Antisense oligomers for treating pathologies and diseases

Abstract

Because alternative splicing events in the SCN1A gene can result in non-productive mRNA transcripts, which can then lead to abnormal protein expression, therapeutic agents that can target alternative splicing events in the SCN1A gene can regulate the expression level of functional protein and / or inhibit abnormal protein expression in Dravet syndrome patients. Such therapeutic agents can be used to treat conditions caused by SCN1A, SCN8A, or SCN5A protein deficiencies.

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 often associated with channelopathies, characterized by the 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 α-pore-forming subunit of neuronal voltage-gated sodium channels, are associated with the development of diseases in the disease classification 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 embodiment of the present invention is a method of regulating expression of SCN1A protein in a cell having an mRNA containing a nonsense-dependent RNA decay-inducing exon (NMD exon mRNA) encoding the SCN1A protein, the method comprising contacting the cell with a therapeutic agent, whereby the therapeutic agent regulates splicing of the NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of processed mRNA encoding the SCN1A protein and regulating expression of the 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 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 about 100 nucleotides downstream of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE. In some embodiments, the therapeutic agent interferes with the binding of factors involved in splicing of NMD exons from the region of the targeting moiety.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 upstream of the 5' 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 upstream of the 5' end of the NIE.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 SCN1A protein. In some embodiments, the exclusion of NMD exons from processed mRNA encoding 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, about 1.1 to about 9-fold, about 1.1 to about 10 ... The increase is 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.In some embodiments, the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in the cell. In some embodiments, the amount of processed mRNA encoding the SCN1A protein in the cell contacted with the 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 An increase of 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.

[0004]

[0004] Disclosed in one aspect of the invention is a method of treating a disease or condition in a subject in need of such treatment by modulating 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 splicing of a nonsense-dependent mRNA decay-inducing exon (NMD exon) from an mRNA encoding SCN1A, containing an NMD exon in the cells, thereby modulating the level of processed mRNA encoding SCN1A protein and modulating 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 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 about 100 nucleotides downstream of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE. In some embodiments, the therapeutic agent interferes with the binding of factors involved in splicing of NMD exons from the region of the targeting moiety.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 upstream of the 5' 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 upstream of the 5' end of the NIE.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 SCN1A protein. In some embodiments, the exclusion of NMD exons from processed mRNA encoding 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, about 1.1 to about 9-fold, about 1.1 to about 10 ... The increase is 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.In some embodiments, the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in the cell. In some embodiments, the amount of processed mRNA encoding the SCN1A protein in the cell contacted with the 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 The increase is 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. In some embodiments, the disease or condition is Na. v1.1. In some embodiments, the disease or condition is associated with haploinsufficiency of the SCN1A gene, where 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. In some embodiments, the disease or condition is encephalopathy. In some embodiments, the encephalopathy is epileptic encephalopathy. In some embodiments, the disease or condition is Dravet syndrome (DS); Severe Myoclonic Epilepsy of Infancy (SMEI)-Limbic Type (SMEB); Febrile Seizures (FS); Generalized Epilepsy Febrile Seizures Plus (GEFS+); Epileptic Encephalopathy, Early Infancy, 13; Latent Generalized Epilepsy; Latent Focal Epilepsy; Myoclonic Astatic Epilepsy; Lennox-Gast Syndrome; West Syndrome; Idiopathic Seizures; Early Myoclonic Encephalopathy; Progressive Myoclonic Epilepsy; Alternating Hemiplegia of Infancy; Unclassifiable Epileptic Encephalopathy; Sudden Unexpected Death in Epilepsy (SUDEP); Sick Sinus Syndrome 1; Autism; or Malignant Migratory Partial Seizures of Infancy. In some embodiments, the GEFS+ is Generalized Epilepsy Febrile Seizures Plus Type 2. In some embodiments, the febrile seizures are Familial Febrile Seizures 3A. In some aspects, the SMEB is SMEB without generalized spike-and-waves (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O), or intractable epilepsy of childhood with generalized tonic-clonic seizures (ICEGTC).

[0005]

[0005] Disclosed in one embodiment of the present invention is a method of regulating expression of SCN1A protein in a cell having an mRNA containing a nonsense-dependent RNA decay-inducing exon (NMD exon mRNA) encoding the SCN1A protein, the method comprising contacting the cell with a therapeutic agent whereby the therapeutic agent regulates splicing of the NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of processed mRNA encoding the SCN1A protein and regulating the expression of 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 approximately 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to approximately 1000 nucleotides downstream of the 3' end of the NIE.

[0006]

[0006] Disclosed in one aspect of the invention is a method of treating a disease or condition in a subject in need of such treatment by modulating 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 splicing of a nonsense-dependent mRNA decay-inducing exon (NMD exon) from an mRNA encoding SCN1A containing an NMD exon in the cells, thereby modulating the level of processed mRNA encoding SCN1A protein and modulating 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 approximately 1000 nucleotides upstream from the 5' end of the NMD-inducing exon (NIE) to approximately 1000 nucleotides downstream of the 3' end of the NIE.

[0007]

[0007] Disclosed in one aspect 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] Disclosed in one aspect of the present invention is an antisense oligomer (ASO) consisting of a sequence selected from SEQ ID NOs: 12-731.

[0009] Disclosed in one aspect of the invention is a method of treating a disease or condition in a subject in need of such treatment by regulating expression of SCN1A protein in cells of the subject, the method comprising contacting cells of the subject with 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: 12-731; or an ASO consisting of a sequence selected from SEQ ID NOs: 12-731.

[0009]

[0010] Disclosed in one aspect of the invention is 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: 12-731; or a kit comprising an ASO consisting of a sequence selected from SEQ ID NOs: 12-731.

[0010] Reference

[0011] All publications, patents, and patent applications mentioned in this specification are herein 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 set forth with particularity 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 that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which: [Brief description of the drawings]

[0012] [Figure 1AB]

[0013] FIG. 1 illustrates a schematic of a target mRNA containing a nonsense-dependent RNA decay-induced exon (NMD exon mRNA) and therapeutic drug-mediated elimination of the nonsense-dependent mRNA decay-induced exon, which increases the expression of a full-length target protein or functional RNA. FIG. 1A shows a cell divided into nuclear and cytoplasmic fractions. In the nucleus, the pre-mRNA transcript of the target gene is spliced ​​to produce an mRNA, which is transported to the cytoplasm and translated into a target protein. For this target gene, a fraction of its mRNA does not result in target protein production because it contains a nonsense-dependent mRNA decay-induced exon (NMD exon mRNA) that is degraded in the cytoplasm. FIG. 1B shows an example of the same cell divided into nuclear and cytoplasmic fractions. Treatment with a therapeutic drug such as an antisense oligomer (ASO) promotes the elimination of the nonsense-dependent mRNA decay-induced exon, resulting in an increase in the mRNA, which is then translated into higher levels of the target protein. [Figure 1C] FIG. 1C is a schematic diagram of therapeutic ASO-mediated elimination of nonsense-mediated mRNA decay-induced exons, which reduces nonproductive mRNA and increases productive mRNA, resulting in increased expression of full-length target protein from the productive mRNA. [Diagram 2]

[0014] FIG. 2 illustrates the identification of exemplary nonsense-mediated mRNA decay (NMD)-induced exons in the SCN1A gene. Identification of NMD-induced exons in the SCN1A gene using comparative genomics is shown visualized in the UCSC genome browser. The top panel shows a zoomed-in graph of the SCN1A gene. Conservation levels across 100 vertebrate species are shown as peaks. The highest peaks correspond to exons (black boxes), whereas no peaks are observed in the majority of introns (lines with arrowheads). A peak of conservation was identified in intron 20 (NM_006920) and is shown in the middle panel. Inspection of the conserved sequence identified a 64 bp exon-like sequence (lower panel, sequence highlighted in grey) flanked by 3' and 5' splice sites (underlined sequences), which we refer to as exon 20x. Inclusion of this exon results in a frameshift, thus introducing a premature stop codon in exon 21, targeting this transcript for NMD. Figure 2 depicts SEQ ID NO:732. [Diagram 3]

[0015] Figure 3A illustrates the confirmation of the NMD-induced exon via cycloheximide treatment. RT-PCR analysis of cytoplasmic RNA from DMSO-treated (CHX-) or cycloheximide-treated (CHX+) Neuro 2A (mouse neural progenitor cells) using primers for exon 21 and downstream exons confirmed the presence of a band corresponding to the NMD-induced exon (21x). The identity of this product was confirmed by sequencing. Densitometric analysis of this band was performed to calculate the % inclusion of exon 21x in the total SCN1A transcript. Inhibition of NMD by treatment of Neuro 2A with cycloheximide (CHX+) resulted in a two-fold increase in the product corresponding to the NMD-induced exon 21x in the cytoplasmic fraction (compare light gray bar, CHX- with dark gray bar, CHX+).

[0016] Figure 3B illustrates confirmation of the NMD-induced exon via cycloheximide treatment. RT-PCR analysis using cytoplasmic RNA from DMSO-treated (CHX-) or cycloheximide-treated (CHX+) RenCell VM (human neural progenitor cells) and primers in exon 21 and exon 23 confirmed the presence of a band corresponding to the NMD-induced exon (20x). The identity of this product was confirmed by sequencing. Densitometric analysis of this band was performed to calculate the % inclusion of exon 20x in the total SCN1A transcript. Inhibition of NMD by treating RenCell VM with cycloheximide (CHX+) resulted in a two-fold increase in the product corresponding to the NMD-induced exon 20x in the cytoplasmic fraction (compare light grey bar, CHX- with dark grey bar, CHX+). [Figure 4]

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

[0018] Figure 5A illustrates ASOs to the SCN1A exon 20x region selected from the extension ASO walk assessed by RT-PCR. A representative PAGE shows SYBR-safe stained RT-PCR products from SCN1A mock-treated, control ASO-treated (NT), or ASOs to the SCN1A exon 20x region from the extension walk via nucleofection at 1 μM for 24 hours in RenCell. Mock = no ASO; Control NT = non-targeting control; Posctrl = positive control. [Figure 5B]

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

[0020] FIG. 5C illustrates a graph of qPCR results of the extension ASO walk using the samples from FIG. 5A, normalized to the RPL32 internal control, plotting the fold change in SCN1A mRNA compared to mock. Detailed Description of the Invention

[0013] Splicing and nonsense-mediated mRNA decay

[0021] Intervening sequences or introns are removed by a large and highly dynamic RNA-protein complex called the spliceosome, which coordinates interactions between the primary transcript, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome assembles ad hoc in an ordered manner for each intron, starting with recognition of the 5' splice site (5'ss) by U1 snRNA or the 3' splice site (3'ss) by the U2 pathway, which involves 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 65 kD subunit encoding U2AF2 (U2AF65), which binds to the polypyrimidine tract (PPT), and a 35 kD subunit encoding U2AF1 (U2AF35), which interacts with a highly conserved AG dinucleotide at the 3'ss, stabilizing U2AF65 binding. In addition to the BPS / PPT unit and the 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures that activate or repress splice site recognition, known as intron or exon splicing enhancers or silencers. These elements allow authentic splice sites to be recognized among the vast plethora of cryptic or pseudosites (which have the same sequence as authentic sites but are an order of magnitude more numerous) in the genomes of higher eukaryotes. They often have regulatory functions, but the mechanisms of their activation or repression are poorly understood.

[0014]

[0022] Because the decision to splice or not to splice can typically be modeled as a stochastic rather than a deterministic process, even the most well-defined splicing signal can splice incorrectly. However, under normal conditions, pre-mRNA splicing proceeds with surprisingly high fidelity. This is due in part to the activity of adjacent cis-acting auxiliary exonic and intronic splicing regulatory elements (ESRs or ISRs). Typically, these functional elements are classified as either exonic or intronic splicing enhancers (ESEs or ISEs) or silencers (ESSs or ISSs) based on their ability to promote or inhibit splicing, respectively. Although there is now evidence that some auxiliary cis-acting elements may act by affecting the dynamics of spliceosome assembly, such as the positioning of the U1 snRNP complex with the 5'ss, many elements appear highly 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 a key role in defining exons. SR proteins promote exon recognition by recruiting components of the pre-spliceosome to adjacent splice sites or by antagonizing the effects of nearby ESSs. The repressive effect of ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their role in splicing regulation, silencer elements have also been suggested to have a role in suppressing pseudoexons (a set of decoy intron splice sites with typical exonic spacing but no functional open reading frame). ESEs and ESSs, in conjunction with their cognate trans-acting RBPs, are key components in the 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 various strengths that can occur frequently in human genes. In multi-exon genes, various pairs of splice sites can be linked together in many different combinations, creating a wide variety of transcripts from a single gene. This is generally called alternative pre-mRNA splicing. Most mRNA isoforms produced by alternative splicing can be transported from the nucleus and translated into functional polypeptides, but various mRNA isoforms from a single gene can vary greatly in their translation efficiency. mRNA isoforms with a premature stop codon (PTC) 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 lead to aberrant splicing, such as exon skipping or cryptic (or pseudo) exon inclusion or splice site activation, which can cause significant human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by naturally occurring DNA variants in exons and introns.

[0016]

[0024] Given that exon-intron boundaries can occur at any of the three codon positions, only a subset of alternative splicing events can maintain the canonical open reading frame. For example, only exons that are evenly divisible by three can be skipped or included in the mRNA without alteration of the reading frame. Splicing events that do not have compatible phases induce a frameshift. If not reversed by downstream events, the frameshift can certainly result in one or more PTCs, possibly resulting in subsequent degradation by NMD. NMD is a translation-coupled mechanism that removes mRNAs that contain PTCs. NMD can function as a surveillance pathway that exists in all eukaryotes. NMD can suppress errors in gene expression by removing mRNA transcripts that contain premature stop codons. Translation of these aberrant mRNAs can, in some cases, result in deleterious gain-of-function or dominant-negative activity of the resulting protein. Because NMD targets not only certain transcripts of PTCs but also a wide range of mRNA isoforms expressed by many endogenous genes, it has been suggested that NMD is a master regulator driving both fine and coarse regulation of steady-state RNA levels in cells.

[0017]

[0025] An NMD-inducing exon (NIE) is an exon or pseudoexon that is a region within an intron that can activate the NMD pathway when included in a mature RNA transcript. In a constitutive splicing event, an intron containing an NIE is usually spliced ​​out, but the intron or a portion thereof (e.g., the NIE) can be retained during an alternative or aberrant splicing event. A mature mRNA transcript containing such an NIE can be non-productive due to a frameshift that induces the NMD pathway. Inclusion of an NIE in a mature RNA transcript can downregulate gene expression. In this disclosure, an mRNA transcript containing an NIE will be referred to as an "NIE-containing mRNA" or an "NMD exon mRNA."

[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 can be readily identified by one of skill in the art using suitable publicly available algorithms, e.g., those found in Kralovicova, J. and Vorechovsky, I. (2007) "Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition", Nucleic Acids Res., 35, 6399-6413, (http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC2095810 / pdf / gkm680.pdf).

[0020]

[0028] A cryptic splice site or splicing regulatory sequence may compete with the splice site of the NIE for an RNA binding protein such as U2AF. In one embodiment, an agent may be bound to the cryptic splice site or splicing regulatory sequence to prevent binding of the RNA binding protein, thereby favoring utilization of the NIE splice site.

[0021]

[0029] In one embodiment, the cryptic splice site may not include the 5' or 3' splice site of the 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 disclosed method utilizes the presence of NIE in pre-mRNA transcribed from SCN1A gene. Splicing of identified SCN1A NIE pre-mRNA species to produce functional mature SCN1A mRNA can be induced using therapeutic agents such as ASOs that promote exon skipping of NIE. Induction of exon skipping can result in inhibition of NMD pathway. The resulting mature SCN1A mRNA can be translated normally without activating NMD pathway, thereby increasing the amount of SCN1A protein in the patient's cells and alleviating symptoms of pathologies associated with SCN1A deficiency such as Dravet syndrome (DS); generalized epilepsy febrile seizures plus, type 2; familial febrile seizures, 3A; autism; epileptic encephalopathy, premature infantile, 13; sick sinus syndrome 1; Alzheimer's disease; or SUDEP.

[0024]

[0032] In various aspects, the 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 SCN1A pre-mRNA can be targeted by therapeutic agents such as ASOs. In some aspects, the ASO targets SCN1A pre-mRNA transcripts that contain an NIE. In some aspects, the ASO targets a sequence within the NIE of an 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 an 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 an SCN1A pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron flanking the 5' end of the NIE of the SCN1A pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron flanking the 3' end of the NIE of the SCN1A pre-mRNA transcript. In some embodiments, the ASO targets a sequence comprising the NIE-intron boundary of the SCN1A pre-mRNA transcript. The NIE-intron boundary may refer to the junction of an intron sequence and the NIE region. The intron sequence may be flanking the 5' end of the NIE or the 3' end of the NIE. In some embodiments, the ASO targets a sequence within an exon of the SCN1A pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron of the SCN1A pre-mRNA transcript. In some embodiments, the ASO targets a sequence comprising both a portion of an intron and a portion of an exon.

[0025]

[0033] In some embodiments, 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 splicing of the NMD exon mRNA.

[0026]

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

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

[0027]

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

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

[0028]

[0039] In some embodiments, the therapeutic agent targets a targeting moiety that is internal to the NMD exon.

[0040] In some embodiments, the therapeutic agent targets a targeting moiety that comprises 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 that comprises 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 portion 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 consecutive 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 about 1 to about 5000 nucleotides downstream from the 5' end of the intron comprising an NIE. In some embodiments, the ASO targets a sequence 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 100 nucleotides, about 100 to about 20 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 100 nucleotides, about 100 to about 2 ... The target sequence is a sequence that is 0 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. 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 5' end of an intron comprising an NIE.

[0030]

[0043] In some embodiments, the ASO comprises 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 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides, at least about 260 nucleotides, at least about 270 nucleotides, at least about 280 nucleotides, at least about 290 nucleotides, at least about 300 nucleotides, at least about 310 nucleotides, at least about 320 nucleotides, at least about 330 nucleotides, at least about 340 nucleotides, at least about 350 nucleotides, at least about 360 nucleotides, at least about 370 nucleotides, at least about 380 nucleotides, at least about 390 nucleotides, The present invention targets sequences that are 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.

[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 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 1000 to about 2000 nucleotides, about 1500 to about 20 ... The target sequence is a sequence located about 00 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'). 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 comprises 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 11 nucleotides, In some embodiments, the target sequence is 0 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').

[0033]

[0046] In some embodiments, the ASO targets a sequence that is about 1 to about 500 nucleotides downstream from the 5' end of the NIE. 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 downstream from the 5' end of the NIE region.

[0034]

[0047] In some embodiments, the ASO targets a sequence that is about 1 to about 500 nucleotides upstream from the 3' end of the NIE. 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 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 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 1000 to about 2000 nucleotides, about 1500 to about 20 ... The target sequence is a sequence located about 00 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'). 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 comprises 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 11 nucleotides, In one embodiment, the present invention targets a sequence that is 0 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').

[0037]

[0050] In some embodiments, the ASO targets a sequence about 1 to about 5000 nucleotides upstream from the 3' end of the intron comprising an NIE. In some embodiments, the ASO targets a sequence 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 100 nucleotides, about 100 to about 20 nucleotides, about 200 to about 500 nucleotides, about 300 to about 300 nucleotides, about 400 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 100 nucleotides, about 100 to about 20 nucleotides, about 200 to about 500 nucleotides, about 300 to about 300 nucleotides, about 300 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 The target sequence is a sequence located 0 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. 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 an NIE.

[0038]

[0051] In some embodiments, the ASO comprises 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 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides, at least about 260 nucleotides, at least about 270 nucleotides, at least about 280 nucleotides, at least about 290 nucleotides, at least about 300 nucleotides, at least about 310 nucleotides, at least about 320 nucleotides, at least about 330 nucleotides, at least about 340 nucleotides, at least about 350 nucleotides, at least about 360 nucleotides, at least about 370 nucleotides, at least about 380 nucleotides, at least about 390 nucleotides, The present invention targets sequences that are 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.

[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 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, or about 600 nucleotides upstream (or 5') from the 5' end of the NIE region. The ASO targets a sequence that is 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'). In some embodiments, the ASO may 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 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 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 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') from the 5' end of the NIE region. 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 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 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.

[0041]

[0054] 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 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 106 nucleotides, at most about 108 nucleotides, at most about 109 nucleotides, at most about 200 nucleotides, at most about 201 nucleotides, at most about 202 nucleotides, at most about 203 nucleotides, at most about 204 nucleotides, at most about 205 nucleotides, at most about 206 nucleotides, at most about 207 nucleotides, at most about 208 nucleotides, at most about 209 nucleotides, at most about 300 nucleotides, at most about 301 nucleotides, at most about 302 nucleotides, at most about 303 nucleotides, at most about 304 nucleotides, at most about 305 nucleotides, at most about 306 nucleotides, at most about 307 nucleotides, at most about 309 nucleotides, at most about 310 nucleotides, at most about 311 nucleotides, at most about 312 nucleotides, at most about 31 The ASO targets a sequence that is 10 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'). 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 comprises 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 The ASO targets a sequence that is 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, or 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 downstream. 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 about 1 to about 2000 nucleotides upstream (or 5') from the genomic site GRCh38 / hg38:chr2:166007230. 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 from the genomic site GRCh38 / hg38:chr2:166007230. The target sequence is a sequence located 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'). 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') of the genomic site GRCh38 / hg38:chr2:166007230.

[0044]

[0057] 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 from genomic site GRCh38 / hg38:chr2:166007230. The nucleotide sequence may be targeted to a sequence that is 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').

[0045]

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

[0046]

[0059] In some embodiments, the ASO targets a sequence about 1 to about 500 nucleotides upstream from genomic site 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 from genomic site GRCh38 / hg38:chr2:166007293.

[0047]

[0060] In some embodiments, the ASO targets a sequence that is about 1 to about 2000 nucleotides downstream (or 3') from genomic site GRCh38 / hg38:chr2:166007293. 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, or about 1 to about 20 nucleotides downstream (or 3') from genomic site GRCh38 / hg38:chr2:166007293. The target sequence is a sequence located 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'). 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 following genomic site: GRCh38 / hg38:chr2:166007293.

[0048]

[0061] 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 from genomic site GRCh38 / hg38:chr2:166007293. The nucleotide sequence may be targeted to a sequence that is 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').

[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 genomic site 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 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 900 to about 1000 nucleotides, about 1000 to about 150 nucleotides, about 1500 to about 200 nucleotides, about 2000 to about 250 nucleotides, about 2500 to about 300 nucleotides, about 3500 to about 400 nucleotides, about 4500 to about 500 nucleotides, about 5500 to about 600 nucleotides, about 6500 to about 700 nucleotides, about 7500 to about 800 nucleotides, about 8500 to about 900 nucleotides, about 9000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about The target sequence is 50 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. 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 site GRCh38 / hg38:chr2:166002754.

[0051]

[0064] In some embodiments, the ASO comprises 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 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides, at least about 260 nucleotides, at least about 270 nucleotides, at least about 280 nucleotides, at least about 290 nucleotides, at least about 300 nucleotides, at least about 310 nucleotides, at least about 320 nucleotides, at least about 330 nucleotides, at least about 340 nucleotides, at least about 350 nucleotides, at least about 360 nucleotides, at least about 370 nucleotides, at least about 380 nucleotides, at least about 390 nucleotides, The present invention targets sequences that are 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.

[0052]

[0065] In some embodiments, the ASO targets a sequence about 1 to about 5000 nucleotides upstream from genomic site GRCh38 / hg38:chr2:166007229. In some embodiments, the ASO targets a sequence 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 900 to about 1000 nucleotides, about 1000 to about 150 nucleotides, about 1500 to about 200 nucleotides, about 2000 to about 250 nucleotides, about 2500 to about 300 nucleotides, about 3500 to about 400 nucleotides, about 4500 to about 500 nucleotides, about 5500 to about 600 nucleotides, about 6500 to about 700 nucleotides, about 7500 to about 800 nucleotides, about 8500 to about 900 nucleotides, about 9000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about 1000 The target sequence is 50 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. 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 genomic site GRCh38 / hg38:chr2:166007229.

[0053]

[0066] In some embodiments, the ASO comprises 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 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides, at least about 260 nucleotides, at least about 270 nucleotides, at least about 280 nucleotides, at least about 290 nucleotides, at least about 300 nucleotides, at least about 310 nucleotides, at least about 320 nucleotides, at least about 330 nucleotides, at least about 340 nucleotides, at least about 350 nucleotides, at least about 360 nucleotides, at least about 370 nucleotides, at least about 380 nucleotides, at least about 390 nucleotides, The present invention targets sequences that are 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.

[0054]

[0067] In some embodiments, the ASO targets a sequence that is about 1 to about 5000 nucleotides downstream from genomic site GRCh38 / hg38:chr2:166007294. 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 900 to about 1000 nucleotides, about 1000 to about 150 nucleotides, about 1500 to about 200 nucleotides, about 2000 to about 250 nucleotides, about 2500 to about 300 nucleotides, about 3500 to about 400 nucleotides, about 4500 to about 500 nucleotides, about 5500 to about 600 nucleotides, about 6500 to about 700 nucleotides, about 7500 to about 800 nucleotides, about 8500 to about 900 nucleotides, about 9000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about The target sequence is 50 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. 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 site GRCh38 / hg38:chr2:166007294.

[0055]

[0068] In some embodiments, the ASO comprises 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 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides, at least about 260 nucleotides, at least about 270 nucleotides, at least about 280 nucleotides, at least about 290 nucleotides, at least about 300 nucleotides, at least about 310 nucleotides, at least about 320 nucleotides, at least about 330 nucleotides, at least about 340 nucleotides, at least about 350 nucleotides, at least about 360 nucleotides, at least about 370 nucleotides, at least about 380 nucleotides, at least about 390 nucleotides, The present invention targets sequences that are 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.

[0056]

[0069] In some embodiments, the ASO targets a sequence about 1 to about 5000 nucleotides upstream from genomic site GRCh38 / hg38:chr2:166009718. In some embodiments, the ASO targets a sequence 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 900 to about 1000 nucleotides, about 1000 to about 150 nucleotides, about 1500 to about 200 nucleotides, about 2000 to about 250 nucleotides, about 2500 to about 300 nucleotides, about 3500 to about 400 nucleotides, about 4500 to about 500 nucleotides, about 5500 to about 600 nucleotides, about 6500 to about 700 nucleotides, about 7500 to about 800 nucleotides, about 8500 to about 900 nucleotides, about 9000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about 1000 nucleotides, about 10000 to about 1000 The target sequence is 50 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. 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 genomic site GRCh38 / hg38:chr2:166009718.

[0057]

[0070] In some embodiments, the ASO comprises 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 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides, at least about 260 nucleotides, at least about 270 nucleotides, at least about 280 nucleotides, at least about 290 nucleotides, at least about 300 nucleotides, at least about 310 nucleotides, at least about 320 nucleotides, at least about 330 nucleotides, at least about 340 nucleotides, at least about 350 nucleotides, at least about 360 nucleotides, at least about 370 nucleotides, at least about 380 nucleotides, at least about 390 nucleotides, The present invention targets sequences that are 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.

[0058]

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

[0059]

[0072] In some embodiments, the ASO targets a sequence that is about 4 to about 300 nucleotides upstream (or 5') from genomic site GRCh37 / hg19:chr2:166,863,803. 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, or about 500 to about 600 nucleotides from genomic site GRCh37 / hg19:chr2:166,863,803. The ASO targets a sequence that is 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'). In some embodiments, the ASO may target a sequence that is more than 300 nucleotides upstream from genomic site 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 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 The ASO targets a sequence that is 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. 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 about 4 to about 300 nucleotides upstream (or 5') from genomic site 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 10 ... The ASO targets a sequence that is 2 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'). 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 at least about 1 nucleotide, at least about 10 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 96 nucleotides, at least about 97 nucleotides, at least about 98 nucleotides, at least about 99 nucleotides, at least about 100 nucleotides, at least about 101 nucleotides, at least about 102 nucleotides, at least about 103 nucleotides, at least about 104 nucleotides, at least about 105 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, or at least about 1000 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.

[0061]

[0074] In some embodiments, the ASO targets a sequence that is about 4 to about 300 nucleotides upstream (or 5') from genomic site 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 106 nucleotides, at most about 107 nucleotides, at most about 109 nucleotides, at most about 200 nucleotides, at most about 201 nucleotides, at most about 202 nucleotides, at most about 203 nucleotides, at most about 204 nucleotides, at most about 205 nucleotides, at most about 206 nucleotides, at most about 207 nucleotides, at most about 208 nucleotides, at most about 209 nucleotides, at most about 300 nucleotides, at most about 300 nucleotides, at most about 301 nucleotides, at most about 302 nucleotides, at most about 303 nucleotides, at most about 304 nucleotides, at most about 305 nucleotides, at most about 306 nucleotides, at most about 307 nucleotides, at most about 308 nucleotides, at most about 309 The ASO targets a sequence that is 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'). 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 comprises 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, or at most about 106 nucleotides from GRCh37 / hg19:chr2:166,863,740. The ASO targets a sequence that is 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, or 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 downstream. 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, the SCN1A gene (SEQ ID NO:1) was analyzed for NIE and observed for inclusion of a portion of intron 20 (see SEQ ID NO:6, which encodes the intron 20 pre-mRNA) (referred to as exon 23 or exon 20x throughout this disclosure). In some embodiments, the ASOs disclosed herein target an NIE-containing pre-mRNA (SEQ ID NO:2) transcribed from the SCN1A genomic sequence. In some embodiments, the ASOs target an NIE-containing pre-mRNA transcript from the SCN1A genomic sequence that comprises a portion of intron 20. In some embodiments, the ASOs target an NIE-containing pre-mRNA transcript from the SCN1A genomic sequence that comprises exon 23 (or exon 20x) (SEQ ID NO:4). In some embodiments, the ASOs target an NIE-containing pre-mRNA transcript of SEQ ID NO:2 or 9. In some embodiments, the ASOs target an NIE-containing pre-mRNA transcript of SEQ ID NO:2 or 9 that comprises an 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 comprising the NIE (SEQ ID NO:7 or 11). In some embodiments, the ASO targets the SCN1A pre-mRNA sequence according to any one of SEQ ID NOs:6, 7, 10, or 11. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs:12-731. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs:12-371. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs:372-731.

[0063]

[0076] In some embodiments, the SCN1A NIE-containing pre-mRNA transcript is encoded by a gene sequence that has 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 that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs:2-7 and 9-11.

[0064]

[0077] In some embodiments, the SCN1A NIE-containing pre-mRNA transcript (or NMD exon mRNA) comprises a sequence that has 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 that has 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 that has 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 from the 5' end of the NIE. For example, an ASO targeting a sequence upstream from 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 that has 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 targeting a sequence upstream from 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 that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 12-191. In additional examples, an ASO targeting a sequence upstream from the 5' end of an NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence that has 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 targets exon 23 (or exon 20x) in an SCN1A NIE-containing pre-mRNA, which comprises exon 23. In some embodiments, the ASO targets exon 23 sequences downstream (or 3') from the 5' end of exon 23 of the SCN1A pre-mRNA. In some embodiments, the ASO targets exon 23 sequences 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 targeting 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 that has 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 targeting 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 that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 192-371. In additional examples, an ASO targeting a sequence downstream from the 3' end of an NIE (e.g., exon 23 (or exon 20x) in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence that has 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 at least one exon skipping of the NIE 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, resulting in increased SCN1A protein production. In some embodiments, hybridization of the ASO to the targeted portion of the NIE pre-mRNA inhibits or prevents at least one exon skipping of the NIE 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, resulting in a subsequent decrease in SCN1A protein production. In some embodiments, the targeted portion of the SCN1A NIE-containing pre-mRNA is in intron 20. One of 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 connection with the mRNA sequences in NM_006920, NM_001202435, NM_001165964, or NM_001165963. One of skill in the art can also determine the sequence of flanking exons in any SCN1A isoform for targeting 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 embodiments, the methods and compositions of the disclosure are used to modulate (e.g., increase or decrease) expression of SCN1A by inducing or inhibiting exon skipping of pseudoexons of SCN1A NIE-containing pre-mRNA. In some embodiments, the pseudoexon is a sequence within any of introns 1 through 25. In some embodiments, the pseudoexon is a sequence within any of introns 2, 4, 6, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, and 25. In some embodiments, the pseudoexon is a sequence within any of introns 15, 18, and 19. In some embodiments, the pseudoexon can be any SCN1A intron or part thereof. In some embodiments, the pseudoexon is within intron 20. SCN1A intron numbering as used herein corresponds to the RNA sequence in NM_006920. It is understood that this intron numbering may vary in relation to different SCN1A isoform sequences.

[0070] SCN1A protein

[0083] The SCN1A gene encodes the SCN1A (sodium channel, voltage-gated, type I, α subunit) protein, which encodes the voltage-gated sodium channel Na v SCN1A can encode the α-subunit of SCN1A (which may also be referred to as the α-subunit of SCN1.1). As also noted above, SCN1A mutations in DS are spread throughout the protein. More than 100 novel mutations have been identified throughout the gene, with more debilitating ones arising de novo. These include truncations (47%), missense (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 embodiments, 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 that is necessary to eliminate any one or more symptoms of the condition being treated (e.g., Dravet syndrome; generalized epilepsy febrile seizures plus, type 2; familial febrile seizures, 3A; autism; epileptic encephalopathy, premature infantile, 13; sick sinus syndrome 1; Alzheimer's disease; or SUDEP). In some embodiments, the methods are used to increase the production of partially functional SCN1A protein. As used herein, the term "partially functional" refers to any amount of SCN1A protein activity or function that is less than the amount of activity or function that is necessary to eliminate or prevent any one or more symptoms of the disease or condition. In some embodiments, 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 expression of SCN1A protein by cells of a subject having a NIE-containing pre-mRNA encoding SCN1A protein, wherein the subject has Dravet syndrome caused by an insufficient amount of activity of SCN1A protein, wherein the insufficient amount of SCN1A protein is caused by haploinsufficiency of SCN1A protein. In such embodiments, the subject has a first allele that encodes a functional SCN1A protein and a second allele that does not produce SCN1A protein. In another such embodiment, the subject has a first allele that encodes a functional SCN1A protein and a second allele that encodes a non-functional SCN1A protein. In another such embodiment, the subject has a first allele that encodes a functional SCN1A protein and a second allele that encodes 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 from the pre-mRNA, resulting in increased levels of mature mRNA encoding a functional SCN1A protein and increased expression of the SCN1A protein in the cells of the subject.

[0073]

[0086] In a related embodiment, the method is a method of increasing expression of a protein or functional RNA using an ASO. In some embodiments, the ASO is used to increase expression of SCN1A protein in cells of a subject having an NIE-containing pre-mRNA encoding the SCN1A protein, where the subject has a deficiency in the amount or function of SCN1A protein, such as Dravet syndrome (DS) (also known as SMEI); Severe myoclonic epilepsy of infancy (SMEI)-limbic type (SMEB); Febrile seizures (FS); Generalized epilepsy with febrile seizures plus (GE). FS+); 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; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or autism. In some embodiments, the ASO is used to increase expression of SCN1A protein in cells of a subject, where the subject has a deficiency in the amount or function of SCN8A protein, e.g., epileptic encephalopathy, early infantile, 13. In some embodiments, the ASO is used to increase expression of SCN1A protein in cells of a subject, where the subject has a deficiency in the amount or function of SCN5A protein, e.g., sick sinus syndrome 1.

[0074]

[0087] In some embodiments, NIE-containing pre-mRNA transcripts that code for proteins that are causative of a disease or pathology are targeted by ASOs as described herein. In some embodiments, NIE-containing pre-mRNA transcripts that code for proteins that are not causative of the disease are targeted by ASOs. For example, a disease that is the result of a mutation or deficiency in a first protein in a particular pathway may be ameliorated by targeting a NIE-containing pre-mRNA that codes for a second protein to increase production of the second protein. In some embodiments, the function of the second protein can counteract the mutation or deficiency of the first protein (causative of the disease or pathology).

[0075]

[0088] In some embodiments, the subject: (a) a first mutant allele [whereby: (i) SCN1A protein is produced at reduced levels compared to production from a wild-type allele; (ii) the SCN1A protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) neither SCN1A protein nor functional RNA is produced, and (b) a second mutant allele [whereby: (i) SCN1A protein is produced at reduced levels compared to production from a wild-type allele; (ii) the SCN1A protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) 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 a 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 SCN1A protein and an increase in 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 level of expression resulting from exon skipping of the pseudoexon from the NIE-containing pre-mRNA is either in a form that has reduced functionality compared to the equivalent wild-type protein (partially functional) or in a form that has full functionality compared to the equivalent wild-type protein (fully functional).

[0076]

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

[0077]

[0090] In some embodiments, subjects treated using the methods of the present disclosure express a partially functional SCN1A protein at one allele, where the partially functional SCN1A protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, subjects treated using the methods of the present invention express a non-functional SCN1A protein at one allele, where the non-functional SCN1A protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion at one allele. In some embodiments, subjects treated using the methods of the present invention have a deletion of the entire SCN1A gene at one allele.

[0078]

[0091] In some embodiments, the method is a method of decreasing expression of SCN1A protein by cells of a subject having a NIE-containing pre-mRNA encoding the SCN1A protein, wherein the subject has Na v In such embodiments, the subject has an allele in which the SCN1A protein is produced in elevated amounts, or an allele in which the SCN1A protein is produced in elevated amounts. v In some embodiments, the cell has an allele encoding a mutant SCN1A that induces increased activity of Na v The increased activity of 1.1 is characterized by the mutation Na v1.1 channel-mediated prolonged or nearly permanent sodium current, delayed fast inactivation, positive shift in steady-state inactivation, higher channel availability during repeated stimulation, increased non-inactivating depolarization-induced persistent sodium current, delayed entry into inactivation, accelerated recovery from fast inactivation, and / or rescue of folding defects by incubation at low temperature or co-expression of an interacting 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 inhibiting or preventing exon skipping of the pseudoexon from the pre-mRNA, causing a decrease in the level of mature mRNA encoding a functional SCN1A protein and a decrease in expression of SCN1A protein in the cells of the subject.

[0079]

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

[0080]

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

[0081]

[0094] In some embodiments, a subject 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 is Na v In some embodiments, subjects treated using the methods of the present disclosure express elevated amounts of SCN1A protein from one allele due to a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion.

[0082]

[0095] In some embodiments of the present invention, the subject may have a mutation in the SCN1A gene. The mutation in SCN1A may be spread throughout the gene. The SCN1A protein may consist of four domains. The SCN1A domain may have a transmembrane segment. The mutation in the SCN1A protein may occur throughout the protein. The SCN1A protein may consist of at least two isoforms. The mutation in SCN1A may include R931C, R946C, M934I, R1648C, or R1648H. In some cases, the mutation may be observed at the C-terminus of the SCN1A protein. The mutation in the SCN1A protein may also be found in the loop between segment 5 and segment 6 of the first three domains of the SCN1A protein. In some cases, the mutation may be observed at the N-terminus of the SCN1A protein. 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 with the present invention may also code for ion channel pores.

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

[0084]

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

[0085] Exon inclusion

[0098] As used herein, an "NIE-containing pre-mRNA" is a pre-mRNA transcript that contains at least one pseudoexon. Alternative or aberrant splicing may 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. The inclusion of at least one pseudoexon may result in a non-productive mRNA, which may lead to NMD of the mature mRNA. NIE-containing mature mRNAs may sometimes result in aberrant protein expression.

[0086]

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

[0087]

[0100] The degree of exon inclusion can be expressed as % exon inclusion (e.g., the percentage of transcripts in which a given pseudoexon is included). Briefly, % exon inclusion can be calculated as the percentage of the amount of RNA transcripts with the exon inclusion relative to the sum of the average amount of RNA transcripts with exon inclusion + the average amount of RNA transcripts with exon exclusion.

[0088]

[0101] In some embodiments, the included pseudoexons are exons identified as included pseudoexons based on an inclusion determination of at least about 5%, 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 one embodiment, the included pseudoexon is about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, about 10% to about About 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 7 0%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45% , about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35% inclusion of the pseudoexon.ENCODE data (e.g., as described by Tilgner, et al., 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs" Genome Research 22(9): 1616-25) can be used to help identify exon inclusion.

[0089]

[0102] In some aspects, contacting a cell with an ASO that is complementary to a targeted portion of the SCN1A pre-mRNA transcript results in at least a 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% increase in the amount of SCN1A protein produced compared to the amount of protein produced by the cell in the absence / treatment of the ASO. In some embodiments, the total amount of SCN1A protein produced by a cell contacted with an 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, or about 1.5 to about 10 times the amount of target protein produced by a control compound. 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. 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 aspects, contacting a cell with an ASO that is complementary to a targeted portion of the SCN1A pre-mRNA transcript results in at least a 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% decrease in the amount of SCN1A protein produced compared to the amount of protein produced by the cell in the absence / treatment of the ASO. In some embodiments, the total amount of SCN1A protein produced by a cell contacted with an 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, or about 1.5 to about 10 times the amount of target protein produced by a control compound. 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. 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 a cell with an ASO that is complementary to a targeted portion of an SCN1A pre-mRNA transcript results in an increase in the amount of mRNA encoding SCN1A, including mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding SCN1A protein, or mature mRNA encoding SCN1A protein, is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence / treatment of the ASO. In some embodiments, the total amount of mRNA encoding the SCN1A protein or mature mRNA encoding the SCN1A protein produced in a cell contacted with an 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 The increase is 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. The control compound can be, for example, an oligonucleotide that is not complementary to the targeting portion of the SCN1A NIE-containing pre-mRNA.

[0092]

[0105] In some embodiments, contacting a cell with an ASO that is complementary to a targeted portion of an SCN1A pre-mRNA transcript results in a decrease in the amount of mRNA encoding SCN1A, including the mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding SCN1A protein, or mature mRNA encoding SCN1A protein, is decreased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence / treatment of the ASO. In some embodiments, the total amount of mRNA encoding the SCN1A protein or mature mRNA encoding the SCN1A protein produced in a cell contacted with an 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 The control compound is decreased by 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. The control compound can be, for example, an oligonucleotide that is not complementary to the targeting portion of the SCN1A NIE-containing pre-mRNA.

[0093]

[0106] The NIE can be any length. In some embodiments, the NIE comprises the full length sequence of the intron, in which case it can be referred to as intron retention. In some embodiments, the NIE can be a portion of the intron. In some embodiments, the NIE can be the 5' end portion of the intron that includes the 5'ss sequence. In some embodiments, the NIE can be the 3' end portion of the intron that includes the 3'ss sequence. In some embodiments, the NIE can be a portion within the intron that does not include the 5'ss sequence. In some embodiments, the NIE can be a portion within the intron that does not include the 3'ss sequence. In some embodiments, the NIE can be a portion within the intron that does not include the 5'ss sequence or the 3'ss sequence. In some embodiments, the NIE can be 5 to 10 nucleotides in length, 10 to 15 nucleotides in length, 15 to 20 nucleotides in length, 20 to 25 nucleotides in length, 25 to 30 nucleotides in length, 30 to 35 nucleotides in length, 35 to 40 nucleotides in length, 40 to 45 nucleotides in length, 45 to 50 nucleotides in length, 50 to 55 nucleotides in length, 55 to 60 nucleotides in length, 60 to 65 nucleotides in length, 65 to 70 nucleotides in length, 70 to 75 nucleotides in length, 75 to 80 nucleotides in length, 80 to 85 nucleotides in length, 85 to 90 nucleotides in length, 90 to 95 nucleotides in length, or 95 to 100 nucleotides in length. In some embodiments, the NIE 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 in length, 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 greater than 1,000 nucleotides in length.

[0094]

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

[0095] Therapeutic Agents

[0108] In various embodiments of the disclosure, compositions and methods comprising therapeutic agents are provided for modulating the protein expression level of SCN1A. In some embodiments, the present invention provides compositions and methods for modulating alternative splicing of SCNA1 pre-mRNA. In some embodiments, the present invention provides compositions and methods for inducing exon skipping in splicing of SCN1A pre-mRNA (e.g., inducing skipping of pseudoexons during splicing of SCN1A pre-mRNA). In other embodiments, therapeutic agents can be used to induce exon inclusion in order to reduce its protein expression level.

[0096]

[0109] In certain aspects, 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 agent disclosed herein may be an NIE repressor agent.The therapeutic agent may comprise a polynucleic acid polymer.

[0111] According to one aspect of the disclosure, the present invention provides a method for 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 a pre-mRNA transcript and reduces the inclusion of NIE in the mature transcript. For example, the present invention provides a method for 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 suppression of NIE inclusion in mature mRNA, 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, the increase may be clinically significant. The increase may be relative to the active SCN1A protein level in a subject without treatment or relative to the amount of active SCN1A protein in a population of similar subjects. The increase may be at least 10% more active SCN1A protein relative to the average subject or pre-treatment subject. The increase may be at least 20% more active SCN1A protein relative to the average subject or pre-treatment subject. The increase may be at least 40% more active SCN1A protein relative to the average subject or pre-treatment subject. The increase may be at least 50% more active SCN1A protein relative to the average subject or pre-treatment subject. The increase may be at least 80% more active SCN1A protein relative to the average subject or pre-treatment subject. The increase may be at least 100% more active SCN1A protein relative to the average subject or pre-treatment subject. The increase can be at least 200% more active SCN1A protein relative to the average subject, or to a subject prior to treatment. The increase can be at least 500% more active SCN1A protein relative to the average subject, or to a subject prior to treatment.

[0100]

[0114] In the embodiment(s) in which the NIE repressor agent comprises a polynucleic acid polymer, the polynucleic acid polymer can be about 50 nucleotides in length. The polynucleic acid polymer can be about 45 nucleotides in length. The polynucleic acid polymer can be about 40 nucleotides in length. The polynucleic acid polymer can be about 35 nucleotides in length. The polynucleic acid polymer can be about 30 nucleotides in length. The polynucleic acid polymer can be about 24 nucleotides in length. The polynucleic acid polymer can be about 25 nucleotides in length. The polynucleic acid polymer can be about 20 nucleotides in length. The polynucleic acid polymer can be about 19 nucleotides in length. The polynucleic acid polymer can be about 18 nucleotides in length. The polynucleic acid polymer can be about 17 nucleotides in length. The polynucleic acid polymer can be about 16 nucleotides in length. The polynucleic acid polymer can be about 15 nucleotides in length. The polynucleic acid polymer can be about 14 nucleotides in length. The polynucleic acid polymer can be about 13 nucleotides in lengthThe polynucleic acid polymer can be about 12 nucleotides in length. The polynucleic acid polymer can be about 11 nucleotides in length. The polynucleic acid polymer can be about 10 nucleotides in length. The polynucleic acid polymer can be between about 10 and about 50 nucleotides in length. The polynucleic acid polymer can be between about 10 and about 45 nucleotides in length. The polynucleic acid polymer can be between about 10 and about 40 nucleotides in length. The polynucleic acid polymer can be between about 10 and about 35 nucleotides in length. The polynucleic acid polymer can be between about 10 and about 30 nucleotides in length. The polynucleic acid polymer can be between about 10 and about 25 nucleotides in length. The polynucleic acid polymer can be between about 10 and about 20 nucleotides in length. The polynucleic acid polymer can be between about 15 and about 25 nucleotides in length. The polynucleic acid polymer can be between about 15 and about 30 nucleotides in length. The polynucleic acid polymer can be between about 12 and about 30 nucleotides in length.

[0101]

[0115] The sequence of the polynucleic acid polymer can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to a target sequence of an mRNA transcript (e.g., a partially processed mRNA transcript).

[0102]

[0116] The sequence of the polynucleic acid polymer may have 4 or less mismatches to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have 3 or less mismatches to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have 2 or less mismatches to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have 1 or less mismatch to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have no mismatches to the target sequence of the pre-mRNA transcript.

[0103]

[0117] The polynucleic acid polymer can specifically hybridize to the target sequence of the pre-mRNA transcript. For example, the polynucleic acid polymer can have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence complementarity to the target sequence of the pre-mRNA transcript. The hybridization can be under high stringency hybridization conditions.

[0104]

[0118] The polynucleic acid polymer may have a sequence that has 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: 12-731. The polynucleic acid polymer may have a sequence that has 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 12-731. In some cases, the polynucleic acid polymer may have a sequence that has 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: 12-371. In some cases, the polynucleic acid polymer may have a sequence that has 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 12-371. In some cases, the polynucleic acid polymer may have a sequence that has 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: 372-731. In some cases, the polynucleic acid polymer may have a sequence that has 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 372-731.

[0105]

[0119] When referring to a polynucleic acid polymer sequence, one skilled in the art will understand that one or more substitutions, and in some cases two substitutions, can be tolerated in the sequence, provided that it maintains its ability to hybridize to the target sequence or to be recognized as the target sequence (if the substitution is in the target sequence). Reference to sequence identity can be determined by BLAST sequence alignment using standard / default variables. For example, the sequence can have 99% identity and still function according to the present disclosure. In other embodiments, the sequence can have 98% identity and still function according to the present disclosure. In other embodiments, the sequence can have 95% identity and still function according to the present disclosure. In other embodiments, the sequence can have 90% identity and still function according to the present disclosure.

[0106] Antisense oligomers

[0120] Provided herein is a composition comprising an antisense oligomer that induces exon skipping by binding to a targeted portion of an SCN1A NIE-containing pre-mRNA. 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 can 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). The ASO is 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 the ASO 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 that the ASO will bind to other sites and cause "off-target" effects. Any antisense oligomer known in the art, e.g., in PCT Application No. PCT / US2014 / 054151 (published as WO2015 / 035091, entitled "Methods for Inhibiting Nonsense-Mediated mRNA Decay"), which is incorporated herein by reference, can be used to practice the methods described herein.

[0107]

[0121] In some embodiments, the ASO "specifically hybridizes" or is "specific" to the target portion of the target nucleic acid or NIE-containing pre-mRNA. Typically, such hybridization occurs at a T substantially greater than 37° C. mSuch 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 when hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration. A double-stranded polynucleotide can be "complementary" to another polynucleotide when hybridization can occur between one strand of a first polynucleotide and one strand of a second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion (e.g., percentage) of bases in the paired strands that are predicted to form hydrogen bonds with each other according to commonly accepted base-pairing rules. The sequence of an antisense oligomer (ASO) need not be 100% complementary to the sequence of its target nucleic acid to which it hybridizes. In some embodiments, ASOs may contain 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 to which they are targeted. For example, an ASO in which 18 of the 20 nucleobases of the oligomeric compound are complementary to the target region and therefore specifically hybridize would exhibit 90% complementarity. In this example, the remaining non-complementary nucleobases may be grouped together or may be separated by complementary nucleobases and may not be contiguous to each other or to complementary nucleobases. The percent complementarity of an ASO to a region of a target nucleic acid can be routinely determined using BLAST (basic local alignment search tool) and PowerBLAST programs known in the art (Altschul, et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0109]

[0123] An ASO need not hybridize to every nucleobase in a target sequence; the nucleobases to which it does hybridize may be contiguous or discontinuous. An ASO may hybridize across one or more segments of a pre-mRNA transcript such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop or hairpin structure may be formed). In some embodiments, an ASO hybridizes to discontinuous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobases 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 sugar moieties such as peptide nucleic acids (PNAs). ASOs can contain naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of these two or three items. 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. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be apparent to those of skill in the art and can be found, for example, in 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, which are incorporated by reference in their entireties.

[0111]

[0125] One or more nucleobases of the ASO can be any naturally occurring unmodified nucleobase, such as adenine, guanine, cytosine, thymine, and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase so as to be 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 "oligomer linkage" may be used interchangeably and refer to the linkage between the monomers of the ASO. In naturally occurring oligonucleotides, the backbone includes a 3'-5' phosphodiester linkage that links the sugar moieties of the oligomer. The backbone structure or oligomer linkage of the ASOs described herein may include (but is not limited to) phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, phosphoroamidates, and the like. See, e.g., 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, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford, England (1991)); Stec, et al., U.S. Pat. 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, but instead contains peptide bonds (e.g., in peptide nucleic acids (PNAs)) or linking groups including carbamates, amides, 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 embodiments, the stereochemistry at each of the phosphorus-internucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the phosphorus-internucleotide 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 methods for independently selecting the handedness of chirality at each phosphorus atom in a nucleic acid oligomer. In embodiments, the ASOs used in the methods of the invention (including but not limited to any of the ASOs shown herein in Tables 5 and 6) include ASOs having non-random phosphorus-internucleotide linkages. In embodiments, the compositions used in the methods of the invention include pure diastereomeric ASOs. In embodiments, the compositions used in the methods of the invention comprise an ASO 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 embodiments, the ASO has a non-random mixture of Rp and Sp configurations in its phosphorus internucleotide linkages. For example, it has been suggested that a mixture of Rp and Sp in antisense oligonucleotides is necessary to achieve a balance between good activity and nuclease stability (Wan, et al., 2014, "Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages," Nucleic Acids Research 42 (22): 13456-13468, incorporated herein by reference). In some embodiments, the ASOs used in the methods of the invention (including but not limited to any of the ASOs set forth herein in SEQ ID NOs: 12-731) comprise about 5-100% 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, with the remainder Sp, or comprise about 100% Rp.In one or more embodiments, the ASO used in the method of the present invention (including, but not limited to, any of the ASOs set forth in SEQ ID NOs: 12 to 731 herein) is 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 100% 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 with the remainder Sp.

[0115]

[0129] In some embodiments, the ASOs used in the methods of the invention (including but not limited to any of the ASOs set forth herein in SEQ ID NOs: 12-731) comprise about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder Rp, or comprise about 100% Sp. In one or more embodiments, the ASO used in the methods of the present invention (including, but not limited to, any of the ASOs set forth in SEQ ID NOs: 12 to 731 herein) is from 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, with the remainder Rp.

[0116]

[0130] Any of the ASOs described herein may contain a sugar moiety that includes ribose or deoxyribose, as found in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring. 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-guanidiniumethyl, carbamate modified sugars, and bicyclic modified sugars. 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 bridge linkage, as in locked nucleic acids (LNAs). In some embodiments, the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuranosyl or 2' deoxyribofuranosyl modification. In some embodiments, the sugar moiety comprises a 2',4'-constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2',4' constrained 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and are described, for example, in 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 manner, for example, each linkage of the backbone of the ASO contains a phosphorothioate linkage or each ribose sugar moiety contains a 2'O-methyl modification. Such modifications present on each of the monomeric components of the ASO are referred to as "uniform modifications." In some examples, a combination of different modifications may be desired, for example, an ASO may contain a combination of a phosphorodiamidate linkage and a sugar moiety comprising a morpholine ring (morpholino). The combination of various modifications to an ASO is referred to as a "mixed modification" 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 a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a 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 one or more components of any ASO, can be modified to increase binding affinity for a target sequence on a pre-mRNA transcript; to reduce binding to non-target sequences; to reduce degradation by cellular nucleases (i.e., RNase H); to improve uptake of the ASO into cells and / or into the nucleus of cells; to alter the pharmacokinetics or pharmacodynamics of the ASO; and / or to modulate the half-life of the ASO.

[0119]

[0133] In some embodiments, ASOs are composed of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs composed of such nucleotides are particularly well suited for the methods disclosed herein, as oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery 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 of skill in the art will know how to synthesize ASOs. Alternatively, or additionally, ASOs may be obtained from commercial sources.

[0135] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid (e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.) sequence is the 5'-end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5'-direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded 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 termination 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 targeting portion of the SCN1A NIE-containing pre-mRNA that is downstream (in the 3' direction) of the 5' splice site (or the 3' end of the NIE) of an included exon in the SCN1A NIE-containing pre-mRNA (e.g., in the direction specified by a positive number relative to the 5' splice site). In some embodiments, the ASO is complementary to a targeting portion of the SCN1A NIE-containing pre-mRNA that is within a region from 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 targeting portion of the SCN1A NIE-containing pre-mRNA that is within a region between nucleotides +6 and +496 relative to the 5' splice site (or 3' end) of the included exon. In some aspects, the ASO is at 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 +1 or about +1 to about +20.In some aspects, the ASO is complementary to a targeting 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 complementary to (and binds to) a targeting portion of the SCN1A NIE-containing pre-mRNA that is upstream (in the 5' direction) of the 5' splice site (or 3' end) of the included exon in the SCN1A NIE-containing pre-mRNA (e.g., in the direction specified by a negative number relative to the 5' splice site). In some embodiments, the ASO is complementary to a targeting 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 a targeting portion of the SCN1A NIE-containing pre-mRNA that is within the region between nucleotides -1 and -264 relative to the 5' splice site (or 3' end) of the included exon. In some aspects, the ASO is at 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 -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 The ASO is complementary to a targeting moiety within a region of 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. In some aspects, the ASO is complementary to a targeting moiety within a 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 complementary to a targeting portion of the SCN1A NIE-containing pre-mRNA that is upstream (in the 5' direction) of the 3' splice site (or 5' end) of the included exon in the SCN1A NIE-containing pre-mRNA (e.g., in the direction specified by a negative number). In some embodiments, the ASO is complementary to a targeting portion of the SCN1A NIE-containing pre-mRNA that is within a region of about -1 to about -500 relative to the 3' splice site (or 5' end) of the included exon. In some embodiments, the ASO is complementary to a targeting portion of the SCN1A NIE-containing pre-mRNA that is within a region of -1 to -496 relative to the 3' splice site of the included exon. In some aspects, the ASO is at 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 -440, about -1 to about -450, about -1 to about -460, about -1 to about -470, about -1 to about -480, about -1 to about -490, about -1 to about -480, about -1 to about -490, about -1 to about -480, about -1 to about -490, about -1 to about -480, about -1 to about -490, about -1 to about -490, about -1 to about -480, about -1 to about -490, 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 -440, about -1 to about -450, about -1 to about -460, about -1 to about -470, about -1 to about -480, about -1 to about -49 ... 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 -28 0, 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, or about -1 to about -30. In some aspects, the ASO is complementary to a targeting moiety 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 3' splice site of the included exon.

[0124]

[0139] In some embodiments, the ASO is complementary to a targeting 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 SCN1A NIE-containing pre-mRNA (e.g., in the direction specified by a positive number). In some embodiments, the ASO is complementary to a targeting portion of the SCN1A NIE-containing pre-mRNA that is within a 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 a targeting portion that is within a region 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 targeting portion of the SCN1A NIE-containing pre-mRNA is within the region of +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 targeting portion of the SCN1A NIE-containing pre-mRNA is within the NIE. In some embodiments, the targeting portion of the SCN1A NIE-containing pre-mRNA includes the boundary between the pseudoexon and the intron.

[0126]

[0141] ASOs can be of any length suitable for specific binding and effective enhancement of splicing. In some embodiments, ASOs consist of 8-50 nucleobases. For example, ASOs 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, ASOs consist of more than 50 nucleobases. In some embodiments, the ASO comprises from 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 50 nucleobases, 10 to 40 nucleobases, nucleo bases, 10-35 nucleo bases, 10-30 nucleo bases, 10-25 nucleo bases, 10-20 nucleo bases, 10-15 nucleo bases, 11-50 nucleo bases, 11-40 nucleo bases, 11-35 nucleo bases, 11-30 nucleo bases, 11-25 nucleo bases, 11-20 nucleo bases, 11-15 nucleo bases, 12-50 nucleo bases, 12-40 nucleo bases, 12-35 nucleo bases, 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 length.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 are used that are chemically distinct but complementary to the same targeting portion of the NIE-containing pre-mRNA, hi some embodiments, two or more ASOs are used that are complementary to different targeting portions of the NIE-containing pre-mRNA.

[0128]

[0143] In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties (such as, for example, cholesterol moieties, cholesteryl moieties), aliphatic chains (such as, for example, dodecanediol or undecyl residues), polyamine or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides comprising lipophilic moieties and methods for their preparation have been described in the literature. In embodiments, the antisense oligonucleotides are conjugated to moieties including, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as, for example, 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, the conjugate can be linked to one or more of the nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base, or phosphate groups, for example using a linker. The linker can include a bivalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods for making oligonucleotide conjugates are described, for example, in U.S. Pat. No. 8,450,467, entitled "Carbohydrate conjugates as delivery agents for oligonucleotides," which is incorporated herein by reference.

[0129]

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

[0130] Pharmaceutical Compositions

[0145] Pharmaceutical compositions or formulations comprising an agent of the described compositions (e.g., antisense oligonucleotides) 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 public 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 pharma- ceutical acceptable salt, solvate, hydrate, or ester thereof. Pharmaceutical formulations comprising an antisense oligomer may further comprise a pharma- ceutical acceptable excipient, diluent, or carrier.

[0131]

[0146] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, and the like, and are commensurate with a reasonable benefit / risk ratio (see, for example, SM Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose). The salts can be prepared in situ during the final isolation and purification of the compound, or separately by reacting the free base functional group with a suitable organic acid. Examples of pharma-ceutically acceptable, non-toxic acid addition salts are salts of 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 pharma- ceutically 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-hydroxy-ethanesulfonate, Included are 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, and the like. Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like.Further pharma-ceutically acceptable salts include salts of non-toxic 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 embodiments, the compositions are formulated into any of many possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the compositions are formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspensions may also contain stabilizers. In embodiments, the pharmaceutical formulations or compositions of the present invention include, but are not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0133]

[0148] The pharmaceutical compositions or formulations described herein may include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients, as appropriate and as known to those skilled in the art or as described in the known literature. In some embodiments, the liposomal formulation also includes a sterically stabilized liposomal formulation (e.g., a liposomal formulation comprising one or more specialized lipids), which result in a liposomal formulation with enhanced circulation life. In some embodiments, the sterically stabilized liposomal formulation includes one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. In some embodiments, a surfactant is included in the pharmaceutical formulation or composition. The use of surfactants in pharmaceuticals, formulations, and emulsions is well known in the art. In some embodiments, the present invention utilizes a penetration enhancer to enable efficient delivery of antisense oligonucleotides (e.g., to aid in diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs). In embodiments, the penetration enhancer is a surfactant, a fatty acid, a bile salt, a chelating agent, or a non-chelating non-surfactant.

[0134]

[0149] In embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides. In 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 can include small molecules. For example, the one or more additional therapeutic agents can include small molecules described in WO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1, WO201524876A2, WO2013119916A2, and WO2014209841A2, which are incorporated by reference in their entirety. In some embodiments, the one or more additional therapeutic agents include ASOs 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 subjects

[0151] Any of the compositions provided herein may be administered to an individual. An "individual" may be used interchangeably with a "subject" or a "patient." An individual is a mammal, e.g., an animal such as a human or a 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 embodiments, the individual is a human. In embodiments, the individual is a fetus, an embryo, or a child. In other embodiments, the individual may be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to a cell ex vivo.

[0137]

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

[0138]

[0153] The suitable route for administration of the ASO of the present invention may vary depending on the cell type to which the ASO is desired to be delivered.Dravet syndrome; generalized epilepsy febrile seizures plus, type 2; familial febrile seizures, 3A; familial hemiplegic migraine, 3; autism; epileptic encephalopathy, early infancy, 13; sick sinus 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 patients parenterally, for example, by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection, or intravenous injection.

[0139]

[0154] In some embodiments, the disease or condition is Na v 1.1 (the protein encoded by the SCN1A gene). In some cases, the mutation v1.1. In some cases, Na v Loss-of-function mutations in 1.1 are v 1.1 Function of wild-type Na v 1.1 includes one or more mutations that reduce or impair (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more) the function of the Na v Loss-of-function mutations in 1.1 include one or more mutations that result in a disease phenotype. Exemplary loss-of-function mutations include, but are not limited to, R859C, T875M, V1353L, I1656M, R1657C, A1685V, M1841T, and R1916G.

[0140]

[0155] In other cases, the mutation is Na v 1.1. In such cases, the gain-of-function mutation is v 1.1 Activation of wild-type Na v 1.1 includes one or more mutations that lengthen the function of the Na v Gain-of-function mutations in 1.1 include 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 embodiments, the disease or condition is encephalopathy. In some cases, the encephalopathy is caused by Na v 1.1 is induced by a loss-of-function mutation.

[0157] In some embodiments, the encephalopathy is an epileptic encephalopathy. Exemplary epileptic disorders include, but are not limited to, Dravet syndrome (DS) (also known as severe myoclonic epilepsy in infancy or SMEI); severe myoclonic epilepsy in infancy (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); early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or sick sinus syndrome 1. In some aspects, the disease or condition is an epileptic encephalopathy, optionally selected from Dravet syndrome (DS) (also known as severe myoclonic epilepsy of infancy or SMEI); severe myoclonic epilepsy of infancy (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 seizures; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassifiable epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); and sick sinus syndrome 1.

[0142]

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

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

[0143]

[0160] In some cases, 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 intractable epilepsy of childhood with generalized tonic-clonic seizures (ICEGTC).

[0144]

[0161] In some embodiments, Na v Diseases or conditions caused by loss-of-function mutations in 1.1 include, but are not limited to, Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infancy (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; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); autism; or infantile malignant migratory partial seizures.

[0145]

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

[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 Hereditary epilepsy. v 1.1 For hereditary epilepsy, Na v Loss of function mutation in 1.1 or Na v 1.1 may include gain-of-function mutations. In some cases, Na v 1.1 Genetic epilepsy involves one or more genetic mutations. In other cases, Na v 1.1 Genetic epilepsy involves one or more de novo mutations. In some cases, Na v1.1 Genetic epilepsies include Dravet syndrome (DS) (also known as severe myoclonic epilepsy of infancy or SMEI); severe myoclonic epilepsy of infancy (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; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); sudden unexpected death in epilepsy (SUDEP); or malignant migratory partial seizures of infancy. In some cases, Na v Na associated with loss-of-function mutations in 1.1 v 1.1 Genetic epilepsies include Dravet syndrome (DS) (also known as severe myoclonic epilepsy of infancy or SMEI); severe myoclonic epilepsy of infancy (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; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); sudden unexpected death in epilepsy (SUDEP); malignant migratory partial seizures of 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, Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infancy (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; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or infantile malignant migratory partial seizures. In some cases, the disease or condition is Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infancy (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 seizures; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassifiable epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinus syndrome 1; early infantile SCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE); or infantile malignant migratory partial seizures.

[0148]

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

[0167] Dravet syndrome (DS), otherwise known as severe myoclonic epilepsy of infancy (SMEI), is an epileptic encephalopathy that appears in the first year of life. Dravet syndrome is an increasingly recognized epileptic encephalopathy whose clinical diagnosis is supported by the finding of sodium channel gene mutations in roughly 70-80% of patients. Mutations in ion channel genes play an important role in the pathogenesis of a wide range of epilepsy syndromes and give rise to several epilepsies that are considered as channelopathies. As voltage-gated sodium channels (VGSCs) play an essential role in neuronal excitability, it is not surprising that many mutations associated with DS have been identified in genes encoding VGSC subunits. This disease is described, for example, by Mulley, et al., 2005, and a description of this disease is provided 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 harbor abnormalities in the sodium channel α1 subunit gene (SCN1A), with truncating mutations accounting for approximately 40% and significantly correlated with seizure onset at an earlier age. Sequence variants are found in approximately 70% of cases, including truncating mutations (40%) and missense mutations (40%), with the remainder being splice site alterations. Most mutations are de novo, but familial mutations occur in 5-10% of cases, usually missense in nature. The remaining SCN1A mutations include splice site and missense mutations, most of which correspond to the pore-forming domain of the sodium channel. Currently, over 500 mutations have been associated with DS, randomly distributed along the gene (Mulley, et al., Neurol. 2006, 67, 1094-1095).

[0150]

[0169] The SCN1A gene is located in a cluster of sodium channel genes on human chromosome 2q24 and encodes the neuronal voltage-gated sodium channel Na v It encodes the alpha pore-forming subunit known as SCN1A. The SCN1A gene spans approximately 100 kb of genomic DNA and contains 26 exons. The SCN1A protein consists of four domains, each with six transmembrane segments. Two splice variants in exon 11 have been identified that give rise to long and short isoforms that differ in the presence or absence of 11 amino acids in the cytoplasmic loop between domains 1 and 2 (Miller, et al., 1993-2015 and Mulley, et al., 2005, 25, 535-542, incorporated herein by reference).

[0151]

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

[0152]

[0171] One alternative splicing event that may result in a non-productive mRNA transcript is the inclusion of an extra exon in the mRNA transcript, which may induce nonsense-mediated mRNA decay. The present disclosure provides compositions and methods for modulating alternative splicing of SCN1A to increase the production of mature protein-encoding mRNA and the functional SCN1A protein that is translated thereby. These compositions and methods include antisense oligomers (ASOs) that can cause exon skipping to promote 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 pathologies 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 (eg, 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 (eg, familial hemiplegic migraine, 3).

[0155]

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

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

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

[0156]

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

[0181] In some embodiments, antisense oligonucleotides are administered with one or more agents that can enhance the penetration of the antisense oligonucleotides through the blood-brain barrier by any method known in the art.For example, U.S. Patent No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," incorporated herein by reference, describes the delivery of agents to motor neurons by administering adenovirus vectors.For example, U.S. Patent No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference, describes the direct delivery of vectors to the brain (e.g., striatum, thalamus, hippocampus, substantia nigra).

[0157]

[0182] In embodiments, the antisense oligonucleotides are linked or conjugated to agents that provide the desired pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotides are linked to substances known in the art to enhance penetration or transport across the blood-brain barrier (e.g., antibodies against the transferrin receptor). In embodiments, the antisense oligonucleotides are linked to viral vectors, for example, to make the antisense compounds more effective or to enhance transport across the blood-brain barrier. In one or more embodiments, a sugar (e.g., mesoerythritol, 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, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, D(+) arabinose ... In some cases, the osmotic blood-brain barrier is disrupted by infusion of ethanol, 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).For example, U.S. Pat. No. 9,193,969, entitled "Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types," U.S. Pat. No. 4,866,042, entitled "Method for the delivery of genetic material across the blood brain barrier," U.S. Pat. No. 6,294,520, entitled "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, entitled "Parenteral delivery systems," each of which is incorporated herein by reference, describe methods and materials for enhancing blood-brain barrier penetration.

[0158]

[0183] In some embodiments, the ASOs of the invention are conjugated to dopamine reuptake inhibitors (DRIs), selective serotonin reuptake inhibitors (SSRIs), noradrenaline reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs) using the methods described in U.S. Pat. No. 9,193,969, incorporated herein by reference.

[0159]

[0184] In some embodiments, subjects treated using the methods and compositions are assessed for improvement in their condition using methods known and described in the art.

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

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

[0161]

[0186] A first round of screening, called an ASO "walk," can be performed using ASOs designed to hybridize to the target region of the pre-mRNA. For example, the ASOs used in the ASO walk can 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 15 nucleotides in length can 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 (an ASO with a scrambled sequence, i.e., an ASO with a sequence not predicted to hybridize to the target region) are delivered, e.g., by transfection, into a disease-relevant cell line expressing a target pre-mRNA (e.g., an NIE-containing pre-mRNA described herein). The exon skipping efficacy of each of the ASOs can be assessed by any method known in the art, e.g., by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described in Example 4. A reduction or absence of longer RT-PCR products produced using primers that span the region containing the included exon (e.g., including exons flanking the NIE) in ASO-treated cells compared to control ASO-treated cells indicates enhanced splicing of the target NIE. In some embodiments, exon skipping efficiency (or splicing efficiency of splicing NIE-containing introns), the ratio of spliced ​​pre-mRNA to unspliced ​​pre-mRNA, the rate of splicing, or the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for evaluating and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0163]

[0188] A second round of screening, called an ASO "microwalk", can be performed using ASOs designed to hybridize to target regions of the pre-mRNA. The ASOs used in the ASO microwalk are tiled nucleotide by nucleotide to further refine the nucleotide sequence of the pre-mRNA that will result in exon skipping (or enhanced splicing of NIEs) when hybridized with the ASO.

[0164]

[0189] By means of ASO “microwalks” involving ASOs spaced in 1-nucleotide increments, as well as longer ASOs (typically 18–25 nucleotides), we will explore in more detail the regions defined by ASOs that promote splicing of targeted introns.

[0165]

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

[0166]

[0191] ASOs that cause exon skipping (or enhanced splicing of NIE-containing introns) when hybridizing to a region of pre-mRNA, resulting in increased protein production, can be tested in vivo using animal models (e.g., transgenic mouse models with full-length human genes knocked in, or humanized mouse models of disease). The appropriate route of administration of the ASO may vary depending on the disease and / or cell type to which delivery of the ASO is desired. The ASO may be administered, for example, by intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, intravitreal, or intravenous injection. Following administration, cells, tissues, and / or organs of the model animal can be evaluated to determine the efficacy of ASO treatment, for example, by assessing splicing (efficiency, rate, extent) and protein production by methods known in the art and described herein. The animal model may be one that exhibits a phenotype or behavior of a 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 this disclosure are methods for identifying or verifying NMD-induced exons in the presence of an NMD inhibitor (e.g., cycloheximide). Exemplary methods are provided in FIG.

[0168] Specific Aspects

[0194] Embodiment 1. A method of regulating expression of SCN1A protein in a cell having an mRNA containing a nonsense-dependent RNA decay-induced exon (NMD exon mRNA) encoding the SCN1A protein, comprising contacting the cell with a therapeutic agent, whereby the therapeutic agent regulates splicing of the NMD exon from the NMD exon mRNA encoding the SCN1A protein, thereby regulating the level of processed mRNA encoding the SCN1A protein and regulating the expression of the SCN1A protein in the cell, wherein the therapeutic agent binds to a targeting portion of the NMD exon mRNA encoding SCN1A, and the targeting portion is 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 of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE.

[0169]

[0195] Embodiment 2. A method of treating a disease or condition in a subject in need of such treatment by modulating expression of SCN1A protein in the cells of the subject, comprising contacting the cells of the subject with a therapeutic agent that modulates splicing of a nonsense-mediated mRNA decay-induced exon (NMD exon) from an mRNA encoding SCN1A that contains an NMD exon in the cells, 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 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 of the 3' end of the NIE to about 1000 nucleotides downstream of the 3' end of the NIE.

[0170]

[0196] Embodiment 3. The method of embodiment 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.

[0197] Embodiment 4. The method of embodiment 1 or 2, wherein 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, or about 50 nucleotides upstream of the 5' end of the NIE.

[0171]

[0198] Embodiment 5. The method of embodiment 1 or 2, wherein 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, or about 1 nucleotide upstream of the 5' end of the NIE.

[0172]

[0199] Embodiment 6. The method of embodiment 1 or 2, wherein 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, or about 50 nucleotides downstream of the 3' end of the NIE.

[0173]

[0200] Embodiment 7. The method of embodiment 1 or 2, wherein 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, or about 1 nucleotide downstream of the 3' end of the NIE.

[0174]

[0201] Embodiment 8 The method of any one of embodiments 1 to 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 an NMD exon from the processed mRNA encoding the SCN1A protein.

[0176]

[0204] Aspect 11: In a cell contacted with a therapeutic agent, the exclusion of an NMD exon from a processed mRNA encoding an SCN1A protein 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, or about 1.1 to about 9 times, or about 1.1 to about 10 ... The method of embodiment 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.

[0177]

[0205] Embodiment 12 The method of embodiment 10, wherein the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in said cell.

[0206] Aspect 13: The amount of processed mRNA encoding the SCN1A protein in a cell 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 The method of embodiment 10, wherein the increase is from 2 to about 5 fold, from about 2 to about 6 fold, from about 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 vThe method of embodiment 2, which is induced by a loss-of-function mutation in 1.1.

[0208] Embodiment 15. The method of embodiment 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.

[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 method of aspect 14, wherein the disease or condition is 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; 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 infantile malignant migratory partial seizures.

[0180]

[0212] Embodiment 19 The method of embodiment 18, wherein GEFS+ is generalized epilepsy febrile seizures plus type 2.

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

[0181]

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

[0182]

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

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

[0183]

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

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

[0184]

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

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

[0185]

[0221] Aspect 28. A method of treating a disease or condition in a subject in need of such treatment by modulating expression of SCN1A protein in the 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 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 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.

[0186]

[0222] Embodiment 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 to 731.

[0187]

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

[0224] Embodiment 31. A method for treating a disease or condition in a subject in need of such treatment by regulating expression of SCN1A protein in cells of the subject, the method comprising contacting the cells of the subject with an ASO of embodiment 29 or 30.

[0188]

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

[0226] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the spirit of the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. The following claims are intended to define the scope of the invention, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby. EXAMPLES

[0189]

[0227] The present invention will be more specifically illustrated 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-induced exon inclusion events in SCN1A transcripts by RNAseq using next-generation sequencing

[0228] Whole-transcriptome shotgun sequencing using next-generation sequencing was performed to reveal a snapshot of the transcripts produced by the SCN1A gene and to identify NIE inclusion events. To this end, polyA was isolated from nuclear and cytoplasmic fractions of HCN (human cortical neurons). +RNA was isolated and a cDNA library was constructed using Illumina's TruSeq Stranded mRNA library Prep Kit. The library was paired-end sequenced and the resulting 100 nucleotide reads were mapped to the human genome (February 2009, GRCh37 / hg19 assembly). The sequencing results for SCN1A are shown in Figure 2. Briefly, Figure 2 shows the mapped reads visualized using the UCSC Genome Browser (operated by the UCSC Genome Informatics Group (Center for Biomolecular Science and Engineering, University of California, Santa Cruz, 1156 HighStreet, Santa Cruz, CA 95064) and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome Browser database: 2015 Update," Nucleic Acids Research 43, Database Issue, doi: 10.1093 / nar / gku1177), where the extent and number of reads can be estimated by the peak signal. The height of the peak indicates the level of expression given by the density of reads in a particular region. The top panel shows a zoomed-in graphical representation of the SCN1A gene. The conservation level across 100 vertebrate species is shown as a peak. The highest peak corresponds to an exon (black box), whereas no peak is observed in the majority of introns (lines with arrowheads). A conserved peak was identified in intron 20 (NM_006920) and is shown in the middle panel. Inspection of the conserved sequence identified a 64 bp exon-like sequence (lower panel, sequence highlighted in grey) flanked by 3' and 5' splice sites (underlined sequences). Inclusion of this exon results in a frameshift, so a premature stop codon is introduced in exon 21 to target this transcript for NMD.

[0190]

[0229] The sequences of an exemplary SCN1A gene, pre-mRNA, exons, and introns 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 +RT-PCR analysis of cytoplasmic RNA from mouse Neuro 2A cells (Figure 3A) and RenCell VM (human neural progenitor cells) (Figure 3B) using primers in exon 21 and exon 23 confirmed the presence of a band corresponding to the NMD-induced exon (20x). The identity of this product was confirmed by sequencing. Densitometric analysis of this band was performed to calculate the % inclusion of exon 20x in the total SCN1A transcript. RenCell VM were treated with cycloheximide (CHX + ) inhibited NMD, leading to a twofold increase in the product corresponding to the NMD-induced exon 20x in the cytoplasmic fraction (light grey bars, CHX - and dark grey bars, CHX + (Compare

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

[0231] ASOs were designed to cover a region approximately 1000 to approximately 100 nucleotides upstream of the 5' end of the SCN1A exon 23 (exon 20x) gene by shifting 5 nucleotides at a time. ASOs were also 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 by shifting 5 nucleotides at a time. 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 assessed by RT-PCR

[0232] ASO walk sequences can be evaluated, for example, by RT-PCR. In Figure 5A, a representative PAGE shows SYBR-safe stained RT-PCR products of SCN1A mock treated, control ASO treated, targeting exon 20x region, as described herein in Example 3 and in the description of Figure 3, by nucleofection at a concentration of 1 μM in RenCell. Two products (one containing exon 20x and the other excluding exon 20x) are quantified and exon 20x inclusion % is plotted in a bar graph (Figure 5B). Taqman q PCR products are also normalized to RPL32 internal control, and fold change compared to mock is plotted in a bar graph (Figure 5C).

[0211]

[0233] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the spirit of the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. The following claims are intended to define the scope of the invention, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby. This application includes the following inventions. [Item 1] A method for regulating expression of SCN1A protein in a cell having an mRNA that contains a nonsense-dependent RNA decay-inducing exon (NMD exon mRNA) and encodes 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: 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 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, [Item 2] A method for treating a disease or condition in a subject in need of such treatment by regulating expression of 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 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: 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 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, [Item 3] The method of items 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. [Item 4] The method of item 1 or 2, wherein the targeting 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, or about 50 nucleotides upstream of the 5' end of the NIE. [Item 5] The method of item 1 or 2, wherein the targeting 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, or about 1 nucleotide upstream of the 5' end of the NIE. [Item 6] The method of item 1 or 2, wherein the targeting 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, or about 50 nucleotides downstream of the 3' end of the NIE. [Item 7] The method of item 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. [Item 8] The method of 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 processed mRNA encoding the SCN1A protein. [Item 11] In a cell contacted with a therapeutic agent, the elimination of the NMD exon from the processed mRNA encoding the SCN1A protein 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, or The method of item 10, wherein the increase is 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. [Item 12] The method of Item 10, wherein the therapeutic agent increases the level of processed mRNA encoding the SCN1A protein in the cell. [Item 13] The amount of processed mRNA encoding the SCN1A protein in a cell 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 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 increase. [Item 14] If the disease or condition is Na v 1. The method of item 2, which is induced by a loss-of-function mutation in 1.1. [Item 15] The disease or condition is associated with haploinsufficiency of the SCN1A gene, and the subject has The first allele encodes the target SCN1A, and the second allele encodes the target SCN1A but not the target SCN1A. 15. The method of item 14, wherein the second allele is produced at no or reduced levels, or encodes a non-functional or 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 of infancy (SMEI)-limbic type (SMEB); febrile seizures (FS); generalized epilepsy febrile seizures plus (GEFS+); epileptic encephalopathy, early infantile, 13; cryptogenic 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 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 seizures are familial febrile seizures 3A. [Item 21] The method of item 18, wherein the SMEB is SMEB without generalized spike-and-waves (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). [Item 22] The method of item 1 or 2, wherein the ASO consists of an array selected from SEQ ID NO: 72 or 432. [Item 23] The method of item 1 or 2, wherein the ASO consists of an array selected from SEQ ID NO: 73 or 433. [Item 24] The method of item 1 or 2, wherein the ASO consists of an array selected from SEQ ID NO: 76 or 436. [Item 25] The method of item 1 or 2, wherein the ASO consists of an array selected from SEQ ID NO: 181 or 541. [Item 26] The method of item 1 or 2, wherein the ASO consists of an array selected from SEQ ID NO: 220 or 580. [Item 27] ​​A method for regulating expression of SCN1A protein in a cell having an mRNA that contains a nonsense-dependent RNA decay-inducing exon (NMD exon mRNA) and encodes 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 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. [Item 28] A method for treating a disease or condition in a subject in need of such treatment by regulating expression of 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 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 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. [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 to 731. [Item 30] An antisense oligomer (ASO) consisting of a sequence selected from SEQ ID NOs: 12 to 731. [Item 31] A method for treating a disease or condition in a subject in need of such treatment by regulating expression of SCN1A protein in cells of the subject, comprising: The method comprises contacting the ASO of item 29 or 30 with the cells of the subject. [Item 32] A kit comprising the ASO of Item 29 or 30.

Claims

1. 1. A pharmaceutical composition for use in a method for increasing expression of a Navl.1 protein in a cell having a pre-mRNA that contains a nonsense-mediated mRNA decay-induced exon (NMD exon) and encodes a Navl.1 protein, the method comprising: contacting the cell with a therapeutic agent, where the therapeutic agent promotes the exclusion of the NMD exon from the pre-mRNA encoding the Navl.1 protein, thereby increasing the level of processed mRNA encoding the Navl.1 protein and expression of the Navl.1 protein in the cell; wherein the therapeutic agent binds to a target region of the pre-mRNA encoding Navl.1, and the target region comprises: from about 1000 nucleotides upstream from the 5' end of the NMD exon to about 100 nucleotides upstream from the 5' end of the NMD exon; or From about 100 nucleotides downstream of the 3' end of the NMD exon to about 1000 nucleotides downstream of the 3' end of the NMD exon; Located in The therapeutic agent comprises an antisense oligomer (ASO); and The NMD exon comprises a sequence having at least 95% sequence identity to SEQ ID NO:7; The pharmaceutical composition.

2. 1. A pharmaceutical composition comprising a therapeutic agent for treating a disease or condition in a subject in need thereof by increasing expression of Nav1.1 protein in cells of the subject, the therapeutic agent promotes the exclusion of a nonsense-mediated mRNA decay-inducing exon (NMD exon) from a pre-mRNA that contains the NMD exon and encodes a Navl.1 protein in said cell, thereby increasing the level of processed mRNA encoding the Navl.1 protein and expression of the Navl.1 protein in the subject's cell; The therapeutic agent binds to a targeting portion of the pre-mRNA encoding the Nav1.1 protein; The targeting moiety: 1000 nucleotides upstream from the 5' end of the NMD exon to 100 nucleotides upstream from the 5' end of the NMD exon; or 00 nucleotides downstream of the 3' end of the NMD exon to 1000 nucleotides downstream of the 3' end of the NMD exon; Located in The therapeutic agent comprises an antisense oligomer (ASO); and The NMD exon comprises a sequence having at least 95% sequence identity to SEQ ID NO:7; The pharmaceutical composition.

3. The pharmaceutical composition of claim 2 , wherein the disease or condition is induced by a loss-of-function mutation in the Nav1.1 protein.

4. 3. The pharmaceutical composition of claim 2, wherein the disease or condition is associated with haploinsufficiency of the SCN1A gene and the subject has a first allele that encodes a functional Navl.1 protein and a second allele that produces no or reduced levels of Navl.1 protein, or that encodes a non-functional or partially functional Navl.1 protein.

5. 3. The pharmaceutical composition of claim 2, wherein the disease or condition is Dravet syndrome (DS); severe myoclonic epilepsy of infancy (SMEI)-limbic type (SMEB); febrile seizures (FS); familial febrile seizures 3A; generalized epilepsy febrile seizures plus (GEFS+); generalized epilepsy febrile seizures plus type 2; encephalopathy; 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 infancy.

6. The pharmaceutical composition described in claim 2, wherein the disease or condition is Dravet Syndrome (DS).

7. The pharmaceutical composition according to any one of claims 2 to 6, wherein administration to a subject is by intrathecal injection or intracerebroventricular infusion.

8. The pharmaceutical composition according to any one of claims 2 to 7, wherein the subject is a human subject.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the therapeutic agent interferes with the binding of a factor involved in splicing of the NMD exon from the region of the target region.

10. The target region is: (a) at most 500 nucleotides upstream of the 5′ end of the NMD exon; (b) at least 500 nucleotides upstream of the 5′ end of the NMD exon; (c) at most 500 nucleotides downstream of the 3' end of the NMD exon; or (d) at least 500 nucleotides downstream of the 3' end of the NMD exon; The pharmaceutical composition according to any one of claims 1 to 9,

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the target region is contained within a sequence that has at least 95% sequence identity to SEQ ID NO:

6.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the amount of Nav1.1 protein in cells contacted with the therapeutic agent is increased by at least 1.1-fold compared to the total amount of Nav1.1 protein in control cells.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the ASO consists of a sequence selected from the group consisting of SEQ ID NOs: 72, 73, 76, 181, 220, 432, 433, 436, 541, and 580.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the ASO comprises a 2'-O-methoxyethyl moiety.

15. 15. The pharmaceutical composition of claim 14, wherein each nucleotide of the antisense oligomer comprises a 2'-O-methoxyethyl moiety.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the antisense oligomer is 18 to 50 nucleobases in length.

17. 1. A pharmaceutical composition comprising a therapeutic agent, The therapeutic agent binds to a targeted portion of a pre-mRNA that contains a nonsense-mediated mRNA decay-inducing exon (NMD exon) and encodes the Nav1.1 protein; The therapeutic agent promotes the exclusion of the NMD exon from the pre-mRNA encoding Nav1.1; The targeting moiety: from about 1000 nucleotides upstream from the 5' end of the NMD exon to about 100 nucleotides upstream from the 5' end of the NMD exon; or From about 100 nucleotides downstream of the 3' end of the NMD exon to about 1000 nucleotides downstream of the 3' end of the NMD exon; and The therapeutic agent comprises an antisense oligomer (ASO); and The NMD exon comprises a sequence having at least 95% sequence identity to SEQ ID NO:7; The pharmaceutical composition.

18. If the target region is: (a) at most 500 nucleotides upstream of the 5′ end of the NMD exon; (b) at least 500 nucleotides upstream of the 5′ end of the NMD exon; (c) at most 500 nucleotides downstream of the 3' end of the NMD exon; or (d) at least 500 nucleotides downstream of the 3' end of the NMD exon; The pharmaceutical composition of claim 17,

19. 19. The pharmaceutical composition of claim 17 or 18, wherein the target region is contained within a sequence that has at least 95% sequence identity to SEQ ID NO:

6.

20. The pharmaceutical composition of any one of claims 17 to 19, wherein the ASO consists of a sequence selected from the group consisting of SEQ ID NOs: 72, 73, 76, 181, 220, 432, 433, 436, 541, and 580.

21. The pharmaceutical composition of any one of claims 17 to 20, wherein the ASO comprises a 2'-O-methoxyethyl moiety.

22. 22. The pharmaceutical composition of claim 21, wherein each nucleotide of the antisense oligomer comprises a 2'-O-methoxyethyl moiety.

23. The pharmaceutical composition of any one of claims 17 to 22, wherein the antisense oligomer is 18 to 50 nucleobases in length.

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