates exemplary aspects in which the principles of the invention are utilized, and to the following accompanying drawings.

Brief Description of the Drawings

[0012]

Figure 1AB

[0013] Figure 1 illustrates a schematic diagram of therapeutic agent-mediated elimination of nonsense mutation-dependent mRNA degradation-inducing exons that increases the expression of full-length target proteins or functional RNAs from target mRNAs (NMD exon mRNAs) containing nonsense mutation-dependent RNA degradation-inducing exons. Figure 1A shows a cell divided into nuclear and cytoplasmic fractions. In the nucleus, the pre-mRNA transcript of the target gene undergoes splicing to produce mRNA, which is transported to the cytoplasm and translated into the target protein. For this target gene, a fraction of its mRNA contains a nonsense mutation-dependent mRNA degradation-inducing exon (NMD exon mRNA) and is degraded in the cytoplasm, thus not resulting in target protein production. Figure 1B shows an example of the same cell divided into nuclear and cytoplasmic fractions. Treatment with a therapeutic agent such as an antisense oligomer (ASO) promotes the elimination of the nonsense mutation-dependent mRNA degradation-inducing exon, resulting in an increase in mRNA, which is then translated into a higher level of the target protein.

Figure 1C

Figure 2

[0014] Figure 2 illustrates the identification of an exemplary nonsense mutation-dependent mRNA decay (NMD)-inducing exon in the SCN1A gene. Identification of NMD-inducing exons in the SCN1A gene using comparative genomics is visualized and shown in the UCSC Genome Browser. The upper panel shows an enlarged graph of the SCN1A gene. The conservation level across 100 vertebrate species is shown as a peak. While the highest peak corresponds to an exon (black box), no peak is observed in the majority of introns (arrowed lines). The peak of conservation was identified in intron 20 (NM_006920) and is shown in the central panel. Examination of the conserved sequence identified a 64-bp exon-like sequence (lower panel, sequence highlighted in grey) with the 3’ splice site and 5’ splice site (underlined sequences) flanking it, which is designated exon 20x. Inclusion of this exon results in a frameshift, introducing a premature termination codon in exon 21 and targeting this transcript for NMD.

Figure 3

[0015] Figure 3A illustrates 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 Neuro 2A (mouse neural progenitor cells) treated with DMSO (CHX-) or cycloheximide (CHX+), and primers for exon 21 and downstream exons. The uniqueness of this product was confirmed by sequencing. Concentration measurement analysis of this band was performed to calculate the exon 21x inclusion % of total SCN1A transcripts. When Neuro 2A was treated with cycloheximide (CHX+) to inhibit NMD, the product corresponding to the NMD-inducing exon 21x increased 2-fold in the cytoplasmic fraction (compare the light grey bar, CHX- with the dark grey bar, CHX+).

[0016] Figure 3B illustrates the confirmation of NMD-inducing exons via cycloheximide treatment. The presence of bands corresponding to the NMD-inducing exon (20x) was confirmed by RT-PCR analysis using cytoplasmic RNA from RenCell VM (human neural progenitor cells) treated with DMSO (CHX-) or cycloheximide (CHX+), and primers in exons 21 and 23. The uniqueness of this product was confirmed by sequencing. Concentration measurement analysis of this band was performed to calculate the exon 20x inclusion percentage of the total SCN1A transcript. When RenCell VM was treated with cycloheximide (CHX+) to inhibit NMD, the product corresponding to the NMD-inducing exon 20x increased 2-fold in the cytoplasmic fraction (compare the light gray bar, CHX- with the dark gray bar, CHX+).

Figure 4

[0017] Figure 4 illustrates an exemplary graph of an ASO walk performed on the SCN1A exon 20x region targeting two designated regions (region 1 and region 2) upstream of the 3' splice site of exon 20x and two designated regions (region 3 and region 4) downstream of the 5' splice site of exon 20x. The ASOs were designed to cover these regions by shifting 5 nucleotides at a time.

Figure 5A

[0018] Figure 5A illustrates ASOs to the SCN1A exon 20x region selected from the extended ASO walk, as evaluated by RT-PCR. Representative PAGE shows SYBR-Safe stained RT-PCR products from RenCell treated with SCN1A mock treatment, control ASO treatment (NT), or ASOs to the SCN1A exon 20x region from the extended walk via nucleofection at 1 μM for 24 hours. Mock = no ASO; control NT = non-targeted control; Posctrl = positive control.

Figure 5B

[0019] Figure 5B illustrates a graph plotting the exon 20x inclusion percentage from the data in Figure 5A.

Figure 5C

[0020] Figure 5C illustrates a graph of the qPCR results of the extension ASO walk using the sample of Figure 5A, normalized to the RPL32 internal control, and plots the fold change of SCN1A mRNA compared to mock. Detailed Description of the Invention

[0013] Splicing and nonsense mutation-dependent mRNA decay

[0021] Intervening sequences or introns are removed by a large and extremely dynamic RNA - protein complex called the spliceosome, which coordinates interactions between primary transcripts, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome is assembled ad hoc in an ordered manner for each intron, starting from the recognition of the 5' splice site (5'ss) by U1 snRNA or the recognition of the 3' splice site (3'ss) by the U2 pathway, which involves the binding of the U2 auxiliary factor (U2AF) to the 3'ss region to facilitate U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a 65kD subunit (U2AF65) that encodes U2AF2 and binds to the polypyrimidine tract (PPT), and a 35kD subunit (U2AF35) that encodes U2AF1 and interacts with the highly conserved AG dinucleotide at the 3'ss and stabilizes U2AF65 binding. In addition to this BPS / PPT unit and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures that activate or suppress splice site recognition, known as enhancers or silencers of intron or exon splicing. These elements allow true splice sites to be recognized among the large excess of cryptic or pseudo - sites (having the same sequence as the true sites but in much greater numbers) in the genomes of higher eukaryotes. They often have regulatory functions, but little is understood about the mechanisms of their activation or suppression.

[0014]

[0022] The decision of whether to splice or not can typically be modeled as a probabilistic process rather than a deterministic one, so any given splicing signal may splice inaccurately. However, under normal conditions, pre-mRNA splicing proceeds with a surprisingly high degree of fidelity. This is due in part to the activity of adjacent cis-acting auxiliary exon and intron splicing regulatory elements (ESR or ISR). Typically, these functional elements are classified as either exon splicing enhancers or intron splicing enhancers (ESE or ISE) or silencers (ESS or ISS), respectively, based on their ability to promote or inhibit splicing. They are classified. Although there is evidence today that some auxiliary cis - acting elements may act by influencing the dynamics of spliceosome assembly, such as the placement of the U1 snRNP - 5’ss complex, many elements are very likely to function in concert with trans - acting RNA - binding proteins (RBPs). For example, the serine - and arginine - rich RBP family (SR proteins) is a conserved protein family that plays an important role in defining exons. SR proteins promote exon recognition either by recruiting components of the pre - spliceosome to adjacent splice sites or by antagonizing the effect of nearby ESSs. The inhibitory effect of ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their roles in splicing regulation, silencer elements are also suggested to have a role in the suppression of pseudo - exons (a set of decoy intron splice sites that have the spacing of typical exons but no functional open reading frame). ESEs and ESSs, together with their cognate trans - acting RBPs, are important components in a set of splicing controls that characterize how, where, and when mRNAs are assembled from their precursors.

[0015]

[0023] The sequences that mark exon-intron boundaries are degenerate signals of varying strengths that can occur frequently within human genes. In multi-exon genes, pairs of various splice sites can be linked together in many different combinations to create a wide variety of transcripts from a single gene. This is generally referred to as alternative pre-mRNA splicing. Most mRNA isoforms produced by alternative splicing can be transported from the nucleus and translated into functional polypeptides, but the various mRNA isoforms from a single gene can vary greatly in their translation efficiency. mRNA isoforms with premature termination codons (PTCs) that are at least 50 bp upstream of the exon junction complex can be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway. Mutations in traditional (BPS / PPT / 3’ss / 5’ss) and auxiliary splicing motifs can cause abnormal splicing, such as exon skipping or cryptic (or pseudo) exon inclusion or splice site activation, which can significantly contribute to human morbidity and mortality. Both abnormal splicing and alternative splicing patterns can be affected by natural DNA variants in exons and introns.

[0016]

[0024] If exon-intron boundaries can occur anywhere in three positions of a codon, maintaining the canonical open reading frame can be achieved by only a subset of alternative splicing events. For example, only exons that are evenly divisible by 3 can be skipped or included in the mRNA without altering the reading frame. Splicing events that do not have compatible phases Pricing events induce frameshifts. If not reverted by downstream events, frameshifts will surely result in one or more PTCs and may possibly lead to subsequent degradation by NMD. NMD is a translation-coupled mechanism that removes mRNAs containing PTCs. NMD can function as a surveillance pathway present in all eukaryotes. NMD can suppress errors in gene expression by removing mRNA transcripts containing premature stop codons. Translation of these abnormal mRNAs may, in some cases, result in harmful gain-of-function or dominant-negative activities of the resulting proteins. Since NMD targets not only transcripts with PTCs but also a wide range of mRNA isoforms expressed from many endogenous genes, NMD is suggested to be a major regulator that drives both fine-tuning and coarse-tuning of steady-state RNA levels in cells.

[0017]

[0025] The NMD-inducing exon (NIE) is a region within an intron that is an exon or pseudoexon capable of activating the NMD pathway when included in the mature RNA transcript. In constitutive splicing events, introns containing NIEs are usually spliced out, but the intron or a part thereof (e.g., NIE) may be retained during alternative or abnormal splicing events. Such mature mRNA transcripts containing NIEs may be non-productive due to frameshifts that induce the NMD pathway. Inclusion of NIEs in mature RNA transcripts may downregulate gene expression. In the present disclosure, mRNA transcripts containing NIEs will be referred to as "NIE-containing mRNAs" or "NMD exon mRNAs".

[0018]

[0026] Cryptic (or pseudo) splice sites have the same splicing recognition sequences as authentic splice sites, but are not used in the splicing reaction. They outnumber authentic splice sites by an order of magnitude in the human genome and are usually inhibited by molecular mechanisms that are poorly understood to date. Cryptic 5' splice sites have the consensus NNN / GUNNNN or NNN / GCNNNN (where N is any nucleotide and / is the exon-intron boundary). Cryptic 3' splice sites have the consensus NAG / N. Their activation is positively influenced by the surrounding nucleotides that make them more similar to the optimal consensus of the canonical splice sites (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).

[0019]

[0027] Splice sites and their regulatory sequences are described by those skilled in the art, for example in Kralovicova, J. and Vorechovsky, I. (2007) "Aberrant splice junctions due to auxiliary splicing sequences" (Proc. Natl. Soc. 2009). Aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition, Nucleic Acids Res., 35, 6399-6413, (http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC2095810 / pdf / gkm680.pdf), can be readily identified using suitable publicly available algorithms.

[0020]

[0028] Cryptic splice sites or splicing regulatory sequences may compete with the splice sites of NIE for RNA-binding proteins such as U2AF. In one aspect, a drug can be bound to a cryptic splice site or splicing regulatory sequence to prevent the binding of an RNA-binding protein, thereby favoring the utilization of the NIE splice site.

[0021]

[0029] In one aspect, a cryptic splice site may not include the 5' or 3' splice site of NIE. The cryptic splice site may be at least 10 nucleotides upstream of the NIE 5' splice site. The cryptic splice site may be at least 20 nucleotides upstream of the NIE 5' splice site. The cryptic splice site may be at least 50 nucleotides upstream of the NIE 5' splice site. The cryptic splice site may be at least 100 nucleotides upstream of the NIE 5' splice site. The cryptic splice site may be at least 200 nucleotides upstream of the NIE 5' splice site.

[0022]

[0030] The cryptic splice site may be at least 10 nucleotides downstream of the NIE 3' splice site. The cryptic splice site may be at least 20 nucleotides downstream of the NIE 3' splice site. The cryptic splice site may be at least 50 nucleotides downstream of the NIE 3' splice site. The cryptic splice site may be at least 100 nucleotides downstream of the NIE 3' splice site. The cryptic splice site may be at least 200 nucleotides downstream of the NIE 3' splice site.

[0023] Target transcript

[0031] In some embodiments, the methods of the disclosure utilize the presence of NIEs in pre-mRNAs transcribed from the SCN1A gene. Production of a functional mature SCN1A mRNA by splicing of the identified SCN1A NIE pre-mRNA molecular species can be induced using a therapeutic agent such as an ASO that promotes exon skipping of the NIE. Induction of exon skipping can result in inhibition of the NMD pathway. The resulting mature SCN1A mRNA can be translated without normally activating the NMD pathway, thereby increasing the amount of SCN1A protein in the patient's cells and alleviating the symptoms of a disorder associated with SCN1A deficiency such as Dravet syndrome (DS); generalized epilepsy with febrile seizures plus, type 2; familial febrile seizures, 3A; autism; epileptic encephalopathy, early infantile, 13; pore dysplasia syndrome 1; Alzheimer's disease; or SUDEP.

[0024]

[0032] In various aspects, the present disclosure provides therapeutic agents that can target SCN1A mRNA transcripts to modulate (e.g., enhance or inhibit) splicing or protein expression levels. The therapeutic agent can be a small molecule, polynucleotide, or polypeptide. In some aspects, the therapeutic agent is an ASO. Various regions or sequences on the SCN1A pre-mRNA can be targeted by therapeutic agents such as ASOs. In some aspects, the ASO targets SCN1A pre-mRNA transcripts containing NIE. In some aspects, the ASO targets a sequence within the NIE of the SCN1A pre-mRNA transcript. In some aspects, the ASO targets a sequence upstream (or 5') of the 5' end of the NIE (3'ss) of the SCN1A pre-mRNA transcript. In some aspects, the ASO targets a sequence downstream (or 3') of the 3' end of the NIE (5'ss) of the SCN1A pre-mRNA transcript. In some aspects, the ASO targets a sequence within an intron adjacent to the 5' end of the NIE of the SCN1A pre-mRNA transcript. In some aspects, the ASO targets a sequence within an intron adjacent to the 3' end of the NIE of the SCN1A pre-mRNA transcript. In some aspects, the ASO targets a sequence comprising the NIE-intron boundary of the SCN1A pre-mRNA transcript. The NIE-intron boundary can refer to the junction of the intron sequence and the NIE region. This intron sequence can be adjacent to the 5' end or the 3' end of the NIE. In some aspects, the ASO targets a sequence within an exon of the SCN1A pre-mRNA transcript. In some aspects, the ASO targets a sequence within an intron of the SCN1A pre-mRNA transcript. In some aspects, the ASO targets a sequence comprising both a portion of an intron and a portion of an exon.

[0025]

[0033] In some aspects, the therapeutic agents described herein modulate the binding of factors involved in splicing of NMD exon mRNA.

[0034] In some embodiments, the therapeutic agents described herein interfere with the binding of factors involved in the splicing of NMD exon mRNA.

[0026]

[0035] In some embodiments, the therapeutic agents described herein prevent the binding of factors involved in the splicing of NMD exon mRNA.

[0036] In some embodiments, the therapeutic agent targets a targeting moiety located in an intron region between two reference exon regions of an NMD exon mRNA encoding SCN1A, where this intron region contains the NMD exon.

[0027]

[0037] In some embodiments, the therapeutic agent targets a targeting moiety that at least partially overlaps with an NMD exon.

[0038] In some embodiments, the therapeutic agent targets a targeting moiety that at least partially overlaps with an intron upstream of an NMD exon.

[0028]

[0039] In some embodiments, the therapeutic agent targets a targeting moiety located within an NMD exon.

[0040] In some embodiments, the therapeutic agent targets a targeting moiety comprising 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, or 30 or more contiguous nucleotides of an NMD exon. In some embodiments, the therapeutic agent targets a targeting moiety comprising at most 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, or 30 or more contiguous nucleotides of an NMD exon. In some embodiments, the therapeutic agent targets a targeting moiety comprising 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, or 30 or more contiguous nucleotides of an NMD exon.

[0029]

[0041] In some embodiments, the therapeutic agent targets a targeting moiety proximal to the NMD exon.

[0042] In some embodiments, the ASO targets a sequence located about 1 to about 5000 nucleotides downstream from the 5' end of an intron comprising an NIE. In some embodiments, the ASO targets a sequence located about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, about 1950 to about 2000 nucleotides, about 2000 to about 3000 nucleotides, about 3000 to about 4000 nucleotides, or about 4000 to about 5000 nucleotides downstream from the 5' end of an intron comprising an NIE. In some embodiments, the ASO targets a sequence located about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides downstream from the 5' end of an intron comprising an NIE.

[0030]

[0043] In some embodiments, the ASO targets a sequence that is 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 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, or at least about 5000 nucleotides downstream of the 5' end of an intron comprising the NIE.

[0031]

[0044] In some embodiments, the ASO targets a sequence that is about 1 to about 2000 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, or about 1950 to about 2000 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides upstream (or 5') from the 5' end of the NIE.

[0032]

[0045] In some embodiments, the ASO targets a sequence that is 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, or at least about 2000 nucleotides upstream (or 5') of the 5' end of the NIE.

[0033]

[0046] In some embodiments, the ASO targets a sequence that is about 1 to about 500 nucleotides downstream of the 5' end of the NIE. In some embodiments, the ASO is at the 5' end of the NIE region Target sequences that are 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, or at least about 500 nucleotides downstream.

[0034]

[0047] In some embodiments, the ASO targets sequences that are about 1 to about 500 nucleotides upstream from the 3' end of the NIE. In some embodiments, the ASO targets sequences that are 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, or at least about 500 nucleotides upstream from the 3' end of the NIE region.

[0035]

[0048] In some embodiments, the ASO targets a sequence that is about 1 to about 2000 nucleotides downstream (or 3') from the 3' end of the NIE. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, or about 1950 to about 2000 nucleotides downstream (or 3') from the 3' end of the NIE. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides downstream (or 3') from the 3' end of the NIE.

[0036]

[0049] In some embodiments, the ASO is 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 Target sequences that are 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, or at least about 2000 nucleotides downstream (or 3') thereof.

[0037]

[0050] In some embodiments, the ASO targets a sequence that is about 1 to about 5000 nucleotides upstream from the 3' end of an intron comprising the NIE. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, about 1950 to about 2000 nucleotides, about 2000 to about 3000 nucleotides, about 3000 to about 4000 nucleotides, or about 4000 to about 5000 nucleotides upstream from the 3' end of an intron comprising the NIE. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides upstream from the 3' end of an intron comprising the NIE.

[0038]

[0051] In some embodiments, the ASO targets a sequence that is 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, at least about 1000 nucleotides, at least about 1200 nucl eotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, or at least about 5000 nucleotides upstream of the 3’ end of an intron comprising the NIE.

[0039]

[0052] In some embodiments, the ASO targets a sequence that is about 4 to about 300 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, or about 1450 to about 1500 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO can target a sequence that is more than 300 nucleotides upstream from the 5' end of the NIE. In some embodiments, the ASO targets a sequence that is about 4 to about 300 nucleotides downstream (or 3') from the 3' end of the NIE. In some embodiments, the ASO is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, or about 1450 to about 1500 nucleotides downstream from the 3' end of the NIE. In some embodiments, the ASO targets a sequence that is more than 300 nucleotides downstream from the 3' end of the NIE.

[0040]

[0053] In some embodiments, the ASO targets a sequence that is about 4 to about 300 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO is 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 (or 5') from the 5' end of the NIE. In some embodiments, the ASO targets a sequence that is about 4 to about 300 nucleotides downstream (or 3') from the 3' end of the NIE. In some embodiments, the ASO is 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 Target sequences that are downstream of a nucleotide, 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. In some embodiments, the ASO targets a sequence that is more than 300 nucleotides downstream from the 3' end of the NIE.

[0041]

[0054] In some embodiments, the ASO targets a sequence that is from about 4 to about 300 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO is at most about 10 nucleotides, at most about 20 nucleotides, at most about 50 nucleotides, at most about 80 nucleotides, at most about 85 nucleotides, at most about 90 nucleotides, at most about 95 nucleotides, at most about 96 nucleotides, at most about 97 nucleotides, at most about 98 nucleotides, at most about 99 nucleotides, at most about 100 nucleotides, at most about 101 nucleotides, at most about 102 nucleotides, at most about 103 nucleotides, at most about 104 nucleotides, at most about 105 nucleotides, at most about 110 nucleotides, at most about 120 nucleotides, at most about 150 nucleotides, at most about 200 nucleotides, at most about 300 nucleotides, at most about 400 nucleotides, at most about 500 nucleotides, at most about 600 nucleotides, at most about 700 nucleotides, at most about 800 nucleotides, at most about 900 nucleotides, at most about 1000 nucleotides, at most about 1100 nucleotides, at most about 1200 nucleotides, at most about 1300 nucleotides, at most about 1400 nucleotides, or at most about 1500 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO targets a sequence that is from about 4 to about 300 nucleotides downstream (or 3') from the 3' end of the NIE.In some embodiments, the ASO targets a sequence that is up to about 10 nucleotides, up to about 20 nucleotides, up to about 50 nucleotides, up to about 80 nucleotides, up to about 85 nucleotides, up to about 90 nucleotides, up to about 95 nucleotides, up to about 96 nucleotides, up to about 97 nucleotides, up to about 98 nucleotides, up to about 99 nucleotides, up to about 100 nucleotides, up to about 101 nucleotides, up to about 102 nucleotides, up to about 103 nucleotides, up to about 104 nucleotides, up to about 105 nucleotides, up to about 110 nucleotides, up to about 120 nucleotides, up to about 150 nucleotides, up to about 200 nucleotides, up to about 300 nucleotides, up to about 400 nucleotides, up to about 500 nucleotides, up to about 600 nucleotides, up to about 700 nucleotides, up to about 800 nucleotides, up to about 900 nucleotides, or up to about 1000 nucleotides, up to about 1100 nucleotides, up to about 1200 nucleotides, up to about 1300 nucleotides, up to about 1400 nucleotides, or up to about 1500 nucleotides downstream from the 3’ end of the NIE. In some embodiments, the ASO targets a sequence that is more than 300 nucleotides downstream from the 3’ end of the NIE.

[0042]

[0055] In some embodiments, the NIE (exon 23) as described herein is located between GRCh38 / hg38:chr2:166007230 and chr2:166007293. In some embodiments, the 5’ end of the NIE is located at GRCh38 / hg38:chr2:166007230. In some embodiments, the 3’ end of the NIE is located at GRCh38 / hg38:chr2:166007293.

[0043]

[0056] In some embodiments, the ASO targets a sequence that is approximately 1 to approximately 2000 nucleotides upstream (or 5') from the genomic locus: GRCh38 / hg38:chr2:166007230. In some embodiments, the ASO targets a sequence that is 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, 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, approximately 1450 to approximately 1500 nucleotides, approximately 1550 to approximately 1600 nucleotides, approximately 1650 to approximately 1700 nucleotides, approximately 1750 to approximately 1800 nucleotides, approximately 1850 to approximately 1900 nucleotides, or approximately 1950 to approximately 2000 nucleotides upstream (or 5') from the genomic locus: GRCh38 / hg38:chr2:166007230. In some embodiments, the ASO targets a sequence that is 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, 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, or approximately 950 to approximately 1000 nucleotides upstream (or 5') from the genomic locus: GRCh38 / hg38:chr2:166007230.

[0044]

[0057] In some embodiments, the ASO targets a sequence that is 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, or at least about 2000 nucleotides upstream (or 5') of the genomic locus: GRCh38 / hg38:chr2:166007230.

[0045]

[0058] In some embodiments, the ASO targets a sequence that is from about 1 to about 500 nucleotides downstream of the genomic locus; GRCh38 / hg38:chr2:166007230. In some embodiments, the ASO is 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 also 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, or at least about 500 nucleotides downstream of the genomic locus: GRCh38 / hg38:chr2:166007230.

[0046]

[0059] In some embodiments, the ASO targets a sequence that is from about 1 to about 500 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166007293. In some embodiments, the ASO targets a sequence that is 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, or at least about 500 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166007293.

[0047]

[0060] In some embodiments, the ASO targets a sequence that is from about 1 to about 2000 nucleotides downstream (or 3’) from the genomic locus: GRCh38 / hg38:chr2:166007293. In some embodiments, the ASO targets a sequence that is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, or about 1950 to about 2000 nucleotides downstream (or 3’) from the genomic locus: GRCh38 / hg38:chr2:166007293. In some embodiments, the ASO targets a sequence that is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides downstream (or 3’) from the genomic locus: GRCh38 / hg38:chr2:166007293.

[0048]

[0061] In some embodiments, the ASO targets a sequence that is 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 1 00 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, or at least about 2000 nucleotides downstream (or 3’) of genomic locus: GRCh38 / hg38:chr2:166007293.

[0049]

[0062] In some embodiments, the intron comprising the NIE is located between GRCh38 / hg38:chr2:166002754 and chr2:166009718. In some embodiments, the 5’ end of the intron comprising the NIE is located at GRCh38 / hg38:chr2:166002754. In some embodiments, the 3’ end of the intron comprising the NIE is located at GRCh38 / hg38:chr2:166009718.

[0050]

[0063] In some embodiments, the ASO targets a sequence that is about 1 to about 5000 nucleotides downstream from the genomic locus: GRCh38 / hg38:chr2:166002754. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, about 1950 to about 2000 nucleotides, about 2000 to about 3000 nucleotides, about 3000 to about 4000 nucleotides, or about 4000 to about 5000 nucleotides downstream from the genomic locus: GRCh38 / hg38:chr2:166002754. In some embodiments, the ASO targets a sequence that is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides downstream from the genomic locus: GRCh38 / hg38:chr2:166002754.

[0051]

[0064] In some embodiments, the ASO targets a sequence that is 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, or at least about 5000 nucleotides downstream of the genomic locus: GRCh38 / hg38:chr2:166002754.

[0052]

[0065] In some embodiments, the ASO targets a sequence that is from about 1 to about 5000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166007229. In some embodiments, the ASO targets a sequence that is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, about 1950 to about 2000 nucleotides, about 2000 to about 3000 nucleotides, about 3000 to about 4000 nucleotides, or about 4000 to about 5000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166007229. In some embodiments, the ASO targets a sequence that is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166007229.

[0053]

[0066] In some embodiments, the ASO targets a sequence that is 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least also about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, or at least about 5000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166007229.

[0054]

[0067] In some embodiments, the ASO targets a sequence that is from about 1 to about 5000 nucleotides downstream of the genomic locus: GRCh38 / hg38:chr2:166007294. In some embodiments, the ASO targets a sequence that is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides, about 1050 to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, about 1450 to about 1500 nucleotides, about 1550 to about 1600 nucleotides, about 1650 to about 1700 nucleotides, about 1750 to about 1800 nucleotides, about 1850 to about 1900 nucleotides, about 1950 to about 2000 nucleotides, about 2000 to about 3000 nucleotides, about 3000 to about 4000 nucleotides, or about 4000 to about 5000 nucleotides downstream of the genomic locus: GRCh38 / hg38:chr2:166007294. In some embodiments, the ASO targets a sequence that is from about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, or about 950 to about 1000 nucleotides downstream of the genomic locus: GRCh38 / hg38:chr2:166007294.

[0055]

[0068] In some embodiments, the ASO targets a sequence that is 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, or at least about 5000 nucleotides downstream of the genomic locus: GRCh38 / hg38:chr2:166007294.

[0056]

[0069] In some embodiments, the ASO targets a sequence that is from about 1 to about 5000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166009718. In some embodiments, the ASO targets a sequence located 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, 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, approximately 1450 to approximately 1500 nucleotides, approximately 1550 to approximately 1600 nucleotides, approximately 1650 to approximately 1700 nucleotides, approximately 1750 to approximately 1800 nucleotides, approximately 1850 to approximately 1900 nucleotides, approximately 1950 to approximately 2000 nucleotides, approximately 2000 to approximately 3000 nucleotides, approximately 3000 to approximately 4000 nucleotides, or approximately 4000 to approximately 5000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166009718. In some embodiments, the ASO targets a sequence located 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, 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, or approximately 950 to approximately 1000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166009718.

[0057]

[0070] In some embodiments, the ASO targets a sequence that is 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, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1400 nucleotides, at least about 1500 nucleotides, at least about 1600 nucleotides, at least about 1800 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, or at least about 5000 nucleotides upstream of the genomic locus: GRCh38 / hg38:chr2:166009718.

[0058]

[0071] In some embodiments, the NIE as described herein is located between GRCh37 / hg19:chr2:166,863,740 and GRCh37 / hg19:chr2:166,863,803, as illustrated in FIG. 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.

[0059]

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

[0060]

[0073] In some embodiments, the ASO targets a sequence that is from about 4 to about 300 nucleotides upstream (or 5') of the genomic locus: GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence that is 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 (or 5') of the genomic locus: GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence that is from about 4 to about 300 nucleotides downstream (or 3') of the genomic locus: GRCh37 / hg19:chr2:166,863,740. In some embodiments, the ASO targets a sequence that is 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 Target an array that is downstream of tid, 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. In some embodiments, the ASO targets an array that is more than 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.

[0061]

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

[0062]

[0075] As described in the examples herein, NIE was analyzed for the SCN1A gene (SEQ ID NO: 1), and observations were made regarding the inclusion of a portion of intron 20 (see SEQ ID NO: 6, which encodes intron 20 pre-mRNA), which is referred to herein throughout as exon 23 or exon 20x. In some embodiments, the AS disclosed herein O targets the NIE-containing pre-mRNA (SEQ ID NO: 2) transcribed from the SCN1A genomic sequence. In some embodiments, the ASO targets the NIE-containing pre-mRNA transcript derived from the SCN1A genomic sequence, which comprises a part of intron 20. In some embodiments, the ASO targets the NIE-containing pre-mRNA transcript derived from the SCN1A genomic sequence, which comprises exon 23 (or exon 20x) (SEQ ID NO: 4). In some embodiments, the ASO targets the NIE-containing pre-mRNA transcript of SEQ ID NO: 2 or 9. In some embodiments, the ASO targets the NIE-containing pre-mRNA transcript of SEQ ID NO: 2 or 9, which comprises NIE. In some embodiments, the ASO targets the NIE-containing pre-mRNA transcript of SEQ ID NO: 2, which comprises exon 23 (or exon 20x) (SEQ ID NO: 7). In some embodiments, the ASO disclosed herein targets the SCN1A pre-mRNA sequence (SEQ ID NO: 2 or 9). In some embodiments, the ASO targets the SCN1A pre-mRNA sequence, which comprises NIE (SEQ ID NO: 7 or 11). In some embodiments, the ASO targets the SCN1A pre-mRNA sequence by any one of SEQ ID NO: 6, 7, 10, or 11. In some embodiments, the ASO has a sequence by any one of SEQ ID NO: 12 to 731. In some embodiments, the ASO has a sequence by any one of SEQ ID NO: 12 to 371. In some embodiments, the ASO has a sequence by any one of SEQ ID NO: 372 to 731.

[0063]

[0076] In some embodiments, the SCN1A NIE-containing pre-mRNA 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 NO: 1 or 8. In some embodiments, the SCN1A NIE pre-mRNA transcript comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 2 to 7 and 9 to 11.

[0064]

[0077] In some embodiments, the SCN1A NIE-containing pre-mRNA transcript (or NMD exon mRNA) comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs: 2, 6, 7, 9, 10, and 12. In some embodiments, the SCN1A NIE-containing pre-mRNA 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 and 8. In some embodiments, the targeted portion of the NMD exon mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NOs: 2, 6, 7, 9, 10, and 12.

[0065]

[0078] In some embodiments, the ASO targets a sequence upstream of the 5' end of the NIE. For example, an ASO that targets a sequence upstream of the 5' end of the NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may comprise a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 12-191 or 372-551. In another example, an ASO that targets a sequence upstream of the 5' end of the NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may comprise a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 12-191. In an additional example, an ASO that targets a sequence upstream of the 5' end of the NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may comprise a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 372-551.

[0066]

[0079] In some embodiments, the ASO comprises exon 23 and is SCN1A N The IE targets exon 23 (or exon 20x) in the pre-mRNA. In some embodiments, the ASO targets an exon 23 sequence downstream (or 3') from the 5' end of exon 23 of the SCN1A pre-mRNA. In some embodiments, the ASO targets an exon 23 sequence upstream (or 5') from the 3' end of exon 20x of the SCN1A pre-mRNA.

[0067]

[0080] In some embodiments, the ASO targets a sequence downstream from the 3' end of the NIE. For example, an ASO that targets a sequence downstream from the 3' end of the NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may comprise a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 192 - 371 or 552 - 731. In another example, an ASO that targets a sequence downstream from the 3' end of the NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may comprise a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 192 - 371. In an additional example, an ASO that targets a sequence downstream from the 3' end of the NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may comprise a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 552 - 731.

[0068]

[0081] In some embodiments, the targeting portion of the SCN1A NIE-containing pre-mRNA is in intron 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 (intron numbering corresponds to the mRNA sequence in NM_006920). In some embodiments, hybridization of the ASO to the targeting portion of the NIE pre-mRNA results in skipping of at least one exon of the NIE that is within intron 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, subsequently increasing SCN1A protein production. In some embodiments, hybridization of the ASO to the targeting portion of the NIE pre-mRNA inhibits or interferes with skipping of at least one exon of the NIE that is within intron 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, subsequently decreasing SCN1A protein production. In some embodiments, the targeting portion of the SCN1A NIE-containing pre-mRNA is in intron 20. One of ordinary skill in the art can determine the corresponding intron number in any isoform based on the intron sequences provided herein or using the numbers provided in relation to the mRNA sequences in NM_006920, NM_001202435, NM_001165964, or NM_001165963. One of ordinary skill in the art can also determine the sequence of the exons flanking the SCN1A isoform to be targeted using the methods of the invention based on the intron sequences provided herein or using the numbers provided in relation to the mRNA sequences in NM_006920, NM_001202435, NM_001165964, or NM_001165963.

[0069]

[0082] In some aspects, 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 pre-mRNA. In some aspects, the pseudoexon is a sequence that is within any of introns 1-25. In some aspects, the pseudoexon is a sequence that is within any of introns 2, 4, 6, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, and 25. In some aspects, the pseudoexon is a sequence that is within any of introns 15, 18, and 19. In some aspects, the pseudoexon can be part of any SCN1A intron. In some aspects, the pseudoexon is within intron 20. The SCN1A intron numbering used herein corresponds to the RNA sequence in NM_006920. It is understood that this intron numbering can vary in relation to different SCN1A isoform sequences.

[0070] SCN1A protein

[0083] The SCN1A gene can encode an SCN1A (sodium channel, voltage-gated, type I, alpha subunit) protein (which can also be referred to as the alpha-subunit of voltage-gated sodium channel Na v 1.1). As described above, SCN1A mutations in DS spread throughout this protein. More than 100 novel mutations have been identified throughout this gene, and new ones that are more debilitating have emerged. These include truncations (47%), missenses (43%), deletions (3%), and splice site mutations (7%). The percentage of subjects carrying SCN1A mutations varies between 33% and 100%. The majority of mutations (88%) are novel changes.

[0071]

[0084] In some aspects, the methods described herein are used to modulate (e.g., increase or decrease) the production of functional SCN1A protein. As used herein, the term "functional" refers to the amount of SCN1A protein activity or function necessary to abrogate any one or more symptoms of the disorder being treated (e.g., Dravet syndrome; generalized epilepsy with febrile seizures plus, type 2; familial febrile seizures, 3A; autism; epileptic encephalopathy, early infantile, 13; pore dysplasia syndrome 1; Alzheimer's disease; or SUDEP). In some aspects, the method is used to increase the production of a partially functional SCN1A protein. As used herein, the term "partially functional" refers to any amount of SCN1A protein activity or function that is less than the amount of activity or function necessary to abrogate or prevent any one or more symptoms of a disease or disorder. In some aspects, a partially functional protein or RNA has 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% less activity than a fully functional protein or RNA.

[0072]

[0085] In some embodiments, the method is a method of increasing the expression of SCN1A protein by cells of a subject having NIE-containing pre-mRNA encoding SCN1A protein, wherein the subject has Dravet syndrome caused by a deficiency in the activity of SCN1A protein, and wherein the deficiency in the SCN1A protein is caused by haploinsufficiency of the SCN1A protein. In such embodiments, the subject has a first allele encoding a functional SCN1A protein and a second allele from which no SCN1A protein is produced. In another such embodiment, the subject has a first allele encoding a functional SCN1A protein and a second allele encoding a non-functional SCN1A protein. In another such embodiment, the subject 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 binds to a targeted portion of the NIE-containing pre-mRNA transcribed from the second allele, thereby inducing exon skipping of the pseudoexon derived from the pre-mRNA and causing an increase in the level of mature mRNA encoding the functional SCN1A protein and an increase in the expression of SCN1A protein in the cells of the subject.

[0073]

[0086] In related embodiments, the method uses an ASO to a protein or functional RN A method of increasing the expression of A. In some embodiments, an ASO is used to increase the expression of SCN1A protein in the cells of a subject having NIE-containing pre-mRNA encoding the SCN1A protein, where the subject has a defect in the amount or function of the SCN1A protein, such as Dravet syndrome (DS) (also known as SMEI); infantile severe myoclonic epilepsy (SMEI)-borderline type (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic epilepsy; early myoclonic encephalopathy; progressive myoclonic epilepsy; childhood alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pore dysplasia syndrome 1; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or autism. In some embodiments, an ASO is used to increase the expression of SCN1A protein in the cells of a subject, where the subject has a defect in the amount or function of the SCN8A protein, such as epileptic encephalopathy, early infantile, 13. In some embodiments, an ASO is used to increase the expression of SCN1A protein in the cells of a subject, where the subject has a defect in the amount or function of the SCN5A protein, such as pore dysplasia syndrome 1.

[0074]

[0087] In some embodiments, the NIE-containing pre-mRNA transcript encoding the protein that is the cause of the disease or condition is targeted by the ASO described herein. In some embodiments, the NIE-containing pre-mRNA transcript encoding a protein that is not the cause of the disease is targeted by the ASO. For example, a disease that is the result of a mutation or deficiency of a first protein in a particular pathway may be improved by targeting the NIE-containing pre-mRNA encoding a second protein to increase the production of the second protein. In some embodiments, the function of the second protein can offset the mutation or deficiency of the first protein (the cause of the disease or condition).

[0075]

[0088] In some embodiments, the subject has: (a) a first mutant allele [whereby: (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 having a reduced function compared to the equivalent wild-type protein, or (iii) neither the SCN1A protein nor the functional RNA is produced], and (b) a second mutant allele [whereby: (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 having a reduced function compared to the equivalent wild-type protein, or (iii) the SCN1A protein is not produced], wherein the NIE-containing pre-mRNA is transcribed from the first allele and / or the second allele. In these embodiments, the ASO binds to the targeted portion of the NIE-containing pre-mRNA transcribed from the first allele or the second allele, thereby inducing exon skipping of the pseudoexon from the NIE-containing pre-mRNA, resulting in an increase in the level of the mRNA encoding the SCN1A protein and an increase in the expression of the target protein or functional RNA in the cells of the subject. In these embodiments, the target protein or functional RNA having an increased expression level resulting from exon skipping of the pseudoexon from the NIE-containing pre-mRNA is either in a form having a reduced function (partially functional) compared to the equivalent wild-type protein or in a form having a complete function (fully functional) compared to the equivalent wild-type protein.

[0076]

[0089] In some embodiments, the level of mRNA encoding the SCN1A protein is increased 1.1- to 10-fold when compared to the amount of mRNA encoding the SCN1A protein produced in control cells (e.g., cells not treated with an antisense oligomer, or cells treated with an antisense oligomer that does not bind to the targeted portion of the SCN1A NIE-containing pre-mRNA).

[0077]

[0090] In some embodiments, a subject being treated using the methods of the disclosure expresses a SCN1A protein that is partially functional relative to one allele, where the partially functional SCN1A protein is caused by a frameshift mutation, nonsense mutation, missense mutation, or partial gene deletion. In some embodiments, a subject being treated using the methods of the invention expresses a non-functional SCN1A protein relative to one allele, where the non-functional SCN1A protein is caused by a frameshift mutation, nonsense mutation, missense mutation, partial gene deletion in one allele. In some embodiments, a subject being treated using the methods of the invention has a deletion of the entire SCN1A gene in one allele.

[0078]

[0091] In some embodiments, the method is a method of reducing the expression of the SCN1A protein by cells of a subject having NIE-containing pre-mRNA encoding the SCN1A protein, where the subject has a gain-of-function mutation at Na v 1.1. In such embodiments, the subject has an allele in which the SCN1A protein is produced in increased amounts, or an allele encoding a mutant SCN1A that induces increased activity of Na v 1.1 in the cell. In some embodiments, the increased activity of Na v 1.1 is characterized by a mutant Na v1. A long-term or almost permanent sodium current mediated by a channel, a delay in rapid inactivation, a positive shift in steady-state inactivation, a higher channel availability during repetitive stimulation, an increase in non-inactivating depolarization-induced persistent sodium current, a delay in entry into inactivation, an acceleration of recovery from rapid inactivation, and / or rescue of folding defects by incubation at low temperature or co-expression of interacting proteins. In any of these aspects, the antisense oligomer binds to a targeted portion of the NIE-containing pre-mRNA transcribed from the second allele, thereby inhibiting or interfering with exon skipping of the pseudoexon derived from the pre-mRNA, resulting in a decrease in the level of mature mRNA encoding the functional SCN1A protein and a decrease in the expression of the SCN1A protein in the cells of the subject.

[0079]

[0092] In related aspects, the method is a method of reducing the expression of a protein or functional RNA using an ASO. In some aspects, an ASO is used to reduce the expression of the SCN1A protein in the cells of a subject having NIE-containing pre-mRNA encoding the SCN1A protein. In some aspects, the subject has a gain-of-function mutation (e.g., migraine) in Na v 1.1. In some aspects, an ASO is used to reduce the expression of the SCN1A protein in the cells of a subject, but the subject has a gain-of-function mutation (e.g., familial hemiplegic migraine, 3) in Na v 1.1.

[0080]

[0093] In some aspects, the level of mRNA encoding the SCN1A protein is produced in control cells (e.g., cells not treated with an antisense oligomer, or cells treated with an antisense oligomer that does not bind to the targeted portion of the SCN1A NIE-containing pre-mRNA) and is reduced 1.1 to 10-fold when compared to the amount of mRNA encoding the SCN1A protein.

[0081]

[0094] In some embodiments, a subject being treated using the methods of the present disclosure expresses a mutant SCN1A protein from one allele, where the mutant SCN1A protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion, and where the mutant SCN1A protein causes an elevated activity level of Na 1.1. In some embodiments, a subject being treated using the methods of the present disclosure expresses an increased amount of SCN1A protein from one allele due to a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. v 1.1.

[0082]

[0095] In some embodiments of the invention, a subject may have a mutation in the SCN1A gene. Mutations in SCN1A can spread throughout the gene. The SCN1A protein can consist of four domains. The SCN1A domain can have transmembrane segments. Mutations in the SCN1A protein can occur throughout the protein. The SCN1A protein can consist of at least two isoforms. Mutations in SCN1A can include R931C, R946C, M934I, R1648C, or R1648H. In some cases, mutations 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, mutations may be observed at the N-terminus of the SCN1A protein. Exemplary 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 in the present invention may also encode the pore of the ion channel.

[0083]

[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 in infancy (SMEI). In other embodiments, the methods and compositions described herein can be used to treat borderline drave syndrome; generalized epilepsy with febrile seizures plus, type 2; familial febrile seizures, 3A; familial hemiplegic migraine, 3; autism; epileptic encephalopathy, early infantile, 13; pore dysplasia syndrome 1; Alzheimer's disease or SUDEP. The methods and compositions described herein can also be used to treat borderline SMEI. In addition, the methods and compositions described herein can be used to treat generalized epilepsy with febrile seizures plus (GEFS+). GEFS+ may be associated with mutations in seizure-related ion channel subunits such as SCN1B or GABRG2. The methods and compositions described herein can also be used to treat sodium channelopathy. Sodium channelopathy may be associated with mutations in SCN1A. Sodium channelopathy may also be associated with subunits of SCN1A such as the β subunit of SCN1B. In some cases, additional diseases associated with SCN1A mutations can also be treated by the present disclosure. Associated SCN1A diseases associated with SCN1A mutations include, but are not limited to, congenital non-dystrophic myotonia, hyperkalemic periodic paralysis, and congenital paramyotonia.

[0084]

[0097] In some embodiments, the methods and compositions described herein are used to treat subjects having SCN1A mutations known in the art and described in the above references (e.g., by Hamdan, et al., 2009, Mulley, et al., 2005). In some embodiments, this mutation is within an intron or exon of SCN1A.

[0085] Exon inclusion

[0098] As used herein, "NIE-containing pre-mRNA" is a pre-mRNA transcript that contains 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 describe a fully processed mRNA. Inclusion of at least one pseudoexon can result in a non-productive mRNA and may lead to NMD of the mature mRNA. NIE-containing mature mRNA can sometimes result in aberrant protein expression.

[0086]

[0099] In some embodiments, the included pseudoexon is the most abundant pseudoexon in the population of NIE-containing pre-mRNAs transcribed in the cell from the gene encoding the target protein. In some embodiments, the included pseudoexon is the most abundant pseudoexon in the population of NIE-containing pre-mRNAs transcribed in the cell from the gene encoding the target protein, where the population of NIE-containing pre-mRNAs includes two or more included pseudoexons. In some embodiments, an antisense oligomer targeting the most abundant pseudoexon in the population of NIE-containing pre-mRNAs encoding the target protein induces exon skipping of one or more pseudoexons (including the pseudoexon targeted or bound by the antisense oligomer) in the population. In embodiments, the targeting region is within a pseudoexon that is the most abundant pseudoexon in the NIE-containing pre-mRNA encoding the SCN1A protein.

[0087]

[0100] The degree of exon inclusion is exon inclusion % (e.g., for a given pseudoexon It can be expressed as (the percentage of the transcript containing the exon). Briefly, exon inclusion% can be calculated as the percentage of the amount of the RNA transcript with exon inclusion with respect to the total of the average amount of the RNA transcript with exon inclusion + the average amount of the RNA transcript with exon exclusion.

[0088]

[0101] In some embodiments, the included pseudo-exon is at least about 5%, An exon identified as a pseudo-exon included based on a determination of inclusion of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%. In aspects, the included pseudo-exon is from about 5% to about 100%, from about 5% to about 95%, from about 5% to about 90%, from about 5% to about 85%, from about 5% to about 80%, from about 5% to about 75%, from about 5% to about 70%, from about 5% to about 65%, from about 5% to about 60%, from about 5% to about 55%, from about 5% to about 50%, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 10% to about 100%, from about 10% to about 95%, from about 10% to about 90%, from about 10% to about 85%, from about 10% to about 80%, from about 10% to about 75%, from about 10% to about 70%, from about 10% to about 65%, from about 10% to about 60%, from about 10% to about 55%, from about 10% to about 50%, from about 10% to about 45%, from about 10% to about 40%, from about 10% to about 35%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 100%, from about 15% to about 95%, from about 15% to about 90%, from about 15% to about 85%, from about 15% to about 80%, from about 15% to about 75%, from about 15% to about 70%, from about 15% to about 65%, from about 15% to about 60%, from about 15% to about 55%, from about 15% to about 50%, from about 15% to about 45%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 20% to about 100%, from about 20% to about 95%, from about 20% to about 90%, from about 20% to about 85%, from about 20% to about 80%, from about 20% to about 75%, from about 20% to about 70%, from about 20% to about 65%, from about 20% to about 60%, from about 20% to about 55%, from about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, from about 25% to about 100%, from about 25% to about 95%, from about 25% to about 90%, from about 25% to about 85%, from about 25% to about 80%, from about 25% to about 75%, from about 25% to about 70%, from about 25% to about 65%, from about 25% to about 60%, from about 25% to about 55%, from about 25% to about 50%, from about 25% to about 45 An exon identified as a pseudo-exon included based on a determination of inclusion of %, about 25% to about 40%, or about 25% to about 35%. ENCODE data (e.g., 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(Deep sequencing of subcellular RNA fractions shows that splicing is predominantly co-transcriptional in the human genome but inefficient for IncRNAs)") described by Genome Research 22(9): 1616-25 can be used to help identify exon inclusion.

[0089]

[0102] In some embodiments, complementary to the targeted portion of the SCN1A pre-mRNA transcript Contacting ASO with cells results in an increase of at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% in the amount of SCN1A protein produced, compared to the amount of protein produced by the cells in the absence of ASO / absence of treatment. In some embodiments, the total amount of SCN1A protein produced by the cells contacted with the antisense oligomer is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increased compared to the amount of target protein produced by the control compound. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the pre-mRNA.

[0090]

[0103] In some embodiments, complementary to the targeted portion of the SCN1A pre-mRNA transcript Contacting the ASO, which is complementary, with cells results in a decrease of at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% in the amount of SCN1A protein produced, as compared to the amount of protein produced by the cells in the absence of the ASO / absence of treatment. In some embodiments, the total amount of SCN1A protein produced by the 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, 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 less than the amount of the target protein produced by the control compound. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the pre-mRNA.

[0091]

[0104] In some embodiments, contacting cells with an ASO that is complementary to the targeted portion of the SCN1A pre-mRNA transcript results in an increase in the amount of mRNA encoding SCN1A, which includes mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding the SCN1A protein, or the amount of mature mRNA encoding the SCN1A protein, is at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% increased as compared to the amount of protein produced by the cells in the absence of the ASO / absence of treatment. In some embodiments, the mRNA encoding the SCN1A protein, or the mRNA encoding the SCN1A protein produced in the cells contacted with the antisense oligomer The total amount of mature mRNA is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to the amount of mature RNA produced in untreated cells (e.g., untreated cells, or cells treated with a control compound). The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA.

[0092]

[0105] In some embodiments, complementary to the targeted portion of the SCN1A pre-mRNA transcript Contacting the ASO with the cells results in a decrease in the amount of mRNA encoding SCN1A that contains mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding the SCN1A protein, or mature mRNA encoding the SCN1A protein, is at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% less than the amount of protein produced by the cells in the absence of the ASO / absence of treatment. In some embodiments, the total amount of mRNA encoding the SCN1A protein, or mature mRNA encoding the SCN1A protein, produced in the cells contacted with the antisense oligomer is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold less than the amount of mature RNA produced in untreated cells (e.g., untreated cells, or cells treated with a control compound). The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA.

[0093]

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

[0094]

[0107] Inclusion of a pseudoexon can result in a frameshift, leading to premature introduction of a premature termination codon (PIC) into the mature mRNA transcript targets this transcript for NMD. A mature mRNA transcript containing an NIE can be a non-productive mRNA transcript that does not result in protein expression. The PIC can be present at any position downstream of the NIE. In some embodiments, the PIC can be present in any exon downstream of the NIE. In some embodiments, the PIC can be present within the NIE. For example, inclusion of exon 20x in the mRNA transcript encoded by the SCN1A gene can induce a PIC in the mRNA transcript (e.g., a PIC in exon 21 of the mRNA transcript).

[0095] Therapeutic agent

[0108] In various embodiments of the present disclosure, compositions and methods comprising therapeutic agents are directed to SCN1A It is provided to regulate the protein expression level. In some embodiments, what is provided by the present invention are compositions and methods for regulating the alternative splicing of SCNA1 pre-mRNA. In some embodiments, what is provided by the present invention are compositions and methods for inducing exon skipping in the splicing of SCN1A pre-mRNA (e.g., inducing the skipping of a pseudo-exon during the splicing of SCN1A pre-mRNA). In other embodiments, a therapeutic agent can be used to induce exon inclusion to reduce the protein expression level.

[0096]

[0109] In one embodiment, the therapeutic agents disclosed herein are small molecules, polypeptides, or polynucleic acid polymers. 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 a repressor agent. In additional cases, the therapeutic agent is an enhancer agent.

[0097]

[0110] The therapeutic agents disclosed herein can be NIE repressor agents. The therapeutic agent may contain a polynucleic acid polymer.

[0111] According to one aspect of the present disclosure, what is provided by the present invention is a functional SCN1A A method of treating or preventing a condition associated with a functional SCN1A protein deficiency, comprising administering to a subject an NIE repressor agent to increase the level of the protein, wherein the agent binds to a region of the pre-mRNA transcript and reduces the inclusion of NIE in the mature transcript. For example, the present invention provides a method of treating or preventing a condition associated with a functional SCN1A protein deficiency, comprising administering to a subject an NIE repressor agent to increase the level of functional SCN1A protein, wherein the agent binds to a region of an intron (e.g., intron 20 in the human SCN1A gene) containing NIE in the pre-mRNA transcript, or to an NIE activation regulatory sequence in the same intron.

[0098]

[0112] When referring to suppressing the inclusion of an NIE in mature mRNA, this suppression refers to The suppression may be complete (e.g., 100%) or partial. The suppression may be clinically significant. The suppression / correction may be relative to the level of NIE inclusion in a subject without treatment or relative to the amount of NIE inclusion in a population of similar subjects. The suppression / correction may be at least 10% less NIE inclusion relative to the average subject or pre-treatment subject. The suppression may be at least 20% less NIE inclusion relative to the average subject or pre-treatment subject. The suppression may be at least 40% less NIE inclusion relative to the average subject or pre-treatment subject. The suppression may be at least 50% less NIE inclusion relative to the average subject or pre-treatment subject. The suppression may be at least 60% less NIE inclusion relative to the average subject or pre-treatment subject. The suppression may be at least 80% less NIE inclusion relative to the average subject or pre-treatment subject. The suppression may be at least 90% less NIE inclusion relative to the average subject or pre-treatment subject.

[0099]

[0113] When referring to increasing active SCN1A protein levels, this increase Addition can be clinically important. This increase can be relative to the level of active SCN1A protein in untreated subjects or to the amount of active SCN1A protein in a similar population of subjects. This increase can be at least more than 10% of the active SCN1A protein relative to the average subject or the subject before treatment. This increase can be at least more than 20% of the active SCN1A protein relative to the average subject or the subject before treatment. This increase can be at least more than 40% of the active SCN1A protein relative to the average subject or the subject before treatment. This increase can be at least more than 50% of the active SCN1A protein relative to the average subject or the subject before treatment. This increase can be at least more than 80% of the active SCN1A protein relative to the average subject or the subject before treatment. This increase can be at least more than 100% of the active SCN1A protein relative to the average subject or the subject before treatment. This increase can be at least more than 200% of the active SCN1A protein relative to the average subject or the subject before treatment. This increase can be at least more than 500% of the active SCN1A protein relative to the average subject or the subject before treatment.

[0100]

[0114] In embodiments where the NIE repressor agent comprises a polynucleotide polymer, this The polynucleotide polymer can be about 50 nucleotides in length. This polynucleotide polymer can be about 45 nucleotides in length. This polynucleotide polymer can be about 40 nucleotides in length. This polynucleotide polymer can be about 35 nucleotides in length. This polynucleotide polymer can be about 30 nucleotides in length. This polynucleotide polymer can be about 24 nucleotides in length. This polynucleotide polymer can be about 25 nucleotides in length. This polynucleotide polymer can be about 20 nucleotides in length. This polynucleotide polymer can be about 19 nucleotides in length. This polynucleotide polymer can be about 18 nucleotides in length. This polynucleotide polymer can be about 17 nucleotides in length. This polynucleotide polymer can be about 16 nucleotides in length. This polynucleotide polymer can be about 15 nucleotides in length. This polynucleotide polymer can be about 14 nucleotides in length. This polynucleotide polymer can be about 13 nucleotides in length. This polynucleotide polymer can be about 12 nucleotides in length. This polynucleotide polymer can be about 11 nucleotides in length. This polynucleotide polymer can be about 10 nucleotides in length. This polynucleotide polymer can be between about 10 and about 50 nucleotides in length. This polynucleotide polymer can be between about 10 and about 45 nucleotides in length. This polynucleotide polymer can be between about 10 and about 40 nucleotides in length. This polynucleotide polymer can be between about 10 and about 35 nucleotides in length. This polynucleotide polymer can be between about 10 and about 30 nucleotides in length. This polynucleotide polymer can be between about 10 and about 25 nucleotides in length. This polynucleotide polymer can be between about 10 and about 20 nucleotides in length. This polynucleotide polymer can be between about 15 and about 25 nucleotides in length. This polynucleotide polymer can be between about 15 and about 30 nucleotides in length. This polynucleotide polymer can be between about 12 and about 30 nucleotides in length.

[0101]

[0115] The sequence of this polynucleotide polymer is 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 an mRNA transcript (e.g., a partially processed mRNA transcript).

[0102]

[0116] The sequence of this polynucleotide polymer may have 4 or fewer mismatches with the target sequence of a pre-mRNA transcript. The sequence of this polynucleotide polymer may have 3 or fewer mismatches with the target sequence of a pre-mRNA transcript. The sequence of this polynucleotide polymer may have 2 or fewer mismatches with the target sequence of a pre-mRNA transcript. The sequence of this polynucleotide polymer may have 1 or fewer mismatches with the target sequence of a pre-mRNA transcript. The sequence of this polynucleotide polymer may have no mismatches with the target sequence of a pre-mRNA transcript.

[0103]

[0117] This polynucleotide polymer can specifically hybridize to the target sequence of a pre-mRNA transcript. For example, this polynucleotide polymer may have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence complementarity to the target sequence of a pre-mRNA transcript. This hybridization can be under highly stringent hybridization conditions.

[0104]

[0118] This polynucleotide polymer is selected from the group consisting of SEQ ID NOs: 12 to 731. It may have a sequence having 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 the sequence in question. This polynucleotide polymer may have a sequence having 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 12 to 731. In some cases, this polynucleotide polymer may have a sequence having 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 a sequence selected from the group consisting of SEQ ID NOs: 12 to 371. In some cases, this polynucleotide polymer may have a sequence having 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 12 to 371. In some cases, this polynucleotide polymer may have a sequence having 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 a sequence selected from the group consisting of SEQ ID NOs: 372 to 731. In some cases, this polynucleotide polymer may have a sequence having 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 372 to 731.

[0105]

[0119] When referring to a polynucleotide polymer sequence, one of ordinary skill in the art will understand that one or more substitutions may be tolerated in that sequence, and in some cases two substitutions may be tolerated, provided that its ability to hybridize to the target sequence, or (if the substitution is in the target sequence) its ability to be recognized as the target sequence, is maintained. References to sequence identity can be determined by BLAST sequence alignment using standard / default variables. For example, the sequence may function in accordance with the present disclosure even if it has 99% identity. In other embodiments, the sequence may function in accordance with the present disclosure even if it has 98% identity. In another embodiment, the sequence has 95% identity to the target sequence. In another embodiment, the sequence can have 90% identity and still function according to the present disclosure.

[0106] Antisense oligomer

[0120] The present invention provides a targeting portion of the SCN1A NIE-containing pre-mRNA. ASO is a composition comprising an antisense oligomer that induces exon skipping by binding to a target nucleic acid sequence. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably to refer to an oligomer, such as a polynucleotide, that comprises nucleobases that hybridize to a target nucleic acid (e.g., an SCN1A NIE-containing pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (GU). The ASO may have an exact sequence complementary to the target sequence, or close complementarity (e.g., sufficient complementarity 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 a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to sites other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not the target nucleic acid (to a few sites other than the target nucleic acid). The design of ASOs can take into account the nucleic acid sequence of the targeted portion of the pre-mRNA transcript, or the occurrence of sufficiently similar nucleic acid sequences at other locations in the pre-mRNA or transcriptome of the genome or cell, so as to limit the possibility of the ASO binding to other sites and causing "off-target" effects; see, for example, PCT Application No. PCT / US2014 / 054151 ("Reducing Nonsense-Mediated mRNA Decay"), which is incorporated herein by reference. Any of the antisense oligomers described in WO 2015 / 035091, entitled "Methods for inhibiting mutation-dependent mRNA decay," can be used to practice the methods described herein.

[0107]

[0121] In some embodiments, the ASO is a target nucleic acid or a target of a NIE-containing pre-mRNA. Typically, such hybridization occurs at a T substantially greater than 37° C. m Such hybridization preferably occurs at a temperature of at least 50° C., and typically between 60° C. and approximately 90° C. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, T m is the temperature at which 50% of the target sequence hybridizes to a complementary oligonucleotide.

[0108]

[0122] Oligomers such as oligonucleotides are "complementary" to each other. This is when hybridization occurs in an antiparallel arrangement between two single-stranded polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide when hybridization can occur between one strand of the first polynucleotide and one strand of the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the ratio (e.g., percentage) of bases in the paired strands that are predicted to form hydrogen bonds with each other according to the generally accepted base pairing rules. The sequence of an antisense oligomer (ASO) does not need to be 100% complementary to the sequence of its target nucleic acid. In certain embodiments, the ASO may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to the target region within the target nucleic acid sequence that they target. For example, an ASO in which 18 out of 20 nucleobases of the oligomeric compound are complementary to the target region and thus specifically hybridizes would represent 90% complementarity. In this example, the remaining non-complementary nucleobases may be grouped together, interrupted by complementary nucleobases, or not contiguous with each other or with the complementary nucleobases. The % complementarity between the region of the target nucleic acid and the ASO can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul, et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) known in the art. It may be interrupted, and may not be continuous with each other or with the complementary nucleobases. The % complementarity between the region of the target nucleic acid and the ASO can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul, et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) known in the art.

[0109]

[0123] The ASO does not need to hybridize to all nucleobases in the target sequence. Alternatively, the nucleobases with which it actually hybridizes may be continuous or discontinuous. The ASO may hybridize over one or more segments of the pre-mRNA transcript such that intervening or flanking segments do not participate in the hybridization event (e.g., a loop or hairpin structure may form). In certain embodiments, the ASO hybridizes to discontinuous nucleobases in the target pre-mRNA transcript. For example, the ASO can hybridize to nucleobases in the pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.

[0110]

[0124] The ASOs described herein contain nucleobases that are complementary to nucleobases present in the targeted portion of the NIE-containing pre-mRNA. The term ASO embodies oligonucleotides and other oligomeric molecules that contain nucleobases capable of hybridizing to complementary nucleobases on the target mRNA, but do not contain a sugar moiety such as a peptide nucleic acid (PNA). The ASO can include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of these. The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides with modified or substituted sugar groups and / or nucleotides with modified backbones. In some embodiments, all of the nucleotides of the ASO are modified nucleotides. Those skilled in the art will appreciate chemical modifications of the ASO or components of the ASO that are compatible with the methods and compositions described herein, and for example, U.S. Pat. No. 8,258,109 B2, U.S. Pat. No. 5,656,612, U.S. Patent Publication No. 2012 / 0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355 can be found in.

[0111]

[0125] ​One or more of the nucleobases of the ASO may be adenine, guanine, cytosine, or thymine. The modified nucleobase may be any naturally occurring unmodified nucleobase, such as uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase so that it is capable of hydrogen bonding with a nucleobase present on the target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0112]

[0126] The ASOs described herein also include a backbone structure that links the components of the oligomer. The terms "backbone structure" and "oligomeric linkage" may be used interchangeably and refer to the linkages between the monomers of an ASO. In naturally occurring oligonucleotides, the backbone comprises 3'-5' phosphodiester linkages linking the sugar moieties of the oligomer. The backbone structures or oligomeric linkages of the ASOs described herein can include (but are not limited to) phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, phosphoroamidates, and the like. See, for example, La Planche, et al., Nucleic Acids Res. 14: 9081 (1986); Stec, et al., J. Am. Chem. Soc. 106: 6077 (1984); Stein, et al., Nucleic Acids Res. 16: 3209 (1988); Zon, et al., Anti-Cancer Drug Design 6: 539 (1991); Zon, et al., "Oligonucleotides and Analogues: A Practical Approach" (J. "A Practical Approach to Psychology," pp. 87-108 (ed. F. Eckstein, Oxford University Press, See, e.g., Oxford, UK (1991)); Stec, et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorus and instead contains peptide bonds (e.g., in peptide nucleic acid (PNA)), or a linking group containing carbamate, amide, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.

[0113]

[0127] In some embodiments, each of the phosphorus-nucleotide linkages of the ASO backbone has a random stereochemistry. In some embodiments, the stereochemistry at each of the phosphorus-nucleotide linkages of the ASO backbone is controlled and not random. For example, U.S. Patent Application Publication No. 2014 / 0194610, “Methods for the Synthesis of Functionalized Nucleic Acids,” which is incorporated herein by reference " describes a method for independently selecting the chirality of each phosphorus atom in a nucleic acid oligomer. In one or more embodiments, the ASO (including, but not limited to, any of the ASOs shown herein in Tables 5 and 6) used in the methods of the present invention includes an ASO having a non-random phosphorus-nucleotide internucleotide linkage. In one or more embodiments, the compositions used in the methods of the present invention include pure diastereomeric ASOs. In one or more embodiments, the compositions used in the methods of the present invention include ASOs having a diastereomeric 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%.

[0114]

[0128] In one or more embodiments, the ASO has a non-random mixture of Rp and Sp configurations at its phosphorus-nucleotide internucleotide linkages. For example, it has been suggested that achieving a balance between good activity and nuclease stability is required for the mixing of Rp and Sp in antisense oligonucleotides (Wan, et al., 2014, "Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages", Nucleic Acids Research 42 (22): 13456-13468, incorporated herein by reference). In one or more embodiments, the ASO (including, but not limited to, the present) used in the methods of the present invention ​ (including any of the ASOs shown by SEQ ID NOs: 12 to 731 in the specification) exhibits about 5 to 100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, together with the remaining Sp, or contains about 100% Rp. In an aspect(s), the ASO (including, but not limited to, any of the ASOs shown by SEQ ID NOs: 12 to 731 in the specification) used in the method of the present invention exhibits about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 10 0% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, together with the remaining Sp.

[0115]

[0129] In an aspect(s), the ASO (not limited to (including any of the ASOs shown in SEQ ID NOs: 12 to 731 herein) is included with about 5 to 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 includes at least about 95% Sp together with the remaining Rp or includes about 100% Sp. In an aspect(s), the ASO(s) (including, but not limited to, any of the ASOs shown in SEQ ID NOs: 12 to 731 herein) used in the method of the present invention is included with about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp together with the remaining Rp.

[0116]

[0130] Any of the ASOs described herein is in naturally occurring nucleotides It may contain a sugar moiety, a modified sugar moiety or a sugar analog (including a morpholine ring) that contains ribose or deoxyribose as it exists. Non-limiting examples of modified sugar moieties include 2'-substitutions such as 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 sugar, and bicyclic-modified sugar. In some embodiments, the sugar moiety modification is selected from 2'-O-Me, 2'-F, and 2'-MOE. In some embodiments, the sugar moiety modification is an extra cross-link bond as 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 ribofuranosyl or 2'-deoxyribofuranosyl modification. In some embodiments, the sugar moiety includes 2',4'-constrained 2'-O-methyloxyethyl (cM OE) modification. In some embodiments, the sugar moiety includes cEt2',4'-constrained 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety includes tricyclo DNA (tcDNA) modification. In some embodiments, the sugar moiety includes ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety includes MCE modification. Modifications are known in the art and are described, for example, in the literature by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications", Nucleic Acid Therapeutics 24(1): 37-47, which is incorporated herein by reference for this purpose.

[0117]

[0131] In some embodiments, each monomer of the ASO is modified in the same way, for example Each linkage of the ASO backbone contains a phosphorothioate linkage or each ribose sugar moiety contains a 2’O-methyl modification. Such modifications present in each of the monomeric components of the ASO are referred to as “uniform modifications”. In some embodiments, combinations of different modifications may be desired, for example, the ASO may comprise a combination of phosphoramidate linkages and a sugar moiety comprising a morpholine ring (morpholino). Combinations of various modifications to the ASO are referred to as “mixed modifications” or “mixed chemistry”.

[0118]

[0132] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2’MOE modification and a phosphorothioate backbone. 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 the ASO (e.g., nucleobase, sugar moiety, backbone), can be modified to achieve a desired property or activity of the ASO, or to suppress an undesired property or activity of the ASO. For example, the ASO or any one or more components of the ASO can be modified to enhance the binding affinity to a target sequence on a pre-mRNA transcript; to suppress binding to non-target sequences; to suppress degradation by cellular nucleases (i.e., RNase H); to improve cellular uptake of the ASO and / or nuclear uptake into the cell; to alter the pharmacokinetics or pharmacodynamics of the ASO; and / or to regulate the half-life of the ASO.

[0119]

[0133] In some embodiments, the ASO is 2’-O-(2-methoxyethyl) (MO E) Comprised of phosphorothioate-modified nucleotides. ASOs composed of such nucleotides are particularly well suited for the methods disclosed herein, as oligomers bearing such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery, for example, in some embodiments described herein. See, e.g., Geary, et al., J Pharmacol Exp Ther. 2001; 296(3): 890-7; Geary, et al., J Pharmacol Exp Ther. 2001; 296(3):898-904.

[0120]

[0134] Those skilled in the art will know how to synthesize ASOs. Thus, ASOs may be obtained from commercial sources.

[0135] Unless otherwise specified, single-stranded nucleic acids (e.g., pre-mRNA transcripts, oligonucleotides, etc.) The left hand end of a nucleic acid sequence (such as an octade, ASO, etc.) is the 5' end, and the left hand direction of a single 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 referred to as the 3' end or direction. Generally, a region or sequence that is 5' to a reference point in a nucleic acid is referred to as "upstream" and a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream". Generally, the 5' direction or 5' end of an mRNA is where the initiation or start codon is located, whereas the 3' end or 3' direction is where the stop codon is located. In some aspects, nucleotides that are upstream of the reference point in a nucleic acid may be designated by a negative number, while nucleotides that are downstream of the reference point in a nucleic acid may be designated by a positive number. For example, a reference point (e.g., an exon-exon junction in an mRNA) may be specified as the "zero" position, and the nucleotide immediately adjacent to and upstream of that reference point is specified as a "minus one," e.g., "-1," while the nucleotide immediately adjacent to and downstream of that reference point is specified as a "plus one," e.g., "+1."

[0121]

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

[0122]

[0137] In some embodiments, the ASO is the SCN1A NIE-containing pre-mRNA (e.g. For example, it is complementary to (and binds to) the targeted portion of the SCN1A NIE-containing pre-mRNA that is upstream (5' direction) of the 5' splice site (or 3' end) of the included exon in the direction specified by a negative number (e.g., the 5' splice site of the included exon). In some embodiments, the ASO is complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA that is within the region of about -4 to about -270 relative to the 5' splice site (or 3' end) of the included exon. In some embodiments, the ASO can be complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA that is within the region between nucleotide -1 and nucleotide -264 relative to the 5' splice site (or 3' end) of the included exon. In some aspects, the ASO is complementary to the targeted portion within the region of about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20 relative to the 5' splice site (or 3' end) of the included exon. In some aspects, the ASO is complementary to the targeted portion within the region of about -1 to about -50, about -50 to about -100, about -100 to about -150, about -150 to about -200, or about -200 to about -250 relative to the 5' splice site (or 3' end) of the included exon.

[0123]

[0138] In some embodiments, the ASO is the SCN1A NIE-containing pre-mRNA (e.g., For example, it is complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA that is upstream (5' direction) of the 3' splice site (or 5' end) of the included exon in the direction specified by a negative number (e.g., the 3' splice site of the included exon). In some embodiments, the ASO is the 3' splice It is complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA within the region of approximately -1 to approximately -500 relative to the price site (or 5' end). In some embodiments, the ASO is complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA within the region of -1 to -496 relative to the 3' splice site of the included exon. In some aspects, the ASO is within the region of approximately -1 to approximately -500, approximately -1 to approximately -490, approximately -1 to approximately -480, approximately -1 to approximately -470, approximately -1 to approximately -460, approximately -1 to approximately -450, approximately -1 to approximately -440, approximately -1 to approximately -430, approximately -1 to approximately -420, approximately -1 to approximately -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 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, approximately -1 to approximately -160, 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 -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 included exon and is complementary to the targeted portion. In some aspects, the ASO is within the region 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 included exon and is complementary to the targeted portion.

[0124]

[0139] In some embodiments, the ASO is the SCN1A NIE-containing pre-mRNA (e.g. For example, it is complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA that is downstream (in the 3' direction) of the 3' splice site (or 5' end) of the included exon in the direction specified by a positive number. In some embodiments, the ASO is complementary to the targeted portion of the SCN1A NIE-containing pre-mRNA that is within the region of about +1 to about +100 relative to the 3' splice site of the included exon. In some aspects, the ASO is complementary to the targeted portion that is within the regions of about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, about +1 to about +20, or about +1 to about +10 relative to the 3' splice site of the included exon.

[0125]

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

[0126]

[0141] The ASO is of any length suitable for specific binding and effective enhancement of splicing It may also be so. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, the ASO can 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 nucleobases in length. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the ASO is 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to Lengths of 50 nucleobases, 10 - 40 nucleobases, 10 - 35 nucleobases, 10 - 30 nucleobases, 10 - 25 nucleobases, 10 - 20 nucleobases, 10 - 15 nucleobases, 11 - 50 nucleobases, 11 - 40 nucleobases, 11 - 35 nucleobases, 11 - 30 nucleobases, 11 - 25 nucleobases, 11 - 20 nucleobases, 11 - 15 nucleobases, 12 - 50 nucleobases, 12 - 40 nucleobases, 12 - 35 nucleobases, 12 - 30 nucleobases, 12 - 25 nucleobases, 12 - 20 nucleobases, 12 - 15 nucleobases, 13 - 50 nucleobases, 13 - 40 nucleobases, 13 - 35 nucleobases, 13 - 30 nucleobases, 13 - 25 nucleobases, 13 - 20 nucleobases, 14 - 50 nucleobases, 14 - 40 nucleobases, 14 - 35 nucleobases, 14 - 30 nucleobases, 14 - 25 nucleobases, 14 - 20 nucleobases, 15 - 50 nucleobases, 15 - 40 nucleobases, 15 - 35 nucleobases, 15 - 30 nucleobases, 15 - 25 nucleobases, 15 - 20 nucleobases, 20 - 50 nucleobases, 20 - 40 nucleobases, 20 - 35 nucleobases, 20 - 30 nucleobases, 20 - 25 nucleobases, 25 - 50 nucleobases, 25 - 40 nucleobases, 25 - 35 nucleobases, or 25 - 30 nucleobases. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length.

[0127]

[0142] In some embodiments, two or more ASOs that are chemically distinct but complementary to the same target of the NIE-containing pre-mRNA are used. In some embodiments, two or more ASOs that are complementary to different targeting portions of the NIE-containing pre-mRNA are used.

[0128]

[0143] In the embodiment(s), the antisense oligonucleotide of the present invention is 1 or more It is chemically linked to an upper portion or conjugate (e.g., a targeting portion or other conjugate that enhances the activity or cellular uptake of the oligonucleotide). Such portions include, but are not limited to, lipid portions (e.g., cholesterol portions, cholesteryl portions), aliphatic chains (e.g., dodecanediol or undecyl residues), polyamines or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides comprising lipophilic portions and methods of manufacture have been described in the known literature. In some embodiments, the antisense oligonucleotide is conjugated to a portion comprising, but not limited to, non-basic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. As understood in the art and described in the literature, for example, using a linker, a conjugate can be linked to any of several positions on the sugar, base, or phosphate group, relative to one or more of the nucleotides comprising the antisense oligonucleotide. The linker can include a divalent or trivalent branched-chain linker. In some embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods for manufacturing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides" (carbohydrate conjugates as delivery agents for oligonucleotides), which is incorporated herein by reference. of the carbohydrate conjugate)".

[0129]

[0144] In some embodiments, the nucleic acid to be targeted by the ASO is a cell such as a eukaryotic cell. It is an SCN1A NIE-containing pre-mRNA that is expressed in cells. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cells are in a subject. In some embodiments, the cells are isolated from a subject. In some embodiments, the cells are ex vivo. In some embodiments, the cells are cells or cell lines associated with a pathological condition or disease. In some embodiments, the cells are in vitro (e.g., in a cell culture).

[0130] Pharmaceutical composition

[0145] It comprises an agent (e.g., an antisense oligonucleotide) of the described composition and a pharmaceutical composition or formulation for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature. In embodiments, a pharmaceutical composition or formulation for treating a subject comprises an effective amount of an antisense oligomer as described herein, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. A pharmaceutical formulation comprising an antisense oligomer may further comprise a pharmaceutically acceptable excipient, diluent, or carrier.

[0131]

[0146] A pharmaceutically acceptable salt is suitable for use in contact with human and lower animal tissues without undue toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable benefit / risk ratio (e.g., see S. M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), which is incorporated herein by reference for this purpose). The salt can be prepared in situ during the final isolation and purification of the compound or separately by reacting the free base functional groups with a suitable organic acid. Examples of pharmaceutically acceptable, innocuous acid addition salts are salts of the amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with 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 adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, 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, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include salts of harmless ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates, as appropriate.

[0132]

[0147] In one or more embodiments, the composition includes, but is not limited to, tablets, capsules, It can be formulated into any of a number of possible dosage forms, such as gel capsule preparations, liquid syrup preparations, soft gel preparations, suppositories, and enemas. In some embodiments, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. An aqueous suspension may further contain substances that increase the viscosity of the suspension, such as, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer. In some embodiments, the pharmaceutical preparation or composition of the present invention includes, but is not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing preparations (e.g., cationic or non-cationic liposome preparations).

[0133]

[0148] The pharmaceutical compositions or preparations described herein are suitable and may contain one or more penetration enhancers, carriers, excipients, or other active or inactive components as are well known to those skilled in the art or as described in the known literature. In some embodiments, sterically stabilized liposome preparations (e.g., liposome preparations comprising one or more specialized lipids) are also included in the liposome preparations, and these specialized lipids result in liposome preparations with enhanced circulation lifetimes. In some embodiments, the sterically stabilized liposome preparations contain one or more glycolipids or are derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. In some embodiments, surfactants are included in the pharmaceutical preparation or composition. The use of surfactants in pharmaceuticals, preparations, and emulsions is well known in the art. In some embodiments, the present invention utilizes penetration enhancers to effectively deliver antisense oligonucleotides (e.g., to aid in diffusion through cell membranes and / or to enhance the permeability of lipophilic drugs). In some embodiments, the penetration enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.

[0134]

[0149] In some embodiments, the pharmaceutical preparation comprises a number of antisense oligonucle It contains an ootide. In some embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.

[0135] Combination therapy

[0150] In some embodiments, the ASO disclosed in the present disclosure can be used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents may include small molecules. For example, the one or more additional 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, the one or more additional therapeutic agents may include an ASO that can be used to correct intron retention. In some embodiments, the one or more other agents are selected from the ASOs listed in Table 4.

[0136] Treatment of a subject

[0151] Any of the compositions provided by the present invention can be administered to an individual. "Individual" can be used interchangeably with "subject" or "patient". An individual is a mammal, such as an animal like a human or non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep. In some embodiments, the individual is a human. In some embodiments, the individual is a fetus, an embryo, or a pediatric patient. In other embodiments, the individual can be another eukaryote, such as a plant. In some embodiments, the compositions provided by the present invention are administered to cells in vitro. In some embodiments, the compositions provided by the present invention are used to treat a disease or disorder.

[0137]

[0152] In some embodiments, the compositions provided by the present invention are used to treat a disease or disorder. It is administered to an individual as a method. In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is in a risk state of having a disease, such as any of the diseases described herein. In some embodiments, the individual is in a state with an increased risk of having a disease or disorder caused by an insufficient amount of a certain protein or an insufficient activity of a certain protein. If the individual is in a "state with an increased risk" of having a disease or disorder caused by an insufficient amount of a certain protein or an insufficient activity of a certain protein, the method is involved in prophylactic or preventive treatment. For example, an individual may be in a state with an increased risk of having such a disease or disorder due to a family history of the disease. Typically, an individual in a state with an increased risk of having such a disease or disorder benefits more from prophylactic treatment (e.g., by preventing the onset of the disease or disorder or delaying its progression). In some embodiments, for example, the fetus is treated in utero by administering the ASO composition directly or indirectly (e.g., via the mother) to the fetus.

[0138]

[0153] The route suitable for administration of the ASO of the present invention may vary depending on the cell type to which ASO delivery is desired. Dravet syndrome; Generalized febrile seizures plus, type 2; Familial febrile seizures, 3A; Familial hemiplegic migraine, 3; Autism; Epileptic encephalopathy, early infantile, 13; Pore dysplasia syndrome 1; Alzheimer's disease, or SUDEP affects many tissues and organs, but the most significantly affected tissue is the brain. The ASO of the present invention can be administered to a patient parenterally, for example, by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection, or intravenous injection.

[0139]

[0139]

[0154] In some embodiments, the disease or condition is induced by a mutation in Na v 1.1 (the protein encoded by the SCN1A gene son). In some cases, the mutation is in Na vIt is a loss-of-function mutation in 1.1. In some cases, Na v The loss-of-function mutation in 1.1 is Na v The function of 1.1 is compared to wild-type Na v It contains one or more mutations that reduce or impair the function of 1.1 by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more compared to the function of 1.1. In some cases, Na v The loss-of-function mutation in 1.1 contains 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.

[0140]

[0155] In other cases, the mutation is a gain-of-function mutation in Na v in 1.1. In such cases, the gain-of-function mutation is Na In such cases, the gain-of-function mutation is Na v Activation of 1.1 is longer compared to the function of wild-type Na v 1.1 by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more. In such cases, Na v The gain-of-function mutation in 1.1 contains 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.

[0141]

[0156] In some aspects, the disease or disorder is encephalopathy. In some cases, the encephalopathy is induced by a loss-of-function mutation in Na v in 1.1.

[0157] In some aspects, the encephalopathy is epileptic encephalopathy. Exemplary epileptic includes, but is not limited to, Doose syndrome (DS) (also known as severe myoclonic epilepsy in infancy or SMEI); severe myoclonic epilepsy in infancy (SMEI)-borderland type (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; childhood alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or hole-in-the-heart syndrome 1. In some embodiments, the disease or condition is, optionally, Doose syndrome (DS) (also known as severe myoclonic epilepsy in infancy or SMEI); severe myoclonic epilepsy in infancy (SMEI)-borderland type (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; childhood alternating hemiplegia; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); and an epileptic encephalopathy selected from hole-in-the-heart syndrome 1.

[0142]

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

[0159] In some cases, febrile seizures are familial febrile seizures, 3A.

[0143]

[0160] In some cases, SMEB is SMEB without generalized spike-and-slow waves (SM EB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O), or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0144]

[0161] In some embodiments, Na v diseases or conditions induced by loss-of-function mutations in 1.1 include, but are not limited to, Doose syndrome (DS) (also known as SMEI); severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pore dysplasia syndrome 1; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); autism; or malignant migrating partial seizures in infancy.

[0145]

[0145]

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

[0146]

[0146]

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

[0164] In some embodiments, the disease or condition is Na v 1.1 genetic epilepsy. Na v 1.1 genetic epilepsy may include loss-of-function mutations in 1.1 or Na v gain-of-function mutations in 1.1. In some cases, Na v 1.1 genetic epilepsy includes one or more genetic mutations. In other cases, Na v 1.1 genetic epilepsy includes a gain-of-function mutation in 1.1. v1.1 Hereditary epilepsy includes one or more de novo mutations. In some cases, Na v 1 .1 Hereditary epilepsy includes Doose syndrome (DS) (also known as severe myoclonic epilepsy of infancy or SMEI); severe myoclonic epilepsy of infancy (SMEI)-borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; childhood alternating hemiplegia; unclassifiable epileptic encephalopathy; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); sudden unexpected death in epilepsy (SUDEP); or malignant migrating partial seizures in infancy. In some cases, Na v Na associated with loss-of-function mutations in 1.1 v 1.1 Hereditary epilepsy includes Doose syndrome (DS) (also known as severe myoclonic epilepsy of infancy or SMEI); severe myoclonic epilepsy of infancy (SMEI)-borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; childhood alternating hemiplegia; unclassifiable epileptic encephalopathy; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); sudden unexpected death in epilepsy (SUDEP); malignant migrating partial seizures in infancy.

[0147]

[0165] In some embodiments, the disease or condition is associated with haploinsufficiency of the SCN1A gene. Exemplary diseases or conditions associated with haploinsufficiency of the SCN1A gene include, but are not limited to, Doose syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infancy (SMEI)-borderline (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus; Genetic epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic epilepsy; early myoclonic encephalopathy; progressive myoclonic epilepsy; childhood alternating hemiplegia; unclassifiable epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); porencephaly syndrome 1; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or malignant migrating partial seizures in infancy are included. In some cases, the disease or condition is Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy in infancy (SMEI)-borderline type (SMEB); febrile seizures (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic epilepsy; early myoclonic encephalopathy; progressive myoclonic epilepsy; childhood alternating hemiplegia; unclassifiable epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); porencephaly syndrome 1; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or malignant migrating partial seizures in infancy.

[0148]

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

[0167] Dravet syndrome (DS) (or severe myoclonic epilepsy in infancy (SME The Dravet syndrome (also known as severe myoclonic epilepsy of infancy, SMEI) is an epilepsy encephalopathy that appears in the first year of life. The Dravet syndrome is an increasingly recognized epilepsy encephalopathy, and its clinical diagnosis is supported by sodium channel gene mutation findings in approximately 70-80% of patients. Mutations in ion channel genes play an important role in the pathogenesis of a wide range of epilepsy syndromes and cause some epilepsies, which are regarded as channelopathies. Since voltage-gated sodium channels (VGSCs) play an essential role in the excitability of nerve cells, it is not surprising that many mutations related to DS have been identified in genes encoding VGSC subunits. For this disease, for example, it was described by Mulley, et al., 2005 and the description of this disease is available in OMIM #607208 (Online Mendelian Inheritance in Man, Johns Hopkins University, 1966-2015), both of which are incorporated herein by reference.

[0149]

[0168] Between 70% and 80% of patients carry a sodium channel alpha-1 subunit gene ( SCN1A) abnormality, with truncating mutations accounting for approximately 40% and showing a significant correlation with earlier age of seizure onset. Sequence variants are found in approximately 70% of cases, including truncating mutations (40%) and missense mutations (40%), with the remainder being splice-site changes. Most mutations are novel, but familial mutations occur in 5-10% of cases and are usually missense in nature. The remaining SCN1A mutations include splice-site and missense mutations, most of which correspond to the pore-forming region of the sodium channel. Currently, over 500 mutations have been associated with DS and are randomly distributed along the gene (Mulley, et al., Neurol. 2006, 67, 1094-1095).

[0150]

[0169] The SCN1A gene is the sodium channel gene on human chromosome 2q24 Located within the cluster, it encodes the α pore-forming subunit known as Na v 1.1. The SCN1A gene spans approximately 100 kb of genomic DNA and contains 26 exons. The SCN1A protein consists of four domains, each having a six-transmembrane segment. Two splice variants in exon 11 have been identified that result in long and short isoforms differing in the presence or absence of 11 amino acids in the cytoplasmic loop between domain 1 and domain 2 (Miller, et al., 1993 - 2015 and Mulley, et al., 2005, 25, 535 - 542, incorporated herein by reference).

[0151]

[0170] Alternative splicing events in the SCN1A gene can result in non-productive mRNA transcripts, which in turn can lead to abnormal protein expression. Thus, therapeutic agents that can target alternative splicing events in the SCN1A gene can regulate the expression level of functional proteins and / or inhibit abnormal protein expression in DS patients. Such therapeutic agents can be used to treat the pathological conditions caused by SCN1A protein deficiency.

[0152]

[0171] Alternative splicing events that can result in non-productive mRNA One such is the inclusion of an extra exon in an mRNA transcript that may induce nonsense mutation-dependent mRNA decay. The present disclosure provides compositions and methods for modulating the alternative splicing of SCN1A to increase the production of the mature mRNA encoding the protein and thereby the functional SCN1A protein translated therefrom. These compositions and methods include antisense oligomers (ASOs) capable of causing exon skipping and promoting constitutive splicing of SCN1A pre-mRNA. In various embodiments, the methods of the present disclosure can be used to increase functional SCN1A protein to treat conditions caused by SCN1A protein deficiency.

[0153]

[0172] In some cases, the disease or condition is SMEB.

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

[0174] In some cases, the disease or condition is febrile seizures (e.g., familial febrile seizures , 3A).

[0154]

[0175] In some cases, the disease or condition is autism (also known as autism spectrum disorder or ASD).

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

[0155]

[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.

[0156]

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

[0181] In one embodiment, the antisense oligonucleotide is a The antisense oligonucleotides are administered in combination with one or more agents capable of enhancing penetration of the antisense oligonucleotides through the blood-brain barrier by any method known in the art. For example, see U.S. Pat. No. 6,632,427, entitled "Adenoviral-vector-mediated gene transfer into medullary motor neurons," which is incorporated herein by reference. No. 6,756,523, entitled "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," which is incorporated herein by reference, describes the delivery of drugs to motor neurons by administration of adenovirus vectors. The paper "Direct delivery of vectors to the brain (e.g., striatum, thalamus, hippocampus, substantia nigra)" describes the direct delivery of vectors to the brain (e.g., striatum, thalamus, hippocampus, substantia nigra).

[0157]

[0182] In some embodiments, the antisense oligonucleotides are In some embodiments, the antisense oligonucleotide is linked or conjugated to an agent that provides a therapeutic or pharmacodynamic property. In some embodiments, the antisense oligonucleotide is linked to an agent known in the art to enhance penetration or transport across the blood-brain barrier (e.g., an antibody against the transferrin receptor). In some embodiments, the antisense oligonucleotide is linked to an agent that provides a therapeutic or pharmacodynamic property. In some embodiments, the antisense oligonucleotide is linked to an agent known in the art to enhance penetration or transport across the blood-brain barrier (e.g., an antibody against the transferrin receptor). The "Do" is ligated to a viral vector, for example, to make the antisense compound more effective or to enhance transport across the blood-brain barrier. In aspects, sugars (e.g., meso-erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, 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 (e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine) are used to assist in osmotic blood-brain barrier disruption. For example, U.S. Patent No. 9,193,969 "Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types", U.S. Patent No. 4,866,042 "Method for the delivery of genetic material across the blood brain 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", each incorporated herein by reference, describe methods and materials for enhancing blood-brain barrier penetration. ​​

[0158]

[0183] In some embodiments, the ASO of the present invention is conjugated to a dopamine reuptake inhibitor (DRI), selective serotonin reuptake inhibitor (SSRI), norepinephrine reuptake inhibitor (NRI), norepinephrine-dopamine reuptake inhibitor (NDRI), and serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI) using the method described in U.S. Patent No. 9,1 93,969, which is incorporated herein by reference.

[0159]

[0184] In some embodiments, for a subject being treated using the methods and compositions, the improvement of the pathological condition is evaluated using methods known and described in the art.

[0160] Method for identifying additional ASOs that induce exon skipping

[0185] Also within the scope of the present disclosure are methods for identifying or determining an ASO that induces exon skipping of SCN1A NIE-containing pre-mRNA. For example, one method may include identifying or determining an ASO that induces pseudoexon skipping of SCN1A NIE-containing pre-mRNA. To identify or determine an ASO that improves the rate and / or extent of splicing of a target intron, one may screen ASOs that specifically hybridize to various nucleotides within the target region of the pre-mRNA. In some embodiments, the ASO may block or interfere with the binding site(s) of splicing repressor(s) / silencer(s). Any method known in the art may be used to identify (determine) an ASO that produces a desired effect (e.g., pseudoexon skipping, production of a protein or functional RNA) when hybridizing to the target region of an exon. These methods can be used to identify an ASO that induces exon skipping of an included exon by binding to a targeted region in an intron adjacent to the included exon or in an exon that is not included. One example of a method that can be used is provided below.

[0161]

[0186] Using ASOs designed to hybridize to target regions of pre-mRNA A first round of screening, called an ASO "walk," may be performed using ASOs. For example, the ASOs used in the ASO walk may be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the included exon (e.g., a portion of the sequence of the exon located upstream of the target / included exon) to approximately 100 nucleotides downstream of the 3' splice site of the target / included exon, and / or approximately 100 nucleotides upstream of the 5' splice site of the included exon to approximately 100 nucleotides downstream of the 5' splice site of the target / included exon (e.g., a portion of the sequence of the exon located downstream of the target / included exon). For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides at +6 to +20 relative to the 3' splice site of the target / included exon. A second ASO may be designed to specifically hybridize to nucleotides that are +11 to +25 relative to the 3' splice site of the targeted / included exon. The ASO is designed to encompass the target region of the pre-mRNA. In embodiments, the ASO can be tiled more closely, for example, every 1, 2, 3, or 4 nucleotides. Additionally, the ASO can be tiled up to 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 up to about 1,160 nucleotides upstream of the 3' splice site to about 500 nucleotides downstream of the 5' splice site. In some embodiments, the ASO can be tiled up to about 500 nucleotides upstream of the 3' splice site to about 1,920 nucleotides downstream of the 3' splice site.

[0162]

[0187] One or more ASOs or a control ASO (scrambled sequence, i.e., hybridized to the target region) An ASO with an array not predicted to redize is delivered into a disease-related cell line expressing a target pre-mRNA (e.g., the NIE-containing pre-mRNA described herein), for example, by transfection. For the exon skipping effect of each of these ASOs, it can be evaluated by any method known in the art, such as reverse transcriptase (RT)-PCR using primers that cover splice junctions, as described in Example 4. A decrease or absence of a longer RT-PCR product produced using primers that cover regions containing the included exon (e.g., including the exon next to NIE) in ASO-treated cells compared to control ASO-treated cells suggests enhanced splicing of the target NIE. In some embodiments, the exon skipping efficiency (or splicing efficiency of splicing the NIE-containing intron), the ratio of spliced pre-mRNA to unspliced pre-mRNA, the splicing rate, or the degree of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO achieved the desired effect (e.g., enhanced production of functional protein). Any method known in the art for evaluating and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0163]

[0188] An ASO designed to hybridize to the target region of the pre-mRNA can be used to perform a second screening called ASO "micro-walking". The ASOs used in ASO micro-walking are tiled every single nucleotide to further refine the nucleotide sequence of the pre-mRNA where exon skipping (or enhanced splicing of NIE) occurs when hybridized to the ASO.

[0164]

[0189] ASOs spaced one nucleotide apart, as well as longer ASOs (typically 1 8 - 25 nucleotides), more specifically explore the regions delineated by ASOs that promote splicing of the target intron by means of ASO "micro - walks".

[0165]

[0190] As described for the above - mentioned ASO walk, the ASO micro - walk is carried out by delivering one or more ASOs, or control ASOs (scrambled sequences, i.e., ASOs with sequences not predicted to hybridize to the target region), into a disease - related cell line expressing the target pre - mRNA, for example, by transfection. For the splicing - inducing effect of each of these ASOs, it can be evaluated by any method known in the art, as described herein (e.g., see Example 4), for example, by reverse transcriptase (RT) - PCR using primers that span the NIE. A decrease or absence of longer RT - PCR products produced using primers that span the NIE in ASO - treated cells compared to control ASO - treated cells suggests enhanced exon skipping (or splicing of the NIE - containing target intron). In some embodiments, the exon skipping efficiency (or splicing efficiency of splicing the NIE - containing intron), the ratio of spliced pre - mRNA to unspliced pre - mRNA, the splicing rate, or the degree of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre - mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., enhanced production of functional protein). Any method known in the art for evaluating and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0166]

[0191] Exon skipping when hybridizing to a region of pre-mRNA (or enhanced splicing of NIE-containing introns) to produce an ASO that increases protein production can be tested in vivo using an animal model (e.g., a transgenic mouse model in which the full-length human gene is knocked in, or a humanized mouse model of a disease). The route suitable for administration of the ASO may vary depending on the disease and / or the cell type for which delivery of the ASO is desired. The ASO can be administered, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection, or intravenous injection. Following administration, the cells, tissues, and / or organs of the model animal can be evaluated to determine the effect of the ASO treatment by evaluating, for example, splicing (efficiency, rate, degree) and protein production by methods known in the art and described herein. The animal model may exhibit a phenotype or behavior of the disease or disease severity.

[0167]

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

[0193] Also within the scope of the present disclosure is a method for identifying or demonstrating NMD-inducing exons in the presence of an NMD inhibitor (e.g., cycloheximide). Exemplary methods are provided in Figure 3 and Example 2.

[0168] Specific embodiments

[0194] Embodiment 1. mRNA containing a nonsense mutation-dependent RNA degradation-inducing exon A method of regulating expression of SCN1A protein in a cell having an mRNA encoding SCN1A protein, said cell comprising contacting said cell with a therapeutic agent, whereby said therapeutic agent regulates splicing of an NMD exon from the NMD exon mRNA encoding SCN1A protein, thereby regulating the level of processed mRNA encoding SCN1A protein and regulating expression of SCN1A protein in said cell, wherein said therapeutic agent binds to a targeting portion of the NMD exon mRNA encoding SCN1A, and The method, wherein the targeting portion is located between about 1,000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) and about 100 nucleotides upstream from the 5' end of the NIE; or between about 100 nucleotides downstream from the 3' end of the NIE and about 1,000 nucleotides downstream from the 3' end of the NIE.

[0169]

[0195] Aspect 2. SC in cells of a subject in need of treating a disease or condition A method for treating an N1A protein in a subject by regulating expression thereof, comprising contacting the cells of the subject with a therapeutic agent that regulates splicing of a nonsense-mediated mRNA decay-induced exon (NMD exon) from an mRNA encoding SCN1A, the NMD exon being contained in the cells, thereby regulating the level of processed mRNA encoding SCN1A protein and regulating the expression of SCN1A protein in the cells of the subject; wherein the therapeutic agent binds to a targeting portion of the NMD exon mRNA encoding SCN1A, and the targeting portion is located about 1000 nucleotides upstream from the 5' end of the NMD-induced exon (NIE) to about 100 nucleotides upstream from the 5' end of the NIE; or about 100 nucleotides downstream from the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE.

[0170]

[0196] Aspect 3. The therapeutic agent inhibits splicing of an NMD exon from the region of the targeting moiety. The method of embodiment 1 or 2, wherein the binding of a factor involved in cloning is interfered with.

[0197] 4. The targeting moiety is at most about 800 nucleotides from the 5' end of the NIE. 3. The method of embodiment 1 or 2, wherein the sequence is 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 upstream of the amino acid sequence.

[0171]

[0198] Embodiment 5. The targeting moiety is at least about 800 nucleotides from the 5' end of the NIE. 3. The method of embodiment 1 or 2, wherein the sequence is 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, or about 1 nucleotide upstream of the sequence.

[0172]

[0199] 6. The targeting portion is at most about 800 nucleotides from the 3' end of the NIE 3. The method of embodiment 1 or 2, wherein the sequence is 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 downstream.

[0173]

[0200] Embodiment 7. The targeting moiety is at least about 800 nucleotides from the 3' end of the NIE. The method of embodiment 1 or 2 that is downstream by 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, or about 1 nucleotide.

[0174]

[0201] Embodiment 8. The method of any one of embodiments 1- 7, wherein the therapeutic agent is an antisense oligomer (ASO).

[0202] Embodiment 9. The method of embodiment 8, wherein 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: 12-731.

[0175]

[0203] Embodiment 10. The method of any one of embodiments 1-9, wherein the therapeutic agent promotes the exclusion of NMD exons from the processed mRNA encoding the SCN1A protein.

[0176]

[0204] Embodiment 11. In cells contacted with the therapeutic agent, the co- ​and the removal of the NMD exon from the processed mRNA encoding the SCN1A protein in the control cell is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, or about 5 fold greater than the removal of the NMD exon from the processed mRNA encoding the SCN1A protein in the control cell. fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

[0177]

[0205] 12. The therapeutic agent is a processing gene encoding the SCN1A protein. The method of embodiment 10, wherein the level of the expressed mRNA in said cell is increased.

[0206] 13. Co-expression of SCN1A protein in cells contacted with a therapeutic agent. The amount of processed mRNA encoded by the SCN1A protein ... The method of embodiment 10, wherein the increase is from 2 to about 7 fold, from about 2 to about 8 fold, from about 2 to about 9 fold, from about 3 to about 6 fold, from about 3 to about 7 fold, from about 3 to about 8 fold, from about 3 to about 9 fold, from about 4 to about 7 fold, from about 4 to about 8 fold, from about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

[0178]

[0207] 14. The disease or condition is v Loss-of-function mutation in 1.1 The method of embodiment 2, wherein

[0208] 15. The disease or condition is associated with haploinsufficiency of the SCN1A gene, 15. The method of embodiment 14, wherein the subject has a first allele that encodes a functional SCN1A and a second allele in which SCN1A is not produced or is produced at a reduced level, or a second allele that encodes a non-functional SCN1A or a partially functional SCN1A.

[0179]

[0209] The method of embodiment 14, wherein the disease or condition is encephalopathy.

[0210] Embodiment 17 The method of embodiment 16, wherein the encephalopathy is epileptic encephalopathy.

[0211] Aspect 18. The disease or condition is Dravet syndrome (DS); The method of embodiment 14, wherein the condition is epilepsy (SMEI)-limbic type (SMEB); febrile seizures (FS); generalized epilepsy febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; latent generalized epilepsy; latent focal epilepsy; myoclonic astatic epilepsy; Lennox-Gast syndrome; West syndrome; idiopathic convulsions; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassifiable epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome 1; autism; or malignant migratory partial seizures of infantile.

[0180]

[0212] Aspect 19. The aspect in which GEFS+ is generalized epilepsy with febrile seizures plus type 2. 18 ways to do it.

[0213] Embodiment 20 The method of embodiment 18, wherein the febrile seizures are familial febrile seizures 3A.

[0181]

[0214] Aspect 21. SMEB is SMEB without generalized spike-and-wave (SMEB-SW) , the method of embodiment 18, which is SMEB without myoclonic seizures (SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O), or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0182]

[0215] Embodiment 22. The ASO consists of a sequence selected from SEQ ID NO: 72 or 432 , the method of embodiment 1 or 2.

[0216] Embodiment 23. The ASO consists of a sequence selected from SEQ ID NO: 73 or 433 , the method of embodiment 1 or 2.

[0183]

[0217] Embodiment 24. The ASO consists of a sequence selected from SEQ ID NO: 76 or 436 , the method of embodiment 1 or 2.

[0218] Embodiment 25. The ASO consists of a sequence selected from SEQ ID NO: 181 or 541 and is the method of embodiment 1 or 2.

[0184]

[0219] Embodiment 26. The ASO consists of a sequence selected from SEQ ID NO: 220 or 580 and is the method of embodiment 1 or 2.

[0220] Embodiment 27. mRNA containing a nonsense mutation-dependent RNA degradation-inducing exon A method for regulating the expression of SCN1A protein in a cell having an mRNA encoding SCN1A protein and being NA (NMD exon mRNA), comprising contacting the cell with a therapeutic agent, whereby the therapeutic agent regulates the splicing of the NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of the processed mRNA encoding the SCN1A protein, and regulating the expression of the SCN1A protein in the cell, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE.

[0185]

[0221] Embodiment 28. In the cells of a subject in need of treating a disease or disorder, a method for treating the subject by regulating the expression of S CN1A protein, comprising contacting the cells of the subject with a therapeutic agent that regulates the splicing of a nonsense mutation-dependent mRNA degradation-inducing exon (NMD exon) from an mRNA containing the NMD exon and encoding SCN1A in the cells, thereby regulating the level of the processed mRNA encoding the SCN1A protein, and regulating the expression of the SCN1A protein in the cells of the subject, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE.

[0186]

[0222] Embodiment 29. An antisense oligomer (ASO) comprising a sequence that is at least about 8 0%, 85%, 90%, 95%, 97%, or 100% identical to any one of SEQ ID NOs: 12-731.

[0187]

[0223] Aspect 30. An antisense oligomer (ASO) consisting of a sequence selected from SEQ ID NOs: 12 to 731.

[0224] Aspect 31. A method for treating a subject in need of treating a disease or disorder, by regulating the expression of SCN1A protein in the cells of the subject, the method comprising contacting the ASO of Aspect 29 or 30 with the cells of the subject. The said method.

[0188]

[0225] Aspect 32. A kit comprising the ASO of Aspect 29 or 30.

[0226] Preferred aspects of the present invention have been set forth and described herein. It will be apparent to those skilled in the art that such aspects are provided only by way of example. Those skilled in the art will now be able to conceive of numerous variations, changes, and substitutions without departing from the spirit of the present invention. It should be understood that various alternative aspects to the aspects of the present invention described herein may be utilized when practicing the present invention. The following claims serve to define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be included therein.

Examples

[0189]

[0227] The present invention will be illustrated more specifically by the following examples. However, it should be understood that the present invention is not limited in any way by these examples. Example 1: Identification of NMD-inducing exon inclusion events in SCN1A transcripts by RNAseq using next-generation sequencing

[0228] Whole transcriptome shotgun sequencing using next-generation sequencing was performed to clarify the snapshot of the transcript produced by the SCN1A gene and identify NIE inclusion events. For this purpose, polyA + RNA was isolated from the nuclear and cytoplasmic fractions of HCN (human cortical neurons), and a cDNA library was constructed using Illumina’s TruSeq Stranded mRNA library Prep Kit. The 100-nucleotide reads generated by pair-end sequencing of this library were mapped to the human genome (February 2009, GRCh37 / hg19 assembly). The sequencing results for SCN1A are shown in Figure 2. Briefly, Figure 2 was visualized using the UCSC Genome Browser (operated by the UCSC Genome Informatics Group (Genome Informatics Group) (Biomolecular Science and Engineering Center, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064)) and shows the mapped reads, as described by, for example, Rosenbloom, et al., 2015, “The UCSC Genome Browser database (UCSC Genome Browser database): 2015 updated version” Nucleic Acids Research 43, Database Issue, doi: 10.1093 / nar / gku1177), and the range of the reads ​The circle and number can be estimated by the peak signal. The height of the peak indicates the expression level given by the density of the readings in the special region. The upper panel shows an enlarged graph of the SCN1A gene. The conservation levels across 100 vertebrate species are shown as peaks. The highest peak corresponds to the exon (black box), while for most introns (arrowed lines), no peak is observed. A conserved peak was identified in intron 20 (NM_006920) and is shown in the central panel. Examination of the conserved sequence identified a 64bp exon-like sequence (lower panel, sequence highlighted in grey) with the 3' splice site and 5' splice site (underlined sequences) alongside. Since the inclusion of this exon results in a frameshift, an immature stop codon is introduced into exon 21 to target this transcript for NMD.

[0190]

[0229] Sequences of the exemplary SCN1A gene, pre-mRNA, exon, and intron are summarized in Table 1. The sequences of each exon or intron are summarized in Table 2.

[0191]

Table 1

[0192]

Table 2-1

[0193]

Table 2-2

[0194]

Table 2-3

[0195]

Table 2-4

[0196]

Table 2-5

[0197]

Table 2-6

[0198]

Table 2-7

[0199] Example 2: Confirmation of NIE via cycloheximide treatment

[0230] DMSO treatment (CHX - ) or cycloheximide treatment (CHX + ) of mouse cytoplasmic RNAs from Neuro 2A cells (Figure 3A) and RenCell VM (human neural progenitor cells) (Figure 3B), and RT-PCR analysis using primers in exons 21 and 23 confirmed the presence of bands corresponding to the NMD-inducing exon (20x). The uniqueness of this product was confirmed by sequencing. Concentration measurement analysis of this band was performed to calculate the exon 20x inclusion percentage of total SCN1A transcripts. Treatment of RenCell VM with cycloheximide (CHX + ) to inhibit NMD resulted in a 2-fold increase in the product corresponding to the NMD-inducing exon 20x in the cytoplasmic fraction (compare the light gray bar, CHX - and the dark gray bar, CHX + ).

[0200] Example 3: ASO walk in the SCN1A exon 23 (exon 20x) region

[0231] By shifting 5 nucleotides at a time, SCN1A exon 2 ASOs were designed to cover the region approximately 1000 to approximately 100 nucleotides upstream of the 5'-end of the 3 (Exon 20x) gene. Also, by shifting 5 nucleotides at a time, ASOs were designed to cover a second region approximately 1000 to approximately 100 nucleotides downstream of the 3'-end of the SCN1A Exon 23 (Exon 20x) gene. A list of ASOs targeting SCN1A is summarized in Table 3. The sequences of the ASOs are summarized in Table 4.

[0201]

Table 3

[0202]

Table 4-1

[0203]

Table 4-2

[0204]

Table 4-3

[0205]

Table 4-4

[0206]

Table 4-5

[0207]

Table 4-6

[0208]

Table 4-7

[0209]

Table 4-8

[0210] Example 4: Extension ASO walk of the SCN1A exon 23 (exon 20x) region, as evaluated by RT-PCR

[0232] For the ASO walk sequences, evaluation can be done, for example, by RT-PCR. In FIG. 5A, a representative PAGE shows SYBR-Safe stained RT-PCR products that target the exon 20x region, mock-treated, control ASO-treated, by nucleofection at a concentration of 1 μM in RenCell, as described in Example 3 and as described herein in the description of FIG. 3. Quantify two products (one comprising exon 20x and the other excluding exon 20x) and plot the exon 20x inclusion percentage in a bar graph (FIG. 5B). Also normalize the Taqman qPCR products to the RPL32 internal control and plot the fold change compared to the mock in a bar graph (FIG. 5C).

[0211]

[0233] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only by way of example. Those skilled in the art will now be able to conceive of numerous variations, changes, and substitutions without departing from the spirit of the present invention. It is to be understood that when practicing the present invention, various alternative embodiments to the embodiments of the present invention described herein may be utilized. The following claims are intended to clarify the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are intended to be included therein. This application encompasses the following inventions. [Item 1] A method for regulating the expression of SCN1A protein in a cell having an mRNA encoding SCN1A protein, which is an mRNA containing a nonsense mutation-dependent RNA degradation-inducing exon (NMD exon mRNA), comprising Contact the cell with a therapeutic agent, whereby the therapeutic agent regulates the splicing of the NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of the processed mRNA encoding the SCN1A protein, and regulates the expression of the SCN1A protein in the cell, comprising, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is: from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 100 nucleotides upstream of the 5' end of the NIE; or from about 100 nucleotides downstream of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE, the method as described above. [Item 2] A method for treating a subject in need of treating a disease or condition by regulating the expression of the SCN1A protein in the cells of the subject, comprising: contacting the cells of the subject with a therapeutic agent that regulates the splicing of the nonsense mutation-dependent mRNA degradation-inducing exon (NMD exon) from the mRNA containing the NMD exon encoding SCN1A in the cells, thereby regulating the level of the processed mRNA encoding the SCN1A protein, and regulating the expression of the SCN1A protein in the cells of the subject, comprising, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is: from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 100 nucleotides upstream of the 5' end of the NIE; or from about 100 nucleotides downstream of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE, the method as described above. [Item 3] The method of Item 1 or 2, wherein the therapeutic agent interferes with the binding of a factor involved in the splicing of the NMD exon from the region of the targeted portion. [Item 4] The method according to item 1 or 2, wherein the targeted portion is located at most 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 upstream of the 5'-end of the NIE. [Item 5] The method according to item 1 or 2, wherein the targeted portion is located at least 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 upstream of the 5'-end of the NIE. [Item 6] The method according to item 1 or 2, wherein the targeted portion is located at most 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 downstream of the 3'-end of the NIE. [Item 7] The method according to item 1 or 2, wherein the targeted portion is located at least 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 downstream of the 3'-end of the NIE. [Item 8] The method according to any one of items 1 to 7, wherein the therapeutic agent is an antisense oligomer (ASO). [Item 9] The method of Item 8, wherein 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: 12 to 731. [Item 10] The method of any one of Items 1 to 9, wherein the therapeutic agent promotes the exclusion of NMD exons from the processed mRNA encoding the SCN1A protein. [Item 11] The method of Item 10, wherein the exclusion of NMD exons from the processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold as compared to the exclusion of NMD exons from the processed mRNA encoding the SCN1A protein in control cells. [Item 12] The method of Item 10, wherein the therapeutic agent increases the level of the processed mRNA encoding the SCN1A protein in the cell. [Item 13] The method of Item 10, wherein the amount of processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold as compared to the total amount of processed mRNA encoding the SCN1A protein in control cells. [Item 14] The method of Item 2, wherein the disease or condition is induced by a loss-of-function mutation in Na v 1.1. [Item 15] The method of Item 14, wherein the disease or condition is associated with haploinsufficiency of the SCN1A gene, wherein the subject has a first allele encoding a functional SCN1A and a second allele that does not produce SCN1A or produces SCN1A at a reduced level, or a second allele encoding a non-functional SCN1A or a partially functional SCN1A. [Item 16] The method of Item 14, wherein the disease or condition is encephalopathy. [Item 17] The method of Item 16, wherein the encephalopathy is epileptic encephalopathy. [Item 18] The method of Item 14, wherein the disease or condition is Dravet syndrome (DS); severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB); febrile seizure (FS); generalized epilepsy with febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic atonic seizure epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); porencephaly syndrome 1; autism; or malignant migrating partial seizures in infancy. [Item 19] The method of Item 18, wherein GEFS+ is generalized epilepsy with febrile seizures plus type 2. [Item 20] The method of Item 18, wherein the febrile seizure is familial febrile seizure type 3A. [Item 21] The method of Item 18, wherein SMEB is SMEB without generalized spike and slow waves (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O), or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). [Item 22] The method of Item 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 72 or 432. [Item 23] The method of Item 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 73 or 433. [Item 24] The method of Item 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 76 or 436. [Item 25] The method of Item 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 181 or 541. [Item 26] The method of Item 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 220 or 580. [Item 27] A method for regulating the expression of SCN1A protein in a cell having mRNA encoding SCN1A protein and containing a nonsense mutation-dependent RNA degradation-inducing exon (NMD exon mRNA), comprising contacting the cell with a therapeutic agent, 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 cell, wherein the therapeutic agent binds to a targeting portion of the NMD exon mRNA encoding SCN1A, and the targeting portion is from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE. [Item 28] A method for treating a subject in need of treating a disease or disorder by modulating the expression of SCN1A protein in the cells of the subject, comprising: contacting a therapeutic agent that modulates the splicing of a nonsense mutation-dependent mRNA degradation-inducing exon (NMD exon) from an mRNA encoding SCN1A containing an NMD exon in the cell with the cells of the subject, thereby modulating the level of processed mRNA encoding SCN1A protein, and modulating the expression of SCN1A protein in the cells of the subject, wherein the therapeutic agent binds to a targeted portion of the NMD exon mRNA encoding SCN1A, and the targeted portion is from about 1000 nucleotides upstream of the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE; said method. [Item 29] 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: 12-731. [Item 30] An antisense oligomer (ASO) consisting of a sequence selected from SEQ ID NOs: 12-731. [Item 31] A method for treating a subject in need of treating a disease or disorder by modulating the expression of SCN1A protein in the cells of the subject, comprising: contacting the ASO of Item 29 or 30 with the cells of the subject; said method. [Item 32] A kit comprising the ASO of Item 29 or 30.

Claims

1. 1. A method for modulating expression of SCN1A protein in a cell having an mRNA that contains a nonsense-mediated RNA decay-inducing exon (NMD exon mRNA) encoding the SCN1A protein, comprising: contacting the cell with a therapeutic agent, whereby the therapeutic agent modulates splicing of an NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby modulating the level of processed mRNA encoding the SCN1A protein; and Regulating the expression of SCN1A protein in the cell; wherein the therapeutic agent binds to a targeting portion of an NMD exon mRNA encoding SCN1A, and the targeting portion comprises: From about 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to about 100 nucleotides upstream from the 5' end of the NIE; or From about 100 nucleotides downstream of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE; The method according to any one of claims 1 to 4,

2. 1. A method of treating a disease or condition in a subject in need thereof by modulating expression of an SCN1A protein in cells of the subject, comprising: contacting the cells of the subject with a therapeutic agent that modulates splicing of a nonsense-mediated mRNA decay-inducing exon (NMD exon) from an mRNA encoding SCN1A that contains an NMD exon in the cells, thereby modulating the level of processed mRNA encoding the SCN1A protein; and modulating expression of SCN1A protein in said cells of said subject; wherein the therapeutic agent binds to a targeting portion of an NMD exon mRNA encoding SCN1A, and the targeting portion comprises: From about 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to about 100 nucleotides upstream from the 5' end of the NIE; or From about 100 nucleotides downstream of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE; The method according to any one of claims 1 to 4,

3. 3. The method of claim 1 or 2, wherein the therapeutic agent interferes with the binding of a factor involved in splicing of the NMD exon from the region of the targeting moiety.

4. The method of claim 1 or 2, wherein the targeting portion is at most 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 5' end of the NIE.

5. The method of claim 1 or 2, wherein the targeting portion is at least 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, or about 1 nucleotide upstream of the 5' end of the NIE.

6. The method of claim 1 or 2, wherein the targeting portion is at most 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 of the 3' end of the NIE.

7. The method of claim 1 or 2, wherein the targeting portion is at least 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, or about 1 nucleotide downstream of the 3' end of the NIE.

8. The method of any one of claims 1 to 7, wherein the therapeutic agent is an antisense oligomer (ASO).

9. 9. The method of claim 8, wherein 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: 12-731.

10. The method of any one of claims 1 to 9, wherein the therapeutic agent promotes the exclusion of the NMD exon from the processed mRNA encoding the SCN1A protein.

11. the exclusion of the NMD exon from the processed mRNA encoding the SCN1A protein in cells contacted with the therapeutic agent is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, 11. The method of claim 10, wherein the increase is about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

12. The method of claim 10, wherein the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in said cell.

13. The amount of processed mRNA encoding the SCN1A protein in cells contacted with a therapeutic agent 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, about 2 to about 10 times, about 1.1 ...

11. The method of claim 10, wherein the increase is up to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

14. The disease or condition is Na ｖ 3. The method of claim 2, which is induced by a loss-of-function mutation in 1.

1.

15. The method of claim 14, wherein the disease or condition is associated with haploinsufficiency of the SCN1A gene, and wherein the subject has a first allele that encodes a functional SCN1A and a second allele in which SCN1A is not produced or is produced at reduced levels, or a second allele that encodes a non-functional SCN1A or a partially functional SCN1A.

16. 15. The method of claim 14, wherein the disease or condition is encephalopathy.

17. 17. The method of claim 16, wherein the encephalopathy is epileptic encephalopathy.

18. The disease or condition may be Dravet syndrome (DS); severe myoclonic epilepsy of infancy (SMEI)-limbic type (SMEB); febrile seizures (FS); generalized epilepsy febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; cryptogenic generalized epilepsy; cryptogenic 15. The method of claim 14, wherein the condition is focal epilepsy; myoclonic astatic epilepsy; Lennox-Gast syndrome; West syndrome; idiopathic convulsions; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassifiable epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome 1; autism; or malignant migratory partial seizures of infancy.

19. 20. The method of claim 18, wherein GEFS+ is generalized epilepsy with febrile seizures plus type 2.

20. 19. The method of claim 18, wherein the febrile seizures are familial febrile seizures 3A.

21. 20. The method of claim 18, wherein the SMEB is SMEB without generalized spike-and-waves (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking one or more features of SMEI (SMEB-O), or refractory epilepsy of childhood with generalized tonic-clonic seizures (ICEGTC).

22. The method of claim 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 72 or 432.

23. The method of claim 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 73 or 433.

24. The method of claim 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 76 or 436.

25. The method of claim 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 181 or 541.

26. The method of claim 1 or 2, wherein the ASO consists of a sequence selected from SEQ ID NO: 220 or 580.

27. 1. A method for modulating expression of SCN1A protein in a cell having an mRNA that contains a nonsense-mediated RNA decay-inducing exon (NMD exon mRNA) encoding the SCN1A protein, comprising: contacting the cell with a therapeutic agent, whereby the therapeutic agent modulates splicing of an NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby modulating the level of processed mRNA encoding the SCN1A protein; and Regulating the expression of SCN1A protein in the cell; wherein the therapeutic agent binds to a targeting portion of an NMD exon mRNA encoding SCN1A, and the targeting portion is located from about 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE.

28. 1. A method of treating a disease or condition in a subject in need thereof by modulating expression of an SCN1A protein in cells of the subject, comprising: contacting the cells of the subject with a therapeutic agent that modulates splicing of a nonsense-mediated mRNA decay-inducing exon (NMD exon) from an mRNA encoding SCN1A that contains an NMD exon in the cells, thereby modulating the level of processed mRNA encoding the SCN1A protein; and modulating expression of SCN1A protein in said cells of said subject; wherein the therapeutic agent binds to a targeting portion of an NMD exon mRNA encoding SCN1A, and the targeting portion is located from about 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to about 1000 nucleotides downstream of the 3' end of the NIE.

29. 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: 12-731.

30. An antisense oligomer (ASO) consisting of a sequence selected from SEQ ID NOs: 12-731.

31. 1. A method of treating a disease or condition in a subject in need thereof by modulating expression of an SCN1A protein in cells of the subject, comprising: The method comprises contacting the ASO of claim 29 or 30 with the cells of the subject.

32. A kit comprising the ASO of claim 29 or 30.

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