Antisense oligonucleotides for treatment of stxbp1-related developmental epileptic encephalopathy

Abstract

The present disclosure describes antisense oligonucleotides (ASOs) that target regions in a STXBP1 pre-mRNA. In certain embodiments, the ASOs are splice switching oligomers (SSOs) which target specific pre-mRNA splicing regulatory elements encoded in the STXBP1 gene and modify STXBP1 pre-mRNA splicing through steric blocking of the targeted splicing regulatory element. The present disclosure also describes methods for modifying the expression of Syntaxin binding protein 1 (STXBP1) using ASOs of the present disclosure. The present disclosure also describes methods of treating STXBP1 -related diseases, including developmental epileptic encephalopathy (DEE), by restoring canonical STXBP1 splicing and upregulating STXBP1 mRNA production using ASOs of the present disclosure.

Description

DESCRIPTIONANTISENSE OLIGONUCLEOTIDES FOR TREATMENT OF STXBP1- RELATED DEVELOPMENTAL EPILEPTIC ENCEPHALOPATHY

[0001] The present disclosure describes antisense oligonucleotides (ASOs) that target regions in a Syntaxin binding protein 1 (STXBP1) pre-mRNA. In certain embodiments, the ASOs are splice switching oligomers (SSOs) which target specific pre-mRNA splicing regulatory elements encoded in the STXBP1 gene and modify STXBP1 pre-mRNA splicing through steric blocking of the targeted splicing regulatory element. The present disclosure also describes methods for modifying the expression of STXBP1 using ASOs of the present disclosure. The present disclosure also describes methods of treating STXBP1 -related diseases, including developmental epileptic encephalopathy (DEE), by restoring canonical STXBP1 splicing and upregulating STXBP1 mRNA production using ASOs of the present disclosure.

[0002] In certain embodiments, the present disclosure describes antisense oligonucleotides (ASOs) that target splicing regulatory elements in a STXBP1 pre-mRNA. In certain embodiments, the present disclosure describes an ASO comprising at least one target binding sequence, wherein the target binding sequence has at least 80% sequence identity to any one of SEQ ID NOs: 3-100. In certain embodiments, the target binding sequence comprises any one of SEQ ID NOs: 3, 4, 13, 23, 43, 52, 72, and 74. In certain embodiments, the target binding sequence comprises SEQ ID NO: 3 or SEQ ID NO: 52.

[0003] In certain embodiments, the ASO comprises at least one target binding sequence, wherein the target binding sequence has at least 80% sequence identity to any one of SEQ ID NOs: 109-206. hi certain embodiments, the target binding sequence comprises a sequence of any one of SEQ ID NOs: 109, 110, 119, 129, 149, 158, 178, and 180. hi certain embodiments, the target binding sequence comprises SEQ ID NO: 109 or SEQ ID NO: 158.

[0004] In certain embodiments, the ASO comprises a phosphorothioate (PS) modification to all intemucleotide phosphodiester linkages in the ASO. In certain embodiments, the ASO comprises a 2'-O-Methoxyethyl (2'-MOE) modification to all nucleoside sugar moieties in the ASO.

[0005] In certain embodiments, the ASO comprises at least one target binding sequence, wherein the target binding sequence has at least 80% sequence complementarity to a target sequence in Exon 3 (SEQ ID NO: 336), Intron 3 (SEQ ID NO: 337), Exon 4 (SEQ ID NO: 338), Intron 4 (SEQ ID NO: 339), Intron 17 (SEQ ID NO: 342), Exon 18 (SEQ ID NO: 343),Intron 18 (SEQ ID NO: 344), or Exon 19 (SEQ ID NO: 345) of human STXBP1 gene, or a combination thereof. In certain embodiments, the target binding sequence has at least 80% sequence complementarity to a target sequence encoded in GRCh38 / hg38 / chr9: 127654840- 127655139 (SEQ ID NO: 335). hi certain embodiments, the target binding sequence has at least 80% sequence complementarity to a target sequence encoded in GRCh38 / hg38 / chr9:127682536-127682658 (SEQ ID NO: 340). In certain embodiments, the target binding sequence has at least 80% sequence complementarity to a target sequence encoded in GRCh38 / hg38 / chr9: 127684296-127684640 (SEQ ID NO: 341). hi certain embodiments, the target sequence comprises any one of SEQ ID NOs: 101-108. In certain embodiments, the target sequence is SEQ ID NO: 106 or SEQ ID NO: 103.

[0006] In certain embodiments, the target binding sequence has at least 85% sequence complementarity to the target sequence. In certain embodiments, the target binding sequence comprises 15-25 nucleotides.

[0007] In certain embodiments, the present disclosure describes a pharmaceutical composition comprising an ASO of the present disclosure and a pharmaceutically acceptable excipient.

[0008] In certain embodiments, the present disclosure describes a method of modifying the expression of Syntaxin binding protein 1 (STXBP1) by delivering an effective amount of an ASO of the present disclosure to a cell comprising the STXBP1 gene. In certain embodiments, delivery of the ASO to the cell results in an increase in STXBP1 production by the cell of at least 120%, when compared to an untreated cell.

[0009] In certain embodiments, the present disclosure describes a method of treating a disease related to a STXBP1 gene mutation in a subject in need thereof, wherein the methods comprises administering to the subject a therapeutically effective amount of a composition comprising an ASO of the present disclosure. In certain embodiments, the disease related to a STXBP1 gene mutation is a developmental and epileptic encephalopathy (DEE), such as Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE.

[0010] In certain embodiments, the present disclosure describes an ASO or a pharmaceutical composition of the present disclosure for use in the prevention and / or treatment of a disease related to a STXBP1 gene mutation. In certain embodiments, the disease related to a STXBP1 gene mutation is a developmental and epileptic encephalopathy (DEE), such as Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE.

[0011] In certain embodiments, the present disclosure describes the use of an ASO or a pharmaceutical composition of the present disclosure for the treatment of a disease related to a STXBP1 gene mutation. In certain embodiments, the present disclosure describes the use of an ASO or a pharmaceutical composition of the present disclosure for the manufacture of a medicament for the treatment of a disease related to a STXBP1 gene mutation. In certain embodiments, the disease related to a STXBP1 gene mutation is a developmental and epileptic encephalopathy (DEE), such as Ohtahara syndrome, West syndrome, Lennox- Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE.BRIEF DESCRIPTION OF FIGURES

[0012] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the present disclosure, as illustrated in the accompanying figures. The figures are not necessarily to scale or comprehensive, with emphasis instead being placed upon illustrating the principles of various embodiments of the present disclosure.

[0013] FIG. 1 A shows a schematic representation of phosphorothioate (PS) modification of a phosphodiester intemucleoside linkage. FIG. IB shows a schematic representation of a 2'-O-Methoxyethyl (2'- MOE) modification at the T position of a nucleoside sugar moiety.

[0014] FIG. 2 shows images of healthy GABA-ergic neurons cells at days 6, 9, and 12.

[0015] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D present graphs of the results from theSTXBP1 ASO in vitro screening of Example 2. FIG. 3 A and FIG. 3B present the results for ASOs 1-49. FIG. 3C and FIG. 3D present the results for ASOs 50-98.

[0016] FIG. 4A presents a graph of the results from the STXBP1 ASO in vitro microwalk screen with one nucleotide overlap + / - 50 bp around 168966 near Exon 19. The assay conditions and methods from the primary screen were maintained in the microwalk screen.

[0017] FIG. 4B and FIG. 4C present a graph of the results from the STXBP1 ASO in vitro microwalk screen with one nucleotide overlap + / - 50 bp around 168966 in a mouse STXBP1 transcript. The assay conditions and methods from the primary screen were maintained in the microwalk screen.DEFINITIONS

[0018] Approximately / About: As used herein, the terms "approximately" and "about" are used interchangeably herein and refer to a value that is within + / - 10% of the recited value as applied to one or more values of interest. In certain embodiments, the term refers to a rangeof values that fall within + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, or less of the stated reference value, unless otherwise expressly stated or otherwise clearly evident from the context.

[0019] Complementary. As used herein, the terms "complementary" or "complementarity" refer to a structural relationship between two nucleotides, nucleosides, or nucleobases (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand such as a hairpin), wherein the relationship permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. Complementary nucleotides can base pair in the canonical Watson-Crick manner (i.e., adenine pairing with thymine or uracil, and guanine pairing with cytosine), or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity.

[0020] Cryptic exon: As used herein, the terms "cryptic exon" or "pseudoexon" can be used interchangeably to refer to an exon that is absent or not detectable in wild-type pre- mRNA (i.e., intronic), but is present and detectable in a variant isoform (i.e., exonic). Cryptic exons may arise as a result of transposable elements, mutations that create new splice sites, or mutations that remove existing binding sites for splicing repressors.

[0021] Deoxyribonucleotide: As used herein, the term "deoxyribonucleotide" refers to a nucleotide having a hydrogen in place of a hydroxyl at the 2' position of its pentose sugar when compared with a ribonucleotide. A modified deoxyribonucleotide has one or more modifications or substitutions of atoms other than hydroxyl at the 2' position, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0022] Duplex: As used herein, the terms "duplex" and "duplex region" in reference to nucleic acids (e.g., oligonucleotides), refer to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides, whether formed by two covalently separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin). A duplex may form despite not having full complementarity between the two strands, or when an abasic moiety is present.

[0023] Modified internucleotide linkage: As used herein, the term "modified intemucleotide linkage" refers to an intemucleotide linkage having one or more chemical modifications when compared with a reference intemucleotide linkage having a phosphodiester bond. A modified intemucleotide linkage is a non-naturally occurring linkage.

[0024] Modified nucleotide'. As used herein, the term "modified nucleotide" refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide is a non-naturally occurring nucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group (in the intemucleotide linkage). Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide.

[0025] Nucleobase'. As used herein, the term "nucleobase" refers to a heterocyclic moiety of a nucleoside (e.g., adenine in adenosine, cytosine in cytidine, guanine in guanosine, thymine in thymidine, and uracil in uridine). A nucleobase can be a "canonical nucleobase" or "primary nucleobase," which are used interchangeably herein and include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). A nucleobase can be a "noncanonical nucleobase," which include synthetic or natural nucleobases which are not a canonical nucleobase.

[0026] Nucleoside'. As used herein, the term "nucleoside" refers to an organic compound having a nucleobase — for example, adenine, cytosine, guanine, thymine, or uracil — covalently attached to a pentose sugar -- for example, ribose or 2'-deoxyribose.

[0027] Nucleotide'. As used herein, the term "nucleotide" refers to a nucleoside in a phosphorylated form (e.g., a phosphate ester or thiophosphate ester of a nucleoside). A nucleotide can serve as a monomeric unit of nucleic acid polymers (e.g., oligonucleotides) such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleotides includes ribonucleotides (2'-OH sugar ring), deoxyribonucleotides (2'-H sugar ring), and modified nucleotides.

[0028] Oligonucleotide'. As used herein, the term "oligonucleotide" refers to a polymer of linked nucleotides, each of which can include a canonical or noncanonical nucleobase. An oligonucleotide is typically less than about 100 nucleotides in length (e.g., 2-50 nucleotides in length). An oligonucleotide may be single-stranded (ss) or double stranded (ds). An oligonucleotide may or may not have duplex regions. Oligonucleotides can include dinucleotide or trinucleotide polymers. As used herein, the term "polynucleotide" refers to a polymer of linked nucleotides which can be more than about 100 nucleotides in length. As used herein, the term dinucleotide includes two linked nucleosides with one or more phosphate-based linkages (e.g., a dinucleoside with two nucleosides and a cyclic phosphorus,phosphate ester, or thiophosphate ester linkage). As used herein, the term trinucleotide includes three linked nucleosides with two or more phosphate-based linkages (e.g., a trinucleoside with three nucleosides and cyclic phosphorus, phosphate ester, or thiophosphate ester linkages).

[0029] Pharmaceutically acceptable'. As used herein, the phrase “pharmaceutically acceptable” refers to compounds, compositions, carriers, excipients, and / or formulations which are suitable for contact (e.g., injection, ingestion, topical) with the tissues of a subject (e.g., human subject) without creating excessive toxicity, irritation, immuno-response, or other complications, according to reasonable medical judgment.

[0030] Percent identity: As used herein, the terms "percent sequence identity" or "percent identity" refers to the number of identical matched positions shared between two polynucleotide or polypeptide sequences over a comparison window, including gaps, additions, or deletions required for optimal alignment of the two sequences. Percentage of sequence identity can be calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and then converting the value to a percentage of sequence identity. Suitable software programs are available from various sources, including sequence comparison tools from the National Center for Biotechnology Information BLAST web site.

[0031] Poison exon\ As used herein, the terms "poison exon" and "toxic exon" can be used interchangeably to refer to a cryptic exon that contains a premature termination codon (PTC) in the reading frame of the exon when included in an RNA transcript. Poison exons can include cryptic exons which, when included in an RNA transcript, cause a reading frame shift in downstream exons resulting in a premature stop codon (PSC), which was not in frame prior to the frame-shift caused by the inclusion of the poison exon. Poison exons can include exon variants (e.g., mutated), extended forms of a wild-type exon, or truncated forms of a wild-type exon.

[0032] Ribonucleotide'. As used herein, the term "ribonucleotide" refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than hydrogen at the 2' position, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0033] Strand'. As used herein, the term "strand" refers to a single, contiguous sequence of nucleotides linked together through intemucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5' end and a 3' end).

[0034] Subject: As used herein, the terms “subject” or “patient” refer to any organism to which a composition of the present disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Examples of subjects include, but are not limited to, mammals such as humans, non-human primates, monkeys, mice, rats, and rabbits.

[0035] Transfection'. As used herein, the term “transfection” refers to the introduction exogenous molecules (e.g., heterologous nucleotide sequence) into a target cell. Examples of transfection include, but are not limited to, biochemical transfection, physical treatments, lipid-based transfection, vector-based transfection, or combinations thereof.ANTISENSE OLIGONUCLEOTIDES (ASOs)Pre-mRNA Splicing

[0036] Most human protein-coding genes are comprised of coding (exonic) sequences and non-coding (intronic) sequences. After a DNA sequence in a gene is transcribed to RNA, the non-coding, intronic RNA sequences are removed from the sequence by spliceosomes, and the coding exonic RNA sequences are then ligated together by the spliceosomes through pre- mRNA splicing to provide a final mRNA that is translated into a protein.

[0037] One of the ways that biological cells achieve diversity in proteins and protein isoforms is through alternative splicing of the encoding genes. In humans, more than 95% of genes undergo alternative splicing, while in mice (which have a similar number of genes) only about 65% of mouse genes undergo alternative splicing. This is particularly notable in the human brain, where the complexity of the human brain is, in part, related to an uncharactcristically-high amount of alternative splicing events found in brain tissue relative to other human tissues. However, this also leaves brain tissue susceptible to splicing alterations based on the diversity of neuronal and non-neuronal cell types, as well as molecular changes leading to connectivity, activity, and plasticity in the brain.

[0038] Proper transcription of open reading frames by RNA polymerase and pre-mRNA splicing of the resulting exons by spliceosomes is directed by specific sequences and conformational structures within the gene sequence. The spliceosome is a large ribonucleoprotein complex which can precisely excise intron sequences, and ligate the remaining exons, by detecting specific sequences in the intronic and exonic sequences. Forexample, pre-mRNA cleavage and ligations reactions (to remove introns) generally occur at conserved 5' splice site sequences found at the 5' end of an intron and corresponding 3' splice site sequences at the 3' end of an intron. The spliceosome assembles on the pre-mRNA by recognizing and binding to a 5' splice sequence at an exon-intron junction using small nuclear RNAs (snRNAs) which form specific RNA-RNA base-pairs with the splice site sequence. The spliceosome then proceeds with two sequential trans-esterification reactions: (1) the first reaction releases the 3' end of the upstream exon from the 5' end of the intronic sequence, which forms an intronic loop structure through an interaction with the branchpoint sequence at the 3' end of the same intron; and (2) the second reaction releases the intronic loop structure from the 3' end of the intron, and ligates together the 3' end of the upstream exon and the 5' end of the downstream exon to form a ligated exon sequence.

[0039] In principle, a 5' splice site for one exon can be spliced to a 3' splice site for any another exon, giving rise to the possibility of alternative splicing events and multiple alternatively-spliced isoforms from a single gene. Other alternative splicing events, such as retaining a portion of an intron, can likewise result in alternatively-spliced mRNA sequences and protein isoforms. The basic alternative splicing patterns include: (i) exon skipping, (ii) intron retention, (iii) alternative 5'-splice and 3'-splice site selection, (iv) alternative 5'- untranslated regions (UTRs) and 3'-UTRs, and (v) mutually exclusive exons.

[0040] The regulation of splicing is directed, in large part, by differential binding of protein splicing factors to sequences in the pre-mRNA transcript. These sequences, which comprise short-sequence splicing regulatory elements encoded within the gene sequence, act as a binding location for the protein splicing factors that influence the inclusion or exclusion of nearby exons and introns. The splicing regulatory elements can either assist or interfere with the recognition of the splice sites by the spliceosome, or in the binding of inhibitory splicing factor proteins or stimulatory splicing factor proteins. These splicing regulatory elements arc broadly classified into four groups: (i) exon splicing enhancers (ESE), (ii) exon splicing silencers (ESS), (iii) intron splicing enhancers (ISE), and (iv) intron splicing silencers (ISS). The splicing regulator sequences and protein splicing factors can be cellspecific or tissue specific.

[0041] Short-sequence splicing regulatory elements can also act as binding sites for specialized trans-acting splicing factors. Examples of splicing regulatory elements which can be targeted to modulate pre-mRNA include: binding sites for serine arginine (SR) proteins such as SF2 / ASF, SC35, 9G8, SRp20, SRp40 and SRp55; binding sites for polypyrimidine- tract-binding protein PTB; binding sites for small nuclear ribonucleoprotein (snRNPs), suchas U2AF65 or U2AF35; and binding sites for heterogenous nuclear ribonucleoproteins (HNRNPs), such as hnRNP L, hnRNP Al, or hnRNP A2. Other RNA-binding proteins (RBPs), Chromatin binding remodeling factors, and epigenetic factors can also modulate splicing.

[0042] Modulation of alternative splicing is particularly valuable in cases of disease caused by genetic mutations (inherent or stress-induced) that lead to disruptions in canonical and alternative splicing from a gene. Faulty regulation of such alternative splicing events has been associated with a number of neurodevelopmental disorders and neuro degenerative diseases.Splice-switching oligonucleotides (SSOs)

[0043] Antisense oligonucleotides (ASOs) are short, polynucleotide molecules (typically 15 30 nucleotides long) which comprise a sequence of nucleotides or nucleotide analogues that bind to a target complementary sequence, such as a gene sequence or mRNA sequence. The base-pairing (i.e., binding) of the ASO to the complementary target sequence generally results in the ASO arresting transcription of the target gene, altering pre-mRNA splicing, or translation of the target mRNA into a protein. The binding and blocking effect of the ASO is maintained until degradation of the bond and / or oligonucleotide by cellular exonuclease and endonuclease activity.

[0044] Splice-switching oligonucleotides (SSOs) are ASOs which can participate in the pre-mRNA splicing process. SSOs include an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements, thereby blocking RNA RNA basepairing or protein RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. For example, SSO base-pairing to a target RNA can alter the recognition of one or more splice sites by the spliceosome, which leads to an alteration in splicing of the targeted transcript. Likewise, an SSO can base-pair to an exon splicing enhancer (ESE), exon splicing silencer (ESS), intron splicing enhancer (ISE), or intron splicing silencer (ISS) to block the binding of an inhibitory splicing factor protein or stimulatory splicing factor protein, thereby altering the splicing of the targeted transcript.

[0045] In certain embodiments, an ASO of the present disclosure base-pairs to a spliceosome recognition site in the target pre-mRNA, thereby altering recognition of the splice recognition sites by the spliceosome. In certain embodiments, an ASO base-pairs to an exon splicing enhancer (ESE) sequence. In certain embodiments, an ASO base-pairs to an exon splicing silencer (ESS) sequence, hi certain embodiments, an ASO base-pairs to anintron splicing enhancer (ISE) sequence, hi certain embodiments, an ASO base-pairs to an intron splicing silencer (ISS) sequence.

[0046] In certain embodiments, the ASO comprises a sequence which is 15-25 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 16-20 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 15 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 16 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 17 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 18 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 19 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 20 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 21 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 22 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 23 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 24 nucleotides in length. In certain embodiments, the ASO comprises a sequence which is 25 nucleotides in length.Chemical Modifications

[0047] The binding affinity of an ASO base-pairing with a complementary sequence will depend on the base-pairing characteristics in the sequence (including complementarity percentage), as well as the chemistries of both the ASO and the binding location. Chemical modification of an ASO can also affect other characteristics of an ASO, including improved cellular uptake, higher solubility and retention in aqueous environments, improved target specificity and binding affinity, better pharmacokinetics, prolonged half-life and in vivo stability, nuclease resistance, and controlled intracellular activity. Common ASO chemical modifications include modifications to the intemucleotide phosphodiester linkage, modification of the nucleoside sugar moiety, or modification of the nucleobase.

[0048] In certain embodiments, an ASO comprises one or more modifications to one or more intemucleotide phosphodiester linkages in the ASO. In certain embodiments, an ASO comprises one or more modifications to all intemucleotide phosphodiester linkages in the ASO. Examples of modifications to intemucleotide phosphodiester linkages include methyl phosphonates (MP), phosphorothioate (PS), phosphorodithioate (PS2), N3'— »P5' phosphoramidate (NP), b or anopho sphate (BP), phosphonoacetate (PACE), phosphoramidates (PA), mesyl phosphoramidate (MsPA), phosphoramidate morpholino (PMO), methylene methylimino (MMI), methyl phosphonothioate, 5'-N carbamate, triazole, thioformacetal,mercapto acetamide, 5 '-methylurea, and guanidium. In certain embodiments, an ASO comprises a phosphorothioate (PS) modification to one or more intemucleotide phosphodiester linkages in the ASO. In certain embodiments, an ASO comprises a phosphorothioate (PS) modification to all intemucleotide phosphodiester linkages in the ASO. FIG. 1 A shows a schematic representation of phosphorothioate (PS) modification of a phosphodiester intemucleoside linkage.

[0049] In certain embodiments, an ASO comprises one or more modifications to one or more nucleoside sugar moieties in the ASO. In certain embodiments, an ASO comprises one or more modifications to all nucleoside sugar moieties in the ASO. Examples of modifications to nucleoside sugar moieties include: 2'-O-alkyl; 2'-O-methyl (2'-OMe); 2'-O- methoxyethyl (2'- MOE); 2'-amino; 2'-fluoro (2'-F); 2'-deoxy-2'-fluoro-arabino nucleic acid (FANA); 2'-O 4'-C-ethylene-bridged nucleic acid (ENA); 2'-O 4'-C methylene- bridged nucleic acid (LNAs); 4'-constrained 2'-O-methoxyethyl (cMOE); 2',4'-constrained 2'-O-ethyl (cEt); peptide nucleic acid (PNA); threose nucleic acids (TNA); hexitol (HNA); altritol (ANA); bicyclo-DNA (bc-DNA); tricyclo-DNA (tc-DNA); and cyclohexene nucleic acid (CeNA). In certain embodiments, an ASO comprises a 2'- MOE modification to one or more nucleoside sugar moieties in the ASO. In certain embodiments, an ASO comprises a 2'- MOE modification to all nucleoside sugar moieties in the ASO. FIG. IB shows a schematic representation of a 2'-O- Methoxyethyl (2'-MOE) modification at the 2' position of a nucleoside sugar moiety.

[0050] Without being bound by theory, modifications in the 2'-ribose sugar moiety (e.g., 2'-Ome, 2'-MOE) can improve non-specific protein binding, target mRNA binding, and cellular uptake of an ASO. Modification in T ribose can also aid in sterically hindering the translation of proteins or by modulating the splicing of mRNA (rather than eliciting RNase H-mediated degradation of mRNA).

[0051] In certain embodiments, SSO nucleotides can be chemically modified so that the RNA-cleaving enzymes (RNase H) are not recruited to degrade the pre-mRNA+SSO complex. This allows the SSOs to modify the pre-mRNA splicing without degrading the bound pre-mRNA and altering the amount of resulting mRNA produced in the cell. Uniformly modified oligomers usually do not induce the degradation of their target RNAs, whereas "gapmer" designs (i.e., ASOs with a stretch of at least unmodified nucleotides flanked by modified regions) encourage RNase H-mediated cleavage of the complementary RNA target in the "gap" region while also enhancing the ASO's nuclease stability and target binding affinity in the "modified" regions.Formulations and Delivery

[0052] In certain embodiments, ASOs of the present disclosure are delivered to one or more cells in a subject. In certain embodiments, ASOs gain access to cells in vivo as naked / unformulated oligonucleotides. In certain embodiments, ASO's are admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecule structures, or mixtures of compounds (e.g., liposomes, receptor targeted molecules, oral formulations, topical formulations), for assisting in uptake, distribution, and / or absorption. In certain embodiments, ASOs of the present disclosure are included in a physiologically or pharmaceutically acceptable carrier, such as an aqueous carrier. In certain embodiments, ASOs are included in formulations suitable for parenteral, intraperitoneal, intravenous, intraarterial, subcutaneous, intramuscular, intratumoral, subconjunctival, intravitreal, inhalation, topical, ophthalmic, oral, or pulmonary administration. In certain embodiments, ASOs of the present disclosure are delivered by intrathecal, intra-cistema magna, intracerebroventricular, intrap ar enchymal, or intra-cerebral-spinal-fluid administration.

[0053] The formulations may be presented in unit dosage form and may be prepared by any of the methods known in the art.STXBP1Overview

[0054] Syntaxin-binding protein 1 (STXBP1, also known as Seel or Muncl8) is encoded by the STXBP1 gene located on chromosome 9.

[0055] STXBP1 has two common isoforms, STXBP1 Isoform A (SEQ ID NO: 1) and STXBP1 Isoform B (SEQ ID NO: 2)

[0056] MAPIGLKAVVGEKIMHDVIKKVKKKGEWKVLVVDQLSMRMLSSCCKMT DIMTEGITIVEDINKRREPLPSLEAVYLITPSEKSVHSLISDFKDPPTAKYRAAHVFFTD SCPDALFNELVKSRAAKVIKTLTEINIAFLPYESQVYSLDSADSFQSFYSPHKAQMKN PILERLAEQIATLCATLKEYPAVRYRGEYKDNALLAQLIQDKLDAYKADDPTMGEGP DKARSQLLILDRGFDPSSPVLHELTFQAMSYDLLPIENDVYKYETSGIGEARVKEVLL DEDDDLWIALRHKHIAEVSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKKMPQY QKELSKYSTHLHLAEDCMKHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVPI LLDANVSTYDKIRIILLYIFLKNGITEENLNKLIQHAQIPPEDSEIITNMAHLGVPIVTDS TLRRRSKPERKERISEQTYQLSRWTPIIKDIMEDTIEDKLDTKHYPYISTRSSASFSTTA VSARYGHWHKNKAPGEYRSGPRLIIFILGGVSLNEMRCAYEVTQANGKWEVLIGST HILTPTKFLMDLRHPDFRESSRVSFEDQAPTME (Isoform A, SEQ ID NO: 1)

[0057] MAPIGLKAVVGEKIMHDVIKKVKKKGEWKVLVVDQLSMRMLSSCCKMT DIMTEGITIVEDINKRREPLPSLEAVYLITPSEKSVHSLISDFKDPPTAKYRAAHVFFTD SCPDALFNELVKSRAAKVIKTLTEINIAFLPYESQVYSLDSADSFQSFYSPHKAQMKN PILERLAEQIATLCATLKEYPAVRYRGEYKDNALLAQLIQDKLDAYKADDPTMGEGP DKARSQLLILDRGFDPSSPVLHELTFQAMSYDLLPIENDVYKYETSGIGEARVKEVLL DEDDDLWIALRHKHIAEVSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKKMPQY QKELSKYSTHLHLAEDCMKHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVPI LLDANVSTYDKIRIILLYIFLKNGITEENLNKLIQHAQIPPEDSEIITNMAHLGVPIVTDS TLRRRSKPERKERISEQTYQLSRWTPIIKDIMEDTIEDKLDTKHYPYISTRSSASFSTTA VSARYGHWHKNKAPGEYRSGPRLIIFILGGVSLNEMRCAYEVTQANGKWEVLIGST HILTPQKLLDTLKKLNKTDEEISS. (Isoform B, SEQ ID NO: 2)

[0058] STXBP1 participates in synaptic release of neurotransmitters by binding to Syntaxin 1 A and interacting with the SNARE complex directly at the synapse. Genetic mutations that result in loss-of-function variants of STXBP1 protein cause STXBP1 -related diseases, including developmental and epileptic encephalopathy (DEE), which is a group of epilepsies where the epileptic activity, seizures and underlying neurobiology contributes to cognitive and behavioral developmental impairments. STXBP1 -related DEE is a haploinsufficiency disorder usually caused by heterozygous de novo mutations in one of the alleles of the STXBP1 gene. Disease causing mutations are varied and range from nonsense, missense, alternative splicing mutations, large insertions, and deletions. Ohtahara syndrome, West syndrome, Lennox- Gastaut syndrome, Dravet syndrome, Rett syndrome, and other early infantile DEEs are diagnosed by mutations in the STXBP1 gene.

[0059] STXBP1 -related DEE is one of the most severe, frequent, and earliest forms of epilepsy. It is a monogenic DEE, with a predicted incidence of 1 / 26,000. The clinical spectrum includes intellectual disability, epilepsy, and movement disorders such as tremor and ataxia. Current treatments for STXBP1 -related DEE arc limited to seizure control. Additional therapies using micro-RNA blocking and AAV gene replacement therapy are being developed. However, there remains an unmet medical need for therapies and treatments that target the developmental aspects of STXBPl-related DEE diseases.STXBP1 SSOs

[0060] In certain embodiments, ASO's of the present disclosure are splice-switching oligonucleotides (SSOs) which include an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements of STXBP1 mRNA. In certain embodiments, the ASOs upregulate STXBP1 mRNA production. In certain embodiments, theASOs prevent the inclusion of one or more STXBP1 exons, hi certain embodiments, the ASOs encourage the inclusion of one or more STXBP1 exons. In certain embodiments, the ASOs encourage retention of one or more STXBP1 introns. In certain embodiments, the ASOs prevent retention of one or more STXBP1 introns.

[0061] In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in the exon 3-4 region of the STXBP1 gene. In certain embodiments, the ASOs target poison exons encoded in the exon 3-4 region of the STXBP1 gene. The poison exons and failed splicing events, if included in the final mRNA, can shift the open-reading frame of the RNA and introduce premature termination codons downstream. Preventing poison exons and failed splicing events can restore canonical splicing patterns at the site. In certain embodiments, the ASOs block inclusionary donor site attributes and / or prevent the inclusion of the non-productive cryptic exon near exon 4.

[0062] In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in the ENST0000637060 human STXBP1 transcript region in the exon 3-4 region of the STXBP1 gene. In certain embodiments, the ASO comprises an oligonucleotide sequence that can basepair to one or more pre-mRNA splicing regulatory elements encoded in the GRCh38 / hg38 / chr9:127654840-127655139 region ofthe STXBP1 gene (SEQ ID NO: 335). In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 335. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 335.

[0063] In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 336. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 336. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 337. hi certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 337. In certain embodiments, the ASO comprises an oligonucleotide sequence that can basepair to one or more regions in SEQ ID NO: 338. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 338. hi certain embodiments, the ASO comprises anoligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 339. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 339.

[0064] In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in the exon 18-19 region of the STXBP1 gene. In certain embodiments, the ASOs target decoy exons or failed splicing events encoded in the exon 18-19 region of the STXBP1 gene, including decoy exons in the terminal intron of the mRNA transcript between exon 18-19. Without being bound by theory, it is possible that canonical splicing sequences for both decoy and canonical splicing in the terminal intron between exon 18-19 of the STXBP1 gene can result in exon inclusionary and exclusionary events, such that SSOs can be used to modify the expression pattern towards canonical transcript to increase expression STXBP1 by blocking the decoy exon sites. The canonical splice donor and acceptor sites are strong, such that blocking the decoy exon sites is unlikely to change the splicing pattern. In certain embodiments, the ASOs block exclusionary donor site attributes near exon 18 and or promote exon 18 inclusion. In certain embodiments, the ASOs block inclusionary acceptor site attributes near exon 19 and / or promote exon 19 exclusion.

[0065] In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in the ENST0000373299 human STXBP1 transcript region in the exon 18-19 region of the STXBP1 gene. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in the ENST0000639953 human STXBP1 transcript region in the exon 18-19 region of the STXBP1 gene. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in the ENST00000373302 human STXBP1 transcript region in the exon 18-19 region of the STXBP1 gene. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in the GRCh38 / hg38 / chr9:127682536-127682658 region ofthe STXBP1 gene (SEQ ID NO: 340). In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 340. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 340. hi certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatoryelements encoded in the GRCh38 / hg38 / chr9: 127684296- 127684640 region of the SITCBPl gene (SEQ ID NO: 341). In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 341. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 341.

[0066] In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 342. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 342. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 343. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 343. hi certain embodiments, the ASO comprises an oligonucleotide sequence that can basepair to one or more regions in SEQ ID NO: 344. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 344. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more regions in SEQ ID NO: 345. In certain embodiments, the ASO comprises an oligonucleotide sequence that can base-pair to one or more pre-mRNA splicing regulatory elements encoded in SEQ ID NO: 345.

[0067] In certain embodiments, the ASO comprises a sequence having at least 80% identity to a sequence in Table 1. In certain embodiments, the ASO comprises a sequence having at least 85% identity to a sequence listed in Table 1. In certain embodiments, the ASO comprises a sequence having at least 90% identity to a sequence listed in Table 1. In certain embodiments, the ASO comprises a sequence having at least 95% identity to a sequence listed in Table 1. In certain embodiments, the ASO comprises a sequence listed in Table 1.TABLE 1 - ASO Sequences

[0068] In certain embodiments, the ASO comprises TGTGAGTAGAACCTGTGG (SEQ ID NO: 3). In certain embodiments, the ASO comprises AGCACCTCCCACTTTCCG (SEQ ID NO: 52). In certain embodiments, the ASO comprises AAGAGTGACGGAATAATC (SEQ ID NO: 13). In certain embodiments, the ASO comprises CAAAATGGTCAGCCACTT (SEQ ID NO: 43). In certain embodiments, the ASO comprises ATAGCACACATTCCAAAG (SEQ ID NO: 23). hi certain embodiments, the ASO comprises GAATGTGAGTAGAACCTG (SEQ ID NO: 4). In certain embodiments, the ASO comprises GAGGATCACCAGAGAGTG (SEQ ID NO: 72). In certain embodiments, the ASO comprises GTTCTGAAGGGCCATTCC (SEQ ID NO: 74). In certain embodiments, the ASO comprises any one of SEQ ID NOs: 3-100.

[0069] In certain embodiments, the ASO targets AAGTGGCTGACCATTTTG (SEQ ID NO: 101). hi certain embodiments, the ASO targets CTTTGGAATGTGTGCTAT (SEQ ID NO: 102). In certain embodiments, the ASO targets CGGAAAGTGGGAGGTGCT (SEQ ID NO: 103). In certain embodiments, the ASO targets CACTCTCTGGTGATCCTC (SEQ ID NO: 104). hi certain embodiments, the ASO targets GGAATGGCCCTTCAGAAC (SEQ ID NO: 105). hi certain embodiments, the ASO targets CCACAGGTTCTACTCACA (SEQ ID NO: 106). In certain embodiments, the ASO targets GATTATTCCGTCACTCTT (SEQ ID NO: 107). In certain embodiments, the ASO targets CAGGTTCTACTCACATTC (SEQ ID NO: 108).

[0070] In certain embodiments, the ASO comprises a sequence having at least 80% identity to a sequence in Table 2. In certain embodiments, the ASO comprises a sequence having at least 85% identity to a sequence listed in Table 2. In certain embodiments, the ASO comprises a sequence having at least 90% identity to a sequence listed in Table 2. In certain embodiments, the ASO comprises a sequence having at least 95% identity to a sequence listed in Table 2. In certain embodiments, the ASO comprises a sequence listed in Table 2.TABLE 2 - ASO Sequences (Chemically Modified)

[0071] In certain embodiments, the ASO comprises: / 52MOErT / * / i2MOErG / * / i2MOErT / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErT / * / i2MOEr A / * / i2MOErG / * / i2MOEr A / * / i2MOEr A / * / i2MOErC / * / i2MOErC / * / i2MOErT / * / i2MOErG / * / i2MOErT / * / i2MOErG / * / 32MOErG / (SEQ ID NO: 109).

[0072] In certain embodiments, the ASO comprises: / 52MOErA / * / i2MOErG / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErC / * / i2MOErT / * / i2MOErC / * / i2MOErC / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErT / * / i2MOErT / * / i2MOE rT / * / i2MOErC / * / i2MOErC / * / 32MOErG / (SEQ ID NO: 158).

[0073] In certain embodiments, the ASO comprises: / 52MOErA / * / i2MOErA / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErT / * / i2MOErG / * / i2MOErA / * / i2MOErC / * / i2MOErG / * / i2MOErG / * / i2MOErA / * / i2MOErA / * / i2MOErT / * / i2MOErA / * / i2MOErA / * / i2MOErT / * / 32MOErC / (SEQ ID NO: 119).

[0074] In certain embodiments, the ASO comprises: / 52MOErC / * / i2MOErA / * / i2MOErA / * / i2MOErA / * / i2MOErA / * / i2MOErT / * / i2MOErG / * / i2MOErG / * / i2MOErT / * / i2MOErC / * / i2MOErA / * / i2MOErG / * / i2MOErC / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErT / * / 32MOErT / (SEQ ID NO: 149).

[0075] In certain embodiments, the ASO comprises / 52MOErA / * / i2MOErT / * / i2MOErA / * / i2MOErG / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErA / * / i2MOErT / * / i2MOErT / * / i2MOErC / * / i2MOErC / * / i2MOErA / * / i2MOErA / * / i2MOErA / * / 32MOErG / (SEQ ID NO: 129).

[0076] In certain embodiments, the ASO comprises: / 52MOErG / * / i2MOErA / * / i2MOErA / * / i2MOErT / * / i2MOErG / * / i2MOErT / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErT / * / i2MOErA / * / i2MOErG / * / i2MOErA / * / i2MOErA / * / i2MOErC / * / i2MOErC / * / i2MOErT / * / 32MOErG / (SEQ ID NO: 110).

[0077] In certain embodiments, the ASO comprises / 52MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErG / * / i2MOErA / * / i2MOErT / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErC / * / i2MOErA / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErT / * / 32MOErG / (SEQ ID NO: 178).

[0078] In certain embodiments, the ASO comprises: / 52MOErG / * / i2MOErT / * / i2MOErT / * / i2MOErC / * / i2MOErT / * / i2MOErG / * / i2MOErA / * / i2MOErA / * / i2MOErG / * / i2MOErG / * / i2MOErG / * / i2MOErC / * / i2MOErC / * / i2MOErA / * / i2MOErT / * / i2MOErT / * / i2MOErC / * / 32MOErC / (SEQ ID NO: 180).

[0079] In certain embodiments, the ASO comprises any one of SEQ ID NOs: 109-206.26

[0080] In certain embodiments, the ASO comprises a sequence having at least 80% identity to a sequence in Table 3 or Table 4. In certain embodiments, the ASO comprises a sequence having at least 85% identity to a sequence listed in Table 3 or Table 4. In certain embodiments, the ASO comprises a sequence having at least 90% identity to a sequence listed in Table 3 or Table 4. In certain embodiments, the ASO comprises a sequence having at least 95% identity to a sequence listed in Table 3 or Table 4. In certain embodiments, the ASO comprises a sequence listed in Table 3 or Table 4.TABLE 3 — ASO SequencesTABLE 4 — ASO Sequences

[0081] In certain embodiments, the ASO comprises ATGTGAGTAGAACCTGTG (SEQ ID NO: 207). In certain embodiments, the ASO comprises GAATGTGAGTAGAACCTG (SEQ ID NO: 209). In certain embodiments, the ASO comprises AGAATGTGAGTAGAACCT (SEQ ID NO: 210). In certain embodiments, the ASO comprises TAAGAATGTGAGTAGAAC (SEQ ID NO: 212). In certain embodiments, the ASO comprises GAGTAAGAATGTGAGTAG (SEQ ID NO: 215). In certain embodiments, the ASO comprises GGAGTAAGAATGTGAGTA (SEQ ID NO: 216). In certain embodiments, the ASO comprises CTCAGGTCCATGAGAAAT (SEQ ID NO: 222). In certain embodiments, the ASO comprises TCTCAGGTCCATGAGAAA (SEQ ID NO: 223). In certain embodiments, the ASO comprises GTCTCAGGTCCATGAGAA (SEQ ID NO: 224). In certain embodiments, the ASO comprises TGTCTCAGGTCCATGAGA (SEQ ID NO: 225). hi certain embodiments, the ASO comprises AATGTGAGTAGAACCTGT (SEQ ID NO: 249). In certain embodiments, the ASO comprises ATGTGAGTAGAACCTGTG (SEQ ID NO: 250). In certain embodiments, the ASO comprises GAGGCCGGCTGAACGAGG (SEQ ID NO: 260). In certain embodiments, the ASO comprises AGGCCGGCTGAACGAGGC (SEQ ID NO: 261). In certain embodiments, the ASO comprises GGACACGCCAACGGAGGC (SEQ ID NO: 269). In certain embodiments, the ASO comprises GACACGCCAACGGAGGCC (SEQ ID NO: 270).

[0082] In certain embodiments, the ASO comprises any one of SEQ ID NOs: 207-270.

[0083] In certain embodiments, the ASO comprises a sequence having at least 80% identity to a sequence in Table 5 or Table 6. In certain embodiments, the ASO comprises a sequence having at least 85% identity to a sequence listed in Table 5 or Table 6. In certain embodiments, the ASO comprises a sequence having at least 90% identity to a sequence listed in Table 5 or Table 6. hi certain embodiments, the ASO comprises a sequence having at least 95% identity to a sequence listed in Table 5 or Table 6. hi certain embodiments, the ASO comprises a sequence listed in Table 5 or Table 6.TABLE 5 — ASO Sequences (Chemically Modified)TABLE 6 — ASO Sequences (Chemically Modified)

[0084] In certain embodiments, the ASO comprisesZ52MOErA / *Zi2MOErGZ*Zi2MOErAZ* Zi2MOErA / *Zi2MOErTZ*Zi2MOErGZ*Zi2MOErTZ*Zi2MOErGZ*Zi2MOErAZ* Zi2MOErGZ*Zi2MOErTZ*Zi2MOErAZ*Zi2MOErGZ*Zi2MOErA / *Zi2MOErAZ* Zi2MOErCZ*Zi2MOErCZ*Z32MOErTZ (SEQ ID NO:274).

[0085] In certain embodiments, the ASO comprisesZ52MOErTZ*Zi2MOErAZ*Zi2MOErAZ* Zi2MOErGZ*Zi2MOErA / *Zi2MOErAZ*Zi2MOErTZ*Zi2MOErGZ*Zi2MOErTZ* Zi2MOErGZ*Zi2MOErAZ*Zi2MOErGZ*Zi2MOErTZ*Zi2MOErAZ*Zi2MOErGZ* Zi2MOErAZ*Zi2MOErAZ*Z32MOErCZ (SEQ ID NO:276).

[0086] In certain embodiments, the ASO comprisesZ52MOErGZ*Zi2MOErGZ*Zi2MOErAZ* Zi2MOErGZ*Zi2MOErTZ*Zi2MOErAZ*Zi2MOErA / *Zi2MOErGZ*Zi2MOErAZ* Zi2MOErAZ*Zi2MOErTZ*Zi2MOErGZ*Zi2MOErTZ*Zi2MOErGZ*Zi2MOErAZ* Zi2MOErGZ*Zi2MOErTZ*Z32MOErAZ (SEQ ID NO:280).

[0087] In certain embodiments, the ASO comprisesZ52MOErA / *Zi2MOErAZ*Zi2MOErTZ* Zi2MOErGZ*Zi2MOErTZ*Zi2MOErGZ*Zi2MOErA / *Zi2MOErGZ*Zi2MOErTZ* Zi2MOErAZ*Zi2MOErGZ*Zi2MOErAZ*Zi2MOErA / *Zi2MOErCZ*Zi2MOErCZ* Zi2MOErTZ*Zi2MOErGZ*Z32MOErTZ (SEQ ID NO:313).

[0088] In certain embodiments, the ASO comprisesZ52MOErAZ*Zi2MOErTZ*Zi2MOErGZ* Zi2MOErTZ*Zi2MOErGZ*Zi2MOErAZ*Zi2MOErGZ*Zi2MOErTZ*Zi2MOErAZ* Zi2MOErGZ*Zi2MOErA / *Zi2MOErAZ*Zi2MOErCZ*Zi2MOErCZ*Zi2MOErTZ* Zi2MOErGZ*Zi2MOErTZ*Z32MOErGZ (SEQ ID NO:314).

[0089] In certain embodiments, the ASO comprisesZ52MOErGZ*Zi2MOErAZ*Zi2MOErGZ* Zi2MOErGZ*Zi2MOErCZ*Zi2MOErCZ*Zi2MOErGZ*Zi2MOErGZ*Zi2MOErCZ* Zi2MOErTZ*Zi2MOErGZ*Zi2MOErAZ*Zi2MOErA / *Zi2MOErCZ*Zi2MOErGZ* Zi2MOErAZ*Zi2MOErGZ*Z32MOErGZ (SEQ ID NO:324).

[0090] In certain embodiments, the ASO comprisesZ52MOErAZ*Zi2MOErGZ*Zi2MOErGZ* Zi2MOErCZ*Zi2MOErCZ*Zi2MOErGZ*Zi2MOErGZ*Zi2MOErCZ*Zi2MOErTZ* Zi2MOErGZ*Zi2MOErAZ*Zi2MOErAZ*34 / i2MOErC / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErG / * / 32MOErC / (SEQ ID NO: 325).

[0091] In certain embodiments, the ASO comprises / 52MOErG / * / i2MOErG / * / i2MOErA / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErG / * / i2MOErC / * / i2MOErC / * / i2MOErA / * / i2MOErA / * / i2MOErC / * / i2MOErG / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErG / * / 32MOErC / (SEQ ID NO:333).

[0092] In certain embodiments, the ASO comprises / 52MOErG / * / i2MOErA / * / i2MOErC / * / i2MOErA / * / i2MOErC / * / i2MOErG / * / i2MOErC / * / i2MOErC / * / i2MOErA / * / i2MOErA / * / i2MOErC / * / i2MOErG / * / i2MOErG / * / i2MOErA / * / i2MOErG / * / i2MOErG / * / i2MOErC / * / 32MOErC / (SEQ ID NO:334).

[0093] In certain embodiments, the ASO comprises any one of SEQ ID NOs: 271-334. Methods for Treating Encephalopathies by Restoring STXBP1

[0094] In certain embodiments, the present disclosure describes methods for modifying the expression of Syntaxin binding protein 1 (STXBP1), comprising delivering an effective amount of an ASO of the present disclosure to a cell comprising the STXBP1 gene. In certain embodiments, the ASO binds to a pre-mRNA splicing regulatory element encoded in the STXBP1 gene. In certain embodiments, the splicing regulatory element is for an exon splicing enhancer (ESE). In certain embodiments, the splicing regulatory element is for an exon splicing silencer (ESS). In certain embodiments, the splicing regulatory element is for an intron splicing enhancer (ISE). In certain embodiments, the splicing regulatory element is for an intron splicing silencer (ISS).

[0095] In certain embodiments, delivery of the ASO to the cell results in an increase in STXBP1 production by the cell. In certain embodiments, delivery of the ASO to the cell results in an increase from 105-130% in STXBP1 production by the cell. In certain embodiments, delivery of the ASO to the cell results in an increase of at least 105% in STXBP1 production by the cell. In certain embodiments, delivery of the ASO to the cell results in an increase of at least 110% in STXBP1 production by the cell. In certain embodiments, delivery of the ASO to the cell results in an increase of at least 115% in STXBP1 production by the cell. In certain embodiments, delivery of the ASO to the cell results in an increase of at least 120% in STXBP1 production by the cell. In certain embodiments, delivery of the ASO to the cell results in an increase of at least 125% in STXBP1 production by the cell.

[0096] In certain embodiments, the present disclosure describes methods the treating STXBP1 -related diseases in a subject in need thereof. In certain embodiments, the present disclosure describes methods the treating a developmental and epileptic encephalopathy (DEE) in a subject in need thereof. In certain embodiments, the DEE is Ohtahara syndrome, West syndrome, Lennox- Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE. In certain embodiments, the encephalopathy is caused by, or associated with, one or more missense mutations, nonsense mutations, deletions, inversions, insertions, duplications, frameshift mutations, repeat expansions, haploinsufficiencies, or a combination thereof associated with a STXBP1 gene.

[0097] In certain embodiments, the present disclosure describes methods for treating STXBP1 -related diseases in a subject in need thereof, the method comprising administering a therapeutically effective amount an ASO of the present disclosure to the subject. In certain embodiments, the method comprises administering a therapeutically effective amount of a composition comprising an ASO of the present disclosure to the subject. In certain embodiments, administration of the ASO to the subject increases the level of a STXBP1 produced by the subject. In certain embodiments, administration of the ASO to the subject restores the level of STXBP1 in the subject to a level found in a healthy subject (i.e., an individual that does not have an STXBP1 haploinsufficiency or mutation). In certain embodiments, administration of the ASO to the subject reduces or eliminates one or more symptom of the DEE being treated. In certain embodiments, administration of the ASO to the subject restores one or more cognitive abilities in the subject. In certain embodiments, administration of the ASO to the subject reduces the number, frequency, and or severity of seizures in the subject. In certain embodiments, administration of the ASO to the subject improves and / or restores one or more motor functions in the subject. In certain embodiments, administration of the ASO to the subject improves and / or restores one or more psychiatric functions in the subject.EMBODIMENTS

[0098] Certain embodiments of the present disclosure may also be defined according to any one of the following exemplary, non-limiting embodiments:

[0099] Embodiment 1. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% sequence identity to any one of SEQ ID NOs: 3-100.

[0100] Embodiment 2. The ASO of Embodiment 1, wherein the target binding sequence has at least 85% sequence identity to any one of SEQ ID NOs: 3-100; optionally at least 90% sequence identity; optionally at least 95% sequence identity.

[0101] Embodiment 3. The ASO of Embodiment 1, wherein the target binding sequence comprises any one of SEQ ID NOs: 3-100.

[0102] Embodiment 4. The ASO of Embodiment 1, wherein the target binding sequence comprises any one of SEQ ID NOs: 3, 4, 13, 23, 43, 52, 72, or 74.

[0103] Embodiment 5. The ASO of Embodiment 1, wherein the target binding sequence comprises SEQ ID NO: 3 or SEQ ID NO: 52.

[0104] 6. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% sequence identity to any one of SEQ ID NOs: 207-270.

[0105] Embodiment 7. The ASO of Embodiment 6, wherein the target binding sequence has at least 85% sequence identity to any one of SEQ ID NOs: 207-270; optionally at least 90% sequence identity; optionally at least 95% sequence identity.

[0106] Embodiment 8. The ASO of Embodiment 6, wherein the target binding sequence comprises any one of SEQ ID NOs: 207-270.

[0107] Embodiment 9. The ASO of Embodiment 6, wherein the target binding sequence comprises any one of SEQ ID NOs: 207, 209, 210, 212, 215, 216, 222, 223, 224, 225, 249, 250, 260, 261, 269, or 270.

[0108] Embodiment 10. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% sequence complementarity to a target sequence in: (i) Exon 3 (SEQ ID NO: 336), Intron 3 (SEQ ID NO: 337), Exon 4 (SEQ ID NO: 338), or Intron 4 (SEQ ID NO: 339) of human STXBP1 gene; or (ii) Intron 17 (SEQ ID NO: 342), Exon 18 (SEQ ID NO: 343), Intron 18 (SEQ ID NO: 344), or Exon 19 (SEQ ID NO: 345) of human STXBP1 gene, or a combination thereof.

[0109] Embodiment 11. The ASO of Embodiment 10, wherein the target sequence is in Exon 3 (SEQ ID NO: 336), Intron 3 (SEQ ID NO: 337), Exon 4 (SEQ ID NO: 338), or Intron 4 (SEQ ID NO: 339) of human STXBP1 gene.

[0110] Embodiment 12. The ASO of Embodiment 10, wherein the target sequence is encoded in GRCh38 / hg38 / chr9: 127654840-127655139 (SEQ ID NO: 335).[OHl] Embodiment 13. The ASO of Embodiment 10, wherein the target sequence is in Intron 17 (SEQ ID NO: 342), Exon 18 (SEQ ID NO: 343), Intron 18 (SEQ ID NO: 344), or Exon 19 (SEQ ID NO: 345) of human STXBP1 gene.

[0112] Embodiment 14. The ASO of Embodiment 10, wherein the target sequence is encoded in GRCh38 / hg38 / chr9: 127682536-127682658 (SEQ ID NO: 340).

[0113] Embodiment 15. The ASO of Embodiment 10, wherein the target sequence is encoded in GRCh38 / hg38 / chr9: 127684296-127684640 (SEQ ID NO: 341).

[0114] Embodiment 16. The ASO of Embodiment 10, wherein the target sequence is selected from any one of SEQ ID NOs: 101-108.

[0115] Embodiment 17. The ASO of Embodiment 10, wherein the target sequence is SEQ ID NO: 106 or SEQ ID NO: 103.

[0116] Embodiment 18. The ASO of any one of Embodiments 10-17, wherein the target binding sequence has at least 85% sequence complementarity to the target sequence; optionally at least 90% sequence complementarity; optionally at least 95% sequence complementarity: optionally 100% sequence complementarity.

[0117] Embodiment 19. The ASO of any one of Embodiments 1-18, wherein the ASO comprises one or more modifications to one or more intemucleotide phosphodiester linkages in the ASO; optionally wherein the ASO comprises one or more modifications to all intemucleotide phosphodiester linkages in the ASO.

[0118] Embodiment 20. The ASO of Embodiment 19, wherein the ASO comprises a phosphorothioate (PS) modification to one or more intemucleotide phosphodiester linkages in the ASO; optionally wherein the ASO comprises a phosphorothioate (PS) modification to all intemucleotide phosphodiester linkages in the ASO.

[0119] Embodiment 21. The ASO of any one of Embodiments 1-20, wherein the ASO comprises one or more modifications to one or more nucleoside sugar moiety in the ASO; optionally wherein the ASO comprises one or more modifications to all nucleoside sugar moieties in the ASO.

[0120] Embodiment 22. The ASO of Embodiment 21, wherein the ASO comprises a 2'-O- Methoxyethyl (2'-MOE) modification to one or more nucleoside sugar moiety in the ASO; optionally wherein the ASO comprises a 2'-O-Methoxyethyl (2'-MOE) modification to all nucleoside sugar moieties in the ASO.

[0121] Embodiment 23. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% identity to any one of SEQ ID NOs: 109-206.

[0122] Embodiment 24. The ASO of Embodiment 23, wherein the target binding sequence has at least 85% sequence identity to any one of SEQ ID NOs: 109-206; optionally at least 90% sequence identity; optionally at least 95% sequence identity.

[0123] Embodiment 25. The ASO of Embodiment 23, wherein the target binding sequence comprises any one of SEQ ID NOs: 109-206.

[0124] Embodiment 26. The ASO of Embodiment 23, wherein the target binding sequence comprises any one of SEQ ID NOs: 109, 110, 119, 129, 149, 158, 178, and 180.

[0125] Embodiment 27. The ASO of Embodiment 23, wherein the target binding sequence comprises SEQ ID NO: 109 or SEQ ID NO: 158.

[0126] Embodiment 28. The ASO of any one of Embodiments 1-27, wherein the target binding sequence comprises 15-25 nucleotides; optionally 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; optionally 18 nucleotides.

[0127] Embodiment 29. A pharmaceutical composition comprising the ASO of any one of Embodiments 1-28, and a pharmaceutically acceptable excipient.

[0128] Embodiment 30. A method of modifying the expression of Syntaxin binding protein 1 (STXBP1), comprising delivering an effective amount of an ASO of any one of Embodiments 1-28 to a cell comprising the STXBP1 gene.

[0129] Embodiment 31. The method of Embodiment 30, wherein the ASO binds to a pre- mRNA splicing regulatory element encoded in the STXBP1 gene; optionally wherein the splicing regulatory element is for an exon splicing enhancer (ESE), an exon splicing silencer (ESS), an intron splicing enhancer (ISE), or an intron splicing silencer (ISS).

[0130] Embodiment 32. The method of Embodiment 30 or Embodiment 31, wherein delivery of the ASO to the cell results in an increase in STXBP1 production by the cell; optionally an increase of at least 110% in STXBP1 production by the cell; optionally an increase of at least 115% in STXBP1 production by the cell; optionally an increase of at least 120% in STXBP1 production by the cell.

[0131] Embodiment 33. A method of treating a disease related to a STXBP1 gene mutation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising an ASO of any one of Embodiments 1-28 or a pharmaceutical composition of claim 29

[0132] Embodiment 34. The method of Embodiment 33, wherein the disease related to a STXBP1 gene mutation is a developmental and epileptic encephalopathy (DEE); optionally Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE.

[0133] Embodiment 35. The method of Embodiment 33 or Embodiment 34, wherein administering the composition to the subject reduces or eliminates one or more symptom of the disease; optionally wherein administering the composition to the subject results in one ormore of: (i) restores one or more cognitive abilities in the subject, (ii) reduces the number, frequency, and / or severity of seizures in the subject, (iii) improves and / or restores one or more motor functions in the subject, and (iv) improves and / or restores one or more psychiatric functions in the subject.

[0134] Embodiment 36. An ASO according to any one of Embodiments 1-28, or a pharmaceutical composition according to embodiment 29, for use in the prevention and / or treatment of a disease related to a STXBP1 gene mutation.

[0135] Embodiment 37. The ASO or pharmaceutical composition for use according to Embodiment 36, wherein the disease related to a STXBP1 gene mutation is a developmental and epileptic encephalopathy (DEE); optionally Ohtahara syndrome, West syndrome, Lennox- Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE.

[0136] Embodiment 38. Use of an ASO according to any one of Embodiments 1-28 or a pharmaceutical composition according to Embodiment 29 for the treatment of a disease related to a STXBP1 gene mutation.

[0137] Embodiment 39. Use of an ASO according to any one of Embodiments 1-28 or a pharmaceutical composition according to Embodiment 29 for the manufacture of a medicament for the treatment of a disease related to a STXBP1 gene mutation.

[0138] Embodiment 40. The use according to Embodiment 38 or Embodiment 39, wherein the disease related to a STXBP1 gene mutation is selected from a developmental and epileptic encephalopathy (DEE); optionally Ohtahara syndrome, West syndrome, Lennox- Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE.EXAMPLESExample 1: Antisense Oligonucleotide (ASO) design

[0139] Potential poison / decoy exon sequences were identified in: (i) the exon 3-4 region, including exon 3, intron 3, exon 4, and intron 4 (41439..47753 of STXBP1) and (ii) the exon 18-19 region, including intron 17, exon 18, intron 18, and exon 19 (67967..80423 of STXBP1) in the human STXBP1 gene. STXBP1 transcript regions ENST00000373302, ENST0000373299, ENST0000637060, and ENST0000639953 were specifically identified for ASO targeting.

[0140] 98 antisense oligonucleotides (ASOs) were designed to target splicing regulatory elements in the exon 3-4 region (including ENST0000637060) and the in the exon 18-19 region (including ENST00000373302, ENST0000373299, and ENST0000639953). AllASOs were designed as 18-mers with full 2'-O-methoxyethyl (2'-MOE) modification and phosphorothioate intemucleoside linkage.Example 2: STXBP1 ASO in vitro screeningOverview

[0141] 98 ASOs were synthesized as full MOE, 18-mers through IDT (IntegratedTechnologies, Inc.). Commercial GABAergic Neurons (average TPM = 252) were purchased through FujiFilm Cellular Dynamic. Commercial STXBP1 and housekeeping gene qPCR assays were purchased through IDT. Neurons were seeded at 40,000 cells per well (96-well plate) and treated (free-uptake) 3 days post-thaw. 50% media changes were done every 3 days. Cells were pulled on day 11 post-treatment.

[0142] qPCR data was analyzed using the ddCq method. The STXBP1 assay used for this screen had primers spanning exons 5-7. The housekeeping gene used in this screen was HPRT1. The qPCR reaction was carried out in duplex. Data points are derived from 2 biological duplicates and 4 technical replicates.

[0143] LDH-glo analysis was also complete using 2 biological duplicates and 2 technical replicates.ASO Synthesis

[0144] Antisense Oligonucleotides (ASOs) of SEQ ID NOs: 3-100 were synthesized by IDT (Integrated Technologies, Inc.), and supplied at a final quantity of 50 nmol with standard desalting procedures. ASOs were reconstituted to 200 uM in 0.1 x sterile PBS for the study. Media & Components

[0145] iCell Neural Base Medium 1 (FujiFilm Cellular Dynamics #M1010); iCell Neural Supplement A (FujiFilm Cellular Dynamics #M1032); Maintenance medium protocol- 100 mL of iCell Neural Base Medium 1 and 2 mL of iCell Neural Supplement A were mixed together and stored at 4 °C for up to 3 weeks. Medium was equilibrated to room temperature before use.Cell Culture Surface Coating protocol

[0146] For a 96-well plate, 50 uL of 0.01% PLO solution was added to each well and incubated at 37 °C for at least 1 hour, washed 3 times with 200 uL ddH20 and the plate was allowed to dry for 10-20 minutes, laminin was diluted to 3.3 ug / mL in DPBS and 50 uL was added to each well and incubated at 37 °C for at least 1 hour or 4 °C overnight (if incubated at 4 °C, it was then incubated at 37 °C for one hour before use). Laminin was aspirated when cells were ready to be plated.Thawing & Plating

[0147] iCell GABA neurons, 01434 (IPS cell-derived GABAergic inhibitory neurons) were purchased from Fuji Film Cellular Dynamics #C1012.

[0148] Maintenance medium was equilibrated to room temperature and vials were thawed at 37 °C (-3-5 minutes). Cells were gently transferred to a 50 mL conical tube dropwise while swirling the tube, and the vial was rinsed with 1 mL media and gently transferred to the same tube (dropwise and swirling). 8 mL of media was gently added to the same tube (dropwise and swirling). Cells were counted using a hemocytometer and trypan blue exclusion. Cells were then diluted to 40,000 cells per well in a 96 well plate (125,000 viable cells / cm2), laminin solution was aspirated from plates, and cells were dispensed into the empty precoated wells. Plates were then incubated at 37 °C for 24 hours.ASOFree Uptake

[0149] GABAergic Neurons were treated with 20uM of ASOs at day 3 post-thaw in biological duplicate. 50% media changes and observation of cells under the microscope were done every 3 days. RNA was extracted on day 14 post-thaw (day 11 post-treatment). FIG. 2 shows images of healthy GABA-ergic neurons cells at days 6, 9, and 12. mRNA analysis

[0150] Target mRNA levels were detected using the QuantStudio7 Pro with primers in duplex using AgPath-ID One-Step RT-PCR (ThermoFisher #4387391). The samples were normalized to the housekeeping gene HPRT1. The ddCq method was used to calculate %Control (STXBP1).Results

[0151] Results from the STXBP1 ASO in vitro screening of Example 2 are shown in FIG. 3A and FIG. 3B (ASOs 1-49), and FIG. 3C and FIG. 3D (ASOs 50-98).

[0152] Screening results identified three ASOs which had STXBP1 mRNA levels more than 120% of the untreated control (UTC) 168917, 168966, and 168976. Five ASOs were also identified which had STXBP1 mRNA levels from 110-119% of the control 168937, 168939, 168967, 168986, and 169007. 168996 showed STXBP1 mRNA levels of about 109.1% of control, 169007 showed STXBP1 mRNA levels of about 108.8% of control, and sixteen other ASOs were identified which had STXBP1 mRNA levels from 100-109% of the control. Additional, thirty-six ASOs showed 90-99% of the control, thirty-five ASOs showed 80-89% of the control, and one ASOs showed 70-79% of the control.

[0153] A summary of the eight sequences which had STXBP1 mRNA levels more than 110% of the untreated control (UTC) is provided in Table 7.TABLE 7 — ASOs with more than 110% STXBP1 mRNA of the control

[0154] Based on splicing models used to design the initial 98 ASOs for screening: (i) ASOs 168986 and 169006 potentially block inclusionary donor site attributes and prevent inclusion of a non-productive cryptic exon near exon 4 (ENST00000637060 track); (ii) ASO 168917 potentially blocks exclusionary donor site attributes and potentially promotes exon 18 inclusion (ENST00000373299 track); (iii) ASOs 168937 and 168939 potentially block acceptor site inclusionary attributes and potentially promote exon 19 exclusion (ENST00000637953 track); (iv) ASOs 168967 and 168966 possibly block inclusionary acceptor site attributes and potentially promote exon 19 exclusion (ENST00000373302 track); and (v) ASO 168976 potentially blocks inclusionary donor site attributes and potentially promotes exon 19 exclusion (ENST00000373302 track).Example 3: STXBP1 ASO 168966 in vitro microwalk screeningOverview

[0155] ASO ID 168966 (SEQ ID NO: 3) targets CCACAGGTTCTACTCACA (SEQ ID NO: 106) on Exon 19 (ENST00000373302), and was selected for a microwalk study. 64 ASOs (SEQ ID NOs: 207-270) were synthesized and tested according to the protocols of Example 2. The mouse ASO microwalk screen (Table 4) was performed in neuroblastoma cells (Neuro-2a). Cells were electroporated at 200V, 20,000 cells / well, 24 hour incubation, and the samples were normalized to the housekeeping gene Actnlb.Results

[0156] Results from the STXBP1 ASO in vitro microwalk screening of Example 3 are shown in FIG. 4A, FIG. 4B, and FIG. 4C.SEQUENCESGRCh38 / hg38 / chr9: 127654840-127655139 (Exon 3-4)

[0157] actggggtagcgcttatttagacatttgccttatggttatttgtatgtttatgttttattcacgctttggaatgtgtgctataacttt tggtgaataataaggagcaggattgctggatcacatggtaagtatgcttagttttgtaagatactgccaaaccatttttcaaagtggctgac catttgcattcccagcagtagtgaatgagaatcctgttgctccacatccttaccagcatttggagttgtcagtgttctgaattttgagcattc taccagctgtgtggtggtatggtat (SEQ ID NO: 335)Exon 3 (41439..41520) - ENSE00000927170

[0158] GUGCUGGUGGUGGAUCAGUUAAGCAUGAGGAUGCUGUCCUCCUGCU GCAAGAUGACAGACAUCAUGACCGAGGGCAUAACGA (SEQ ID NO: 336)Intron 3 (41521..46098)

[0159] gugagcaugcgcucugcacuugagcagugcuggagccuucucucuguggcgggcuuagcucuuuccuau ucauuauuaucuccuuugauuuuagauucuuccagcaaccugugugcuaagaccagaagcuccugagaggcgcaaggcu aucuuguucaucacugaaucccuagcaccaaauccagggcuagaugcucauucaaugucuguuaaaggaaugccaaaaga aggguuaaaucaagaaagaaaaggcagcccacccaauagaaaaaaguucugagcaggcacuuaacaaaagaagaaauccaa auggccaaucaaucuaugaaaauuguccacugucauuaguggucagggaaaugcaauucaaaccagagugagaugccga uauagauucaaaauauuagaauauuuaaucucugauaaugccaagugacggugaggagguagaaaaauggaaacuccua cgugauggaaugucugcugguguagccccuuugaaaaccacuuggcagugucuaguaaaguugaagguaugcaugucc ugugagccagcaagucuguggucagguguguguccuagacaaccucagguaugugugcccaggauacaugugcaggaau acaugugcaguguugcucauagguuccccaaguggaaaauaaccaaaacguccaucaaguguagaaugguuaaaucagu ugcgugcaauuacacaguagaauacucuacagggaugaaaaugggcgagcccugagcgcucacaguaacaugaaugcauc ugaugaacaacggugagcaaagacagcaagacacgaaagaccacugauagugagccuucauuccacagaguucagaccgc agagcucagcuguguuguuuuaaaaugcagauguaagggguaaaaccucaagagaagagcucaccauucaagggaggau aaugauucuguuugcggggaagagaggaaaugguguugauagaggggaaugugagcuggagccagggcacuuuugugg ugcugauuggguuucguuucuuaacugggguagcgcuuauuuagacauuugccuuaugguuauuuguauguuuaugu uuuauuc acgcuuuggaaugugugcuauaacuuuuggugaauaauaaggagc aggauugcuggaucac augguaagua ugcuuaguuuuguaagauacugccaaaccauuuuucaaaguggcugaccauuuugcauucccagcaguagugaaugaga auuccuguugcuccacauccuuaccagcauuuggaguugucaguguucugaauuuugagcauucuaccagcugugugg ugguaugguauuguuguuuuaauuugcauuucccugaugaccuaggauguggagcaucuuuucacaugcuuauuugcc aucuguauauuguuuuuugguacagugucuguaaggucuuuggcccguuuuuuaauuggguuauuuguuuucuuauu guugaauuuuaagggauauuuguauauuuuggauaacaguucuuuaucagauaugucuuuuggaaauauuugcccuca gucuguggcuugucuuuucauucucuugauggcaucuugcacagagcaguacuuuuuaauuuuaaugaaguccagcuu aucaauucuuguuuucauggauugucacuuuggugucauaucuaaaaagucauugccaaauccaaggacaccuagcuuu ucuuuuauguaauccucuaggaauuuuauaauuucgcauuuuacauuuaggucugugauccauuuugaguuaacauuucuaaaggguauaauguaugugucuguauucauuuugguugcacguggaugugauuccuucauuuuuaaaaaacagauc c aguauaucuaucagc agaagcac aguccuguaaauggcauac acaguuuuuuuguuuguuuguuuguuugagacaga gucuugcugucucccaagcuggagggcaguggcgcaaucucagcucaccacaaccucugccuccuggguucaagggauu auccugccucagccuccuaaguagcugggauuacaggcaccugccaucacgccuggcuaauuuuuguauuuuuaguaga gacaggguuucaccauguuggcuaggcuggucucgaacuccugaccucaagugauccaccugccucggccucccaaagu ggugggauuacaggagugagccacugcacuuggcacacauacacaauuuuuuaugugacuuuuaugucauuuagugacc uaagucaguguguuuuuuguuuguuuguuuuugagacagagucuugcucuguugcccaggauggaguauaguggugc aaucuugacucacugcaaacucugccucugggauucgggcaauucucgugccucagccucccgaguagcugggauuaca ggcaggcgccagcacacccggcuauuuucuuucuuuuuuuuuuuuuuuaguagagacaggguuucaccauguuggcca agcuggucuugaacuccuggccccaaguuaucugcucgccuuggcuucccaaagugcugggauuacagacgugagcuac ugugcccugccccaagucaguuuuaagacuaggcuugccccuauuuugaguuuaugauguuauuuucuucauugaauu uuugaaacuauuuuuuucgucuauc aaagc aaaaacagacaaaaaac ac aaaaguaaaggagaaaaac acuaaauggagcu aacaaggaucuaagaaaagagcagugaccuccuguccuccuaucucccauccucggggucuauuaccccagaggcagcca ccucgcccuucauucuggacaucuuuuuuauuuuucuacauaaaugcuuguucuauuuucucuugauucagugauauu agauauuauuaacugacuuccugugggagauggggauucagagcucuccacuauacacagacucuugccuacccccaaaa gugguuuuuaaaaaauguccuuauggccgggcaugguggcucacaccuguaaucccagcacuuugggaggcagaggcag gcggcucacaaggucaggaauuugagaccagccuguccaacagagugaaacccugucucuacuaaaaauacaaaaauuag ccaagcauggucgcacgugccuguagucccagcuacucaggaggcugaggugggagaaucacuugaaccugggaggugg agguugcagugagccaagauugugccacugcacuccagccugggcgacagagcaagacugucucaaaaagaaaaagaaag aaagaaggaaagaaaaaguguucuuacuuaacaaaauuuaaaaguuagcaacccuauguugaagcucugaaacauuuugg ggggagauauuaguggccuaugaaauuaaacaaaucuauaaccacugguaaaguagauguuuggggauuuuugguauu aauuugaacauuauuuaaggugaaagucuuuauaaauauuccaagucuuuauaaauguuacaauccucuaaagugaugc uuuuaucugcuuaaaugaucugcauagaaagaauggcaccuguauaauugccuugcucucucaaugccagaggacagua gguaacacauuuuaagcauuaccgggugauugguaagauucucccaaaagcuguucuuuccaggugaagucccugacau aacuguagaggaaac aagc aaacc aaagc cuaugguuuguuuuaacc aaauagaacauuuaaucuuugc acuauugaagg aagcaauauuguauagagaaugugguuggaaugcccucauuugauaugcagaggagagcaaugaaacuauccagcuguu gauaaauaaacccaacauggaaauuccaaaggaauugggauuaucuagggcagaugagacaaagcugaagaagaggcaaa ggacccauucuugacagaauuagauaucauuuuccauuuucucucagaacagaucaaugaauuuacuuaaacugaaaug ggaagugagaacuaucuagugugaagauuucagauuuuucuuuguuguuguuuuugucguuguuuuugagauggagu cucacucugucacccaggcuggagugcagugguacuaucuuggcuuacugcagcuuccaccucccagguucaaucgauu cucccaccucagccuccugaguagcuggaauuauaggcacauuccaacaugccuggcuaauuuuuguaguuuuuuagua gagaugagguuucaccauguuggccaggcuggucuugaacuccugacaucaagugaugugcccaccucagccucccaaa gugcugggauuacaggcaugagccaccaugcccagcccagauuucagauucuaacaucuugggcauguugagaccaugg aauucacucccaaggaacgugguaaccuuaaggaaugagugaaugauuccccccggucccccacuguagauauuuaucugcaacagucugaagguagccugucuauauugcuucugguuuuauggucacccaucauguuuucuuauucacuguggguu guuuuguugucuag (SEQ ID NO: 337)Exon 4 (46099..46175) ENSE00000927171

[0160] UUGUGGAAGAUAUCAAUAAGCGCAGAGAGCCGCUCCCCAGCCUGGAGGCUGUGUAUCUCAUCACUCCAUCCGAGAAG (SEQ ID NO: 338)Intron 4 (46176..47753)

[0161] guaaaccuuccaccagaggaggacucuggcuaagguuagauggacuggcuugggauuuauguggcacaga cugucugacauugcuagguuaagcacgcauuuaacacacugcucuguuuaagcaugcaauccacaaugggguggggucg ggggugguguuuaugcagugagaagugaugaauguauuaagcguucuuaaacacagcagcugugcagaaaaccagagcc uguguuccguggcccuggaucauucuaguuuucccccuugcacucccucuucagguaggacccacucugucuaauuagc aguccaccuccagacuuugguauuuuaugcuuucauuuuauuuuaucaauauccaguuacuguucacauuuaccugucu uauaauuuuggagguuacuucaaaucacuauccaaacaagguccaacuccuugcuucuggguaauaguugucuuaggca uguuuucuuuuguuuuguuuuguugguuuuguuuuguuuuuugagacgaagucuagcucuguugcccaggcuggagu gcaguggcacgaucucggcucagugcaaccucugccucccagguucaagagauucuccugccucagccucccaaguagcu gggauuacaggcacccccccgccacacccagcuaauuuuuguauuuuugguagagacaggguuucaccauguuggccag gcuggucucgaacuccugaccucaagugaucuaccugucuuggucucccaaagugcugggauuacaagcaugagccacc acucucagccuuuuuuguuuuuuuggguuguuguuguuguuuuuuguuuguuuguuuuuguuuuuguuuuuuuuug uuuuuuuuuugagacaggucucucuccaucacccaggcuggagugcaguguugugaucacaguucacuacagccccgac cuccggggcuuaagugauccucccaccucagccucccaaacagcugggacuacaggcacaugccaccaugcccagcuaau uuuugguagagauggagaucuugcuauguugcccaggcuaguggcaaacuccugagcucaagugauccaccugccuugg ccucccaaaguguugggauuacaguugugagccaccaugccagccuuaggugucuuagucuauauaaauuuccuccaau uuucacacuuuuuuuuuucacuuugaaaacuggcuuacauaugaauuucuacagaugcaauuaaauugccaaaagguca ucaauaugaaucuccuggaagguuucugccggguuuccuuuuuugugggugucuuccuaacaacuaauuacaguggcu ccaaagaacugauacugucauuccagaaugagcuaagaaggccuuuguucaucuaaacucugaaauaguacuaauuuuaa guccucaggacuugguuauggccaaacaugaguguuagaauuaugcaucaaaaauccccugauuuugugcaaguggcuu gaagccauuggauuuaacucaguucagauacaggucccauuuggcucuagaauuggauucaggucccucuuuuuccccc ccauccacag (SEQ ID NO: 339)GRCh38 / hg38 / chr9: 127682536-127682658 (Exon 18-19)

[0162] AACGGAAAGTGGGAGGTGCTGATAGgtgagtggccgtgcttccagcggaaggcgccgccg catcgcacctcaactccattccacgctttggtgtcgcattctgtcatagactcccttgaccca (SEQ ID NO: 340)GRCh38 / hg38 / chr9: 127684296-127684640 (Exon 18-19)

[0163] gtaggcctaagttggcatgttcctgtgacgtacctttgttcaagcagtcagctggcctctgttctcccacaggttctactca catcttactcccaccaaatttctcatggacctgagacaccccgacttcagggagtcctctagggtatcttttgaggatcaggctccaacaatggagtgagagcc aaagaaac aaagtaaaagcagcttattac agaaaagaaactcttcc actctgaagggctttctttgattatc cgtc actctttttcttattcttccttttagttattattattgattttgtttcaacaagaaaatgcttgcagttctcaac (SEQ ID NO: 341)Intron 17 (67967..70129)

[0164] guaggugaaaaucccuuagcaaggacagaggggagccagcucugguuggaugucagcucugcggcagacc cacuguacggcugugugaucuaccagagcccucucgcugcugcuccccacagcugcagcgugggccaggucugcacagu ggucccacggcccuucccccuccagcaucccguuccucucugauucaucccucacaccuccucccucgcucacucugcau cagccaugccagcuuccucgcacuucucaaacagucaggcuuacgucugccucaggaccacugcacuugcuuuucccucu gccugaaauuaucuguccccagauaugucugcaugggcugggcacaguagcucacaccuguaaucccagaacuuuggga ggccaaggcgggugaaucacuugaggucaggcauuugagaccagccuggcgaacauggugaaacccugucucuacuaaa aauacaaaaauuagcugggcaugguggcggacgccuguagucccagcuacucaggaggcugaggcaggagaauugcuug aacccaggaggcggagguuguggugagcuaagaucgcaucacugcacuccagucugggugacagagcgagacuccaugu caaacaaagaaaaaauaugucugcauggcuugccucuuaucuccuuuggucuuuaaucaaguguuaccuucuccaaucu ucccucaccacccuguuuaaaauugcagugcaucagcguucacagcuuuacucaugcaguccaaguguggagcagcccac augucccccagcugacgaguggguaacacacguggucucuccacacaguggcuccugagaacagucagggcugagcacgc acucgggaugguagaguuagcagcucgagagggcgacccuggcacugcagcugugccacaccuggcaccccaggaugug cacaucucugucccuccucuccugcuuuccccaccugucuucucuuuugcgucucuucucucuguguucacuuuuggcu ucagcugggccagaggccugugguggccucugugucauugugcagcccuucucauggcagcagagguaaaacguuuuu ugccaaauauggcauaaaauacaaaaucccaaguagcgccacagucaggcugggugaugagaaugggauaucaaaugauu gcac aaggaaagaaagcaacugugagauucucuguuggccc aaacggugc ccc aaacggugucuuc cugguuuggaaag ugacuguaaagcugucaugagucaggaagaaaggcgacgcauugcaguggccagaaaggacuugcuccuuucaucucau uuuucucauuuucuuaaccugggaacaacccauuuauuccuguuucucagucaaagguaaagagcuauugaaggaagcc cagguucagagggaggccccucugggagcagccuugccacugaaguacucuuccucccuggcuucgccgcaauuuccag gaggcagcucagggauggagaagccuccccaguggugcccggacaggguuccagggcuucccugugcuuggcccuauag cacagagcucuucaucagaccaggauuugggcucacuuccuucuccccaccugccccauucaaugaugaccucuugguca aggccugugucuccuuaggaugugguacuggggaggcccuggccuccaagaggcacucacucaguacauguuuucaagg gaucuaugugccagccugaaggcagaucuuagacugucaucacugugcacugacagcagagugucccaggcaagcucag aggcaugugugacuugggguagacgucugcaugagccucuagcagggggaugcuacagaacuugacccagcaaacguca gagcugaacugauccugucuguugccugcauguuucuaagagugacaaggucacagcagcacaggcauucauagugacu cuucuccugggaaccaauccacuugaaaauugcauugcagugaaauuucagccagcacucacuaagcaucuacacuguuu ggggcac aaguguac agac cuaauaaguaaac aaaccacugggguaugaagcacuagaugc cgugagugcagauaaacug acugacauucucuuucucucucucuuucucag (SEQ ID NO: 342)Exon 18 (70130..70284) - ENSE00003760296

[0165] CGCCCGCUAUGGGCACUGGCAUAAGAACAAGGCCCCAGGCGAGUACCGCAGUGGCCCCCGCCUCAUCAUUUUCAUCCUUGGGGGUGUGAGCCUGAAUGAGAUGCGCUGCGCCUACGAGGUGACCCAGGCCAACGGAAAGUGGGAGGUGCUGAU AG (SEQ ID NO: 343)Intron 18 (70285..78498)

[0166] gugaguggccgugcuuccagcggaaggcgccgccgcaucgcaccucaacuccauuccacgcuuugguguc gcauucugucauagacucccuugacccacaaaacuuagucuuuagguaacuguggacagcagaagcuaggggcaaaggg uucccuccuuggucugcccugcugcaucucucaauggaaggcagccucaugagguaggaagugccugaucccagggucc ggcagacucaggcucagauuccugcucugcaaacugcaagugggggaccuugagcaagugguucgagcucuccaaccau uuccuuacucagcaaaacaggaucagucauacucauucacagccuugaacuaggcccaagaugaggucugucagucuccu ggcauuggccucaugcccaggcaggaaugggcagugaauggcagucaaucgcugcccugucagguggagauggacagac agcugugagcaggugcugccugaaacuacugccugaccgcuggacucugcccagggcucccccagccaccuccaaccacu uucuaggucugccaugcccuuucucguauuuccuguucguuucccaaaagagaggaagcgacuuguaggagcuagcuua acgcagcaggagccuuuucuccuuuuucgugccuuuugggggcuucuaugauguuuagggcuggaagaagaugaacga gcuaagaaaagucuugcuuucuccagaugccucccuuuuuguccuguuuugcccuuaacaaaauggaggguaccugcca gccugggagucccucuc aacacugucuggc aagaagcuguc auguucacuacc cuggcugggcucuguccuc aguggaa acaggcucacgcugagcaaggagugaggccagggcucuuggccuccuuuuggaagggggugugcucugcacagugggg aauuauacucuccagggccaaggauggccucucaggccacuuacacaugaagugcuacagggagaggcccugagcugccc cagcugggaggcaucgcugaccuucucccauaggugcccaccacuggucuuaaucccaggcacagucaugaucucgauaa caaguggugguuuuauaccuccuauuuggggugagcaagucgcagcacccccccccucccaagcuauccaagugagcaga cuccauagaccaguccacauggcuguuucuggguuuuucaagauggaucuggggcugucagccgccucucugauagcac aauccaguggggagacuguaccuccccaccgcagcccucaagcuucccgugucaggucaccagcucuccacauaacuguu uuucccuucuguuguguagccccggugauaaagcucucugcucccugcuugcauauguauugcaugaauguagugagu aauucugagcccaaacacagcuuuucuggacagaagcugucacagccaggcuuuggguguaaaucuuccccuucaucuu uagaccugacuugagguuccauauaucgggaggugggaaccuggggguaugcaguugcccaugaagauucauggaggg gccucgcgcagacagcugacguccgcuguguggccuccccugcuuugugcaugccagccuccuugcuaccauguguguu caugcgcgcuuguguguguguguaugcaugcgcacacaugugugugugucuugcuuucucauaccucauuggcugcau aaaauguaggccuaaguuggcauguuccugugacguaccuuuguucaagcagucagcuggccucuguucucccacaggu ucuacucacauucuuacucccaccaaauuucucauggaccugagacaccccgacuucagggaguccucuaggguaucuuu ugaggaucaggcuccaacaauggagugagagccaaagaaacaaaguaaaagcagcuuauuacagaaaagaaacucuucca cucugaagggcuuucuuugauuauuccgucacucuuuuucuuuauuucuuccuuuuuaguuuauuauuuauuugauu uuguuucaacaagaaaaugcuugcaguucucaacaccgauugaaaauguguccagugcugcccucacagcccugccauga acccucugggcucugaccugcuggcucacccaugccuuggguuccgcacugcuuagucagacgccugcaggcugcuggg uggaaacuaaaaaugauaaugacugcugugaacaguugucagagagggagcugaaguuuugggggaugccugaggcaca gugcccaacccugcuggucugggaugaauuuguggcccccacaaggaauguagaguguccugguugugcugggugugg gggucugagagcgaggccaguggacauucagggaacaccauguccaggaaggcaggcggugguggagauggcuggucgugagggaagcuuggcaggcaauguagaauagac cc cagguc aguggcagcuggacggauggugugaauac cuuuuuc cu aggacaggcacuucucaaggaauaaguuaaaagggguaauugggucacaagccgccaucugcuuggggaaccccaaucu uuagcgggaagagcuuuuguuucauguuccucugcaggaaacggcacuuaacagucauuugcucuuuuacuaauuacuu uuaauucuguggauccuuucaagaguugaguagucauaccccucucucucccacugaagucccuugguaaggucugaga gccugucaguaguaauguauuuuuuuuuuuuugagacggggucucgcuauauugcccaugcuggagugcaguggcucu ucacaggugugaucacaacacacugcagccucaaacuccuggccucaagagauccuccuaucucagccuccugaguagcu ggcaccauaggcccauaccacugugcccagcauaaaccuauuauuaaugcucugugugguuuuagagagcaucuucugu cucccaccaagcuuguuacccagaaccuauugucugcuuaguagacuauccaaauuuuauuuuauuuauuuauuuuuau uuuuauuuauuauuauuauuauuuuuuuugagauagaguuucgcucuuguugcccaggcuggagugcaauggcaugau cucagcucaccacaaccucugccuccuggguucaagugauccuccugccuuagucucccagguugcugggauuacaggc augcgccaccacaccuggcuaauuuuuuuuuuuguauuuuuaguagggaugggguuucuccauguuggucaggcuggu cucgaacucccaaccucaggugauccaccugccucagccucccaaagugcugggauuauaggcgugagccaccgcgccca gccgacuauccaaauuuugaucuucaaaacuguccucauucccuccucucucuuuccauccuucagauacgauagaaccu ucuauguguuuaaauggcagccuuccaucaucacaucagugacuccuuagcuuguuacauauucuuucaugaggccaua uuuuguguaccugacuuuguguauaucauugacaauggacccuggaaaagccccuucaccugagcugagauguggccca gugagucacagagcaugaguccccugugagcauuggagagagaccuggacucagagccaggaggacuggccucuacucc agcuacuccaugggcaccuguguggcugugggaagauacccucgcucucuggccauagcauaggagcacagggagggug gaucgugugcccccaggcacgcaggaccucugacauucuguauauucagugauuacgauuugugccucaaaccaugcag uccugcucacucucucacucucuggugauccucaggggaauggcccuucagaaccuuggcuaggugacaucagaagcug cuuguguauguaaggaaaauggggcuuc cue cuaagguaggaac aggaggcuac gcuguuuaucuccuccucucucc cu uccuugauaugaaugucagggcagcagugugcuaaggaacugaaaguaguugggaacccagaaaguggaccugguuaua gcccuaggcuauagcucccauccugccugggcuggcccucccuagaaagcaaggccaguagguggugaauaaaagccauu guuuuaaaaaaaaucauuccuuagaagcacugauagguuguaaaucagagacaucugguucugugugcagacugcaggu agguggauggauuugcugcaguauguauuuuaagacuucuagaaagacaaauguucagucgagucugcagcaauagcag accacuugc aggaucugaaagagc acagacuc aaacugcaaaggcuc cugcuaggaugaguucugagcuucc cugcuuc a gaacaagcuucuccuuugcagaguauuuuccuuugcguuauauuugcucagcagaggucucagcugccaucaaaacaaa gagaacguaaacagacuccguauauuccuggcuucugaaaucauuugcugagugguguuggguuuuagagcauucagu uaagcaaggcacagagcccuaauaccagugaggucagaaagcagauuaaggucugacuugcccucuucuguugugaucg uccaggugaccaaaguacaggacagugcagacugucagaaaacuugaguguccuaaagaccaguaucauggcagcauauc uuuugugucuuuuuuucuuucuuucuuuugagucaggguuucacucuguuacccaggcuggagugcaguggcaugau uuuagcucacuuc age cuggac cuucuggguuc aagugauc cuuc cac cue agcuc ac cuaguagcugggac cacaggc augcgccaccaugccuggcuaauuguuuuguauuuuugguagagacuggguuucaccacguugcccaggcuggucuug aacuccugagcucaaguuaucccccagccucggccuaccaagugcugggauuacaggcaugagccacugcaccuggccca acauaucuuuuaggagaggaugaguuuuccugcagcaaaugagaggguucucagcagacaccgugcaugggugauggugccaugcccugucuucccagcuccucucccuccuguagcugacgcuggcucccccgaggucaggguuaaccaaeaacucu cacuaaauccucaccagucaucccuccacauugucuaaaaaaugaaaucuuuugauuagauuuucaccucucugcucugg uuaguuuaugauaaaaguacucuucuuuggggaaugcccaagagacugugagaagcaaaugugagagagcugaagacug gaucaagucuuugcaguacaugggggccacuugggagagaguugguggguuuuuauauugauuauuuugcuucauucc agauuggggucagguauguuucauuuuucuuugggacucagauuugaaucugaauucagagcccaccuauugccuggc ugcuauucaagcauccaccugcccauccccuccccucccaggggccacagggucccgagaaucaagagccuggaggcacu gcuucuggaccugucugugccacccacccugagcaagcaccugacugcucuggccuucacgugucccauguguaggaug agaagggugggccggaucacaguuuuccaaaccuggcuaaugaucauacucaccugggcugcucuuuaaauguacacau uuucuggcgccgcccugcauauucugagucaguaggucugcuucagccaaacucaagaaugauuggaccagcuagccug caagaacacuuuugguuuucagauucuguccguguagaaagaaggauuuugucccaucagagcuugcggugucuggccc acagcuuugaaaacauguagacagucauguagaugggccucaacuuaguuuuuagagaccacuguuuacauuucauaau cguccaguuauagggacuaaauaggacccucagagcaauagggcccacuugcugcuuuauucuggaaaaccaacaugguc agccaucuguggauucaaaaugagaacugugcaaaauguggauuauggacucacucagcuuaauuugcaauguuuaugg uggugaaaagcuugacc aauuuauuaaaac aacac agaugacugacuuugaagaauuggcuuucugaugauc agcauuu acaccagcuagucugguccccuuaaagaaaccacguaggugaaggccuaggccacagccuguugcugagggucuucgag gcuucacugauugucuccaauacucaguccuagaauuauuguucuucagucaagacugggagcaaugaauggucaguga uuaaauaauaucagagacuucuagugcauuaauucccuaaccucucuuuggccucucccaaauccuagauuuacauauca gcaaagauagggaggacuuuggaaucagcaucugaucagaggaaccccuucggaugccuccuuuaaaaugagucugccc ucuuauuuccuggagucauuuggauggcuaaggagggccuuggggacauuuucucuggcacgugcgcuccuugcuguu gcuuguaacgaggaagcuc ccaagc gaauc cue ac gcucugcaggacaggcugucuuuc age aucuggac ac gugggcu acauggaguugcaauuuggagggcccccauccuggcaaugguucacucuucagaguuuggggcaagccuuguaauaugc guccuuucuc cue auuuguacagcagc cuuuggccucuuuuaggacuagcaggaggaguggugagguc ac agaaaugag uggacuucaggggaaaggagugcuauaaaaaagcccagggacacacuccugaaaccugcaucgcauuugcugaaggggcc ucaggucugcagaaugaugagucagcuuuccagcgcagggccccuacaggcugcgugcacagggcaucaugccgaggac aaguuccucacucagcugggagugggcagcuggggccugaacugcagagaaagggauguuggaagaacagagacggcuu uccaaguuucugcuagucuggagugaccaggaagggcugcaguuccagcagucuucggucagcugagcgaguaguuag uggucaaaggcauagggacccugguauuggagucaugcccagugagccagccaaagccuucuuuugccugcccuugggg uggaaauccagcauccggugguuuccgcuccccugcucccaugaaggcccauuccuuugccacgucuccagggaggcaca uuuucaaguacgugueucuuuuguucaguucuugucucccaccaggaccuuucuuugaagcacacugcugcaugcgugc aggccucaugcucacccacacaccaccccggucaccucuguucuguuuaaguccauguccaaaccacacgacccaugcauc uuuuuuuacugcacuuacuagaucucucucucucuccuuaugacaaagacuuucauuuauuuuuauuuuuauuuuugc aguagaaguauaucuaauuccauuagcuuaaauuuuugcaagcauuucuaaucucuuacaauacgcggucucugcaagg uaaggccucagguccccuggcccuuucccucucacaccugaggucccacccaccagggcaguaccaacuggcuggcuccc agaggucaguggauueucccagggggcuuugccuaucaacuuccucucccaggcaccaugcuuuccacacauggucccccuaccaacucuggccccauuuucagccuuucccugccuugccauguuuggaauuucucucccuggcuucaccuggaguuu aagagugaauuagaaacgcagcugcccaucaccugcagauggcucugcacccugauggugaggcagcucguuccuuggu gcagccugggacccugccugagccagaggauguguuuuguuuauccacagcccccaaauaucucccaggccuauugcga cgggaccccaaccccaauucugacuccccucccugcuuccacuauacccagaccaggguuggggaggggggugggugca gaucugggaaucgagaagcgcaggaaaggaaguguuggaaaccagaacuggcaagcucacugucuucucugccuccucc ag (SEQ ID NO: 344)Exon 19 (78499..80423)

[0167] GAUCCACACACAUCCUCACCCCACAGAAACUGCUGGACACACUGAAGAAACUGAAUAAAACAGAUGAAGAAAUAAGCAGUUAAAAAAAUAAGUCGCCCCUCCAAAACACGCCCCCAUCCCACAGCGCUCCGCAGCUUCCCACCACCGCCCGCCUCAGUUCCUUUGCGUCUGUUGCCUCCCCAGCCCUGCACGCCCUGGCUGGCACUGUUGCCGCUGCAUUCUCGUGUUCAGUGAUGCCCUCUUCUUGUUUGAAACAAAAGAAAAUAAUGCAUUGUGUUUUUUAAAAAGAGUAUCUUAUACAUGUAUCCUAAAAAGAGAAGCUCAUGUGCAAUUGGUGCACAGCAGGAGAAAUUUCUGGACUGUUAGGAUGAAUGGACGCCUUCUCCCCGUUAUUUAAGAUUUGUGACCUUGUACAUAACCCUGGGUGACGUGCACAUUGCUUGGGUAUGGAACGGUAGAAAUUUGGGUGUUUUUAAAACCUUGUUUGGGGUUGUUCCUGUCCUUGUUGAGAAUCAUAGAGAUGUCUGUGUUCUUGGAGUAUUUCACACUGAGGACUAAUCUGCUAUCUUCAUUCCAGUCCCUACCCCUCAGUGCCUGCUCUCAUCCAAAUAACCUGGGAGGUGACAAUCAGGAUAUCUCAGGAGGUCCAAGGUGGAACAGACCUCUUUGCCUUUCCCAGCGUCUCAUACCCCCGGUAGUGCAGCUGUGGGUGGAGGCUGGGGUGUCUGCACGAAGUCAGGCCAGCGUCCUCCUCCACAGCCUGUCACUGCCCCCUCCCCAGCCUGUGUCCACAGUGCUGUGAUCCCGAGGGAAGUCCUCCAGUCUAAGUCACAGUGCCCUGACAGGUGAGAAGCAAACUCCCGCUGGAAGCCUCCAUCUCUUUGGAAAAACAGUUAGUCUGGAGCCUGUGGCCCAGGCCCUUCUGUCCCCAGGCAUCAUCCCAACAGCUCAUUUUCCCUAGUCCGCCUUCGUUCAAGGGUCAGGAAUGGACCAGAACAGAUGGGUUCUGGAGGCCCCUGAACAGAGGGCUAUGGCUGUGGAGAAGGUUCUUGGCCCGUUGGACUCACACAGACCCUGUACCCUCUCGGCAAGCAUCUUCAGUCAGAUUAUCCUCAGUUUCAGAUACUUCAUAAUACCUUGUGUUGUGUGGGGUCAUACAUCAUCGUGUUUGUAAGAGAAGAUGGUCAUUUUAUUCUCUGUAUAAAACUUAGCUCUAAAGCAGAAACUAAAGCAGCAAAUGCAGGAAGGCUGUCUCGCCAUCCUCAAGACUCAGCAGCUCUCAUUCUCCAGUGGUGAGCACACCAUUUGUGCUGCUGCUGUUGUCGUGAAAUAUAAUAACAGUGGAAGUCACAAAAAUGUCCCCUGCCCAGCCCCCUCGCCGCCCUUGACCUCCUGCAGGCCAUGUGUGUAUUACUUGUCUAGUGAUGUCCUCUCAAAGUGCUGUACGCGAGCUCGGCGCCACCUCCGCCUCCCUUUCAGAGCCUGCUCCCCGCCCUCUCUGCUCGCUGCAUUGUGGUGUUCUCUUCUCAAGGCUUUGAAAUCUCCCCUUGCACUGAGAUUAGUCGUCAGAUCUCUCCCCGUCUCCCUCCCAACUUAUACGACCUGAUUUCCUUAGGACGGAACCGCAGGCACCUGCGCCGGGCGUCUUACUCCCGCUGCUUGUUCUGUCCCCUCCCUCGGACCAAACAGUGCUCAUGCUUCAGGACCUUGUUUGUCGAAGAUGUUGGUUUCCCUUUCUCUGUUAUUUAUAUAAAAAUAAUUUAUCAAAAGGAUAUUUUAAAAAAGCUAGUCUGUCUUGAAACUUGUUUACCUUAAAAUUAUCAGAAUCUCAGUGUUUGAAAGUACUGAAGCACAAACAUAUAUCAUCUCUGUACCAUUCUGUACUAAAGCACUUGAGUCUAAUAAAUAAAGAAAUCAGCACCCCU (SEQ ID NO: 345)

Claims

CLAIMS1. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% sequence identity to any one of SEQ ID NOs: 3-100.

2. The ASO of claim 1, wherein the target binding sequence has at least 85% sequence identity to any one of SEQ ID NOs: 3-100.

3. The ASO of claim 1, wherein the target binding sequence has at least 90% sequence identity to any one of SEQ ID NOs: 3-100.

4. The ASO of claim 1, wherein the target binding sequence has at least 95% sequence identity to any one of SEQ ID NOs: 3-100.

5. The ASO of claim 1, wherein the target binding sequence comprises any one of SEQ ID NOs: 3-100.

6. The ASO of claim 1, wherein the target binding sequence comprises any one of SEQ ID NOs: 3, 4, 13, 23, 43, 52, 72, and 74.

7. The ASO of claim 1, wherein the target binding sequence comprises SEQ ID NO: 3.

8. The ASO of claim 1, wherein the target binding sequence comprises SEQ ID NO: 52.

9. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% sequence identity to any one of SEQ ID NOs: 207-270.

10. The ASO of claim 9, wherein the target binding sequence has at least 85% sequence identity to any one of SEQ ID NOs: 207-270.

11. The ASO of claim 9, wherein the target binding sequence has at least 90% sequence identity to any one of SEQ ID NOs: 207-270.

12. The ASO of claim 9, wherein the target binding sequence has at least 95% sequence identity to any one of SEQ ID NOs: 207-270.

13. The ASO of claim 9, wherein the target binding sequence comprises any one of SEQ ID NOs: 207-270.

14. The ASO of claim 9, wherein the target binding sequence comprises any one of SEQ ID NOs: 207, 209, 210, 212, 215, 216, 222, 223, 224, 225, 249, 250, 260, 261, 269, or 270.

15. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% sequence complementarity to a target sequence in Exon 3 (SEQ ID NO: 336), Intron 3 (SEQ ID NO: 337), Exon 4 (SEQ ID NO: 338), Intron 4 (SEQ ID NO: 339), Intron 17 (SEQ ID NO: 342), Exon 18 (SEQ ID NO: 343), Intron 18 (SEQ ID NO: 344), or Exon 19 (SEQ ID NO: 345) of human STXBP1 gene, or a combination thereof.

16. The ASO of claim 15, wherein the target sequence is in Exon 3 (SEQ ID NO: 336), Intron 3 (SEQ ID NO: 337), Exon 4 (SEQ ID NO: 338), or Intron 4 (SEQ ID NO: 339) of human STXBP1 gene, or a combination thereof.

17. The ASO of claim 15, wherein the target sequence is encoded in GRCh38 / hg38 / chr9:127654840-127655139 (SEQ ID NO: 335).

18. The ASO of claim 15, wherein the target sequence is in Intron 17 (SEQ ID NO: 342), Exon 18 (SEQ ID NO: 343), Intron 18 (SEQ ID NO: 344), or Exon 19 (SEQ ID NO: 345) of human STXBP1 gene, or a combination thereof.

19. The ASO of claim 15, wherein the target sequence is encoded in GRCh38 / hg38 / chr9:127682536-127682658 (SEQ ID NO: 340).

20. The ASO of claim 15, wherein the target sequence is encoded in GRCh38 / hg38 / chr9:127684296-127684640 (SEQ ID NO: 341).

21. The ASO of claim 15, wherein the target sequence is selected from any one of SEQ ID NOs: 101-108.

22. The ASO of claim 15, wherein the target sequence is SEQ ID NO: 106.

23. The ASO of claim 15, wherein the target sequence is SEQ ID NO: 103.

24. The ASO of any one of claims 15-23, wherein the target binding sequence has at least 85% sequence complementarity to the target sequence.

25. The ASO of any one of claims 15-23, wherein the target binding sequence has at least 90% sequence complementarity to the target sequence.

26. The ASO of any one of claims 15-23, wherein the target binding sequence has at least 95% sequence complementarity to the target sequence.

27. The ASO of any one of claims 15-23, wherein the target binding sequence has 100% sequence complementarity to the target sequence.

28. The ASO of any one of claims 1-27, wherein the ASO comprises one or more modifications to one or more intemucleotide phosphodiester linkages in the ASO.

29. The ASO of claim 28, wherein the ASO comprises one or more modification to all intemucleotide phosphodiester linkages in the ASO.

30. The ASO of claim 28, wherein the ASO comprises a phosphorothioate (PS) modification to one or more intemucleotide phosphodiester linkages in the ASO.

31. The ASO of claim 28, wherein the ASO comprises a phosphorothioate (PS) modification to all intemucleotide phosphodiester linkages in the ASO.

32. The ASO of any one of claims 1-31, wherein the ASO comprises one or more modifications to one or more nucleoside sugar moieties in the ASO.

33. The ASO of claim 32, wherein the ASO comprises one or more modifications to all nucleoside sugar moieties in the ASO.

34. The ASO of claim 32, wherein the ASO comprises a 2'-O-Methoxyethyl (2'-MOE) modification to one or more nucleoside sugar moieties in the ASO.

35. The ASO of claim 32, wherein the ASO comprises a 2'-O-Methoxyethyl (2'-MOE) modification to all nucleoside sugar moieties in the ASO.

36. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% identity to any one of SEQ ID NOs: 109-206.

37. The ASO of claim 36, wherein the target binding sequence has at least 85% sequence identity to any one of SEQ ID NOs: 109-206.

38. The ASO of claim 36, wherein the target binding sequence has at least 90% sequence identity to any one of SEQ ID NOs: 109-206.

39. The ASO of claim 36, wherein the target binding sequence has at least 95% sequence identity to any one of SEQ ID NOs: 109-206.

40. The ASO of claim 36, wherein the target binding sequence comprises any one of SEQ ID NOs: 109-206.

41. The ASO of claim 36, wherein the target binding sequence comprises any one of SEQ ID NOs: 109, 110, 119, 129, 149, 158, 178, and 180.

42. The ASO of claim 36, wherein the target binding sequence comprises SEQ ID NO:109.

43. The ASO of claim 36, wherein the target binding sequence comprises SEQ ID NO:158.

44. An antisense oligonucleotide (ASO) comprising at least one target binding sequence, wherein the target binding sequence has at least 80% identity to any one of SEQ ID NOs: 271-334.

45. The ASO of claim 44, wherein the target binding sequence has at least 85% sequence identity to any one of SEQ ID NOs: 271-334.

46. The ASO of claim 44, wherein the target binding sequence has at least 90% sequence identity to any one of SEQ ID NOs: 271-334.

47. The ASO of claim 44, wherein the target binding sequence has at least 95% sequence identity to any one of SEQ ID NOs: 271-334.

48. The ASO of claim 44, wherein the target binding sequence comprises any one of SEQ ID NOs: 271-334.

49. The ASO of claim 44, wherein the target binding sequence comprises any one of SEQ ID NOs: 274, 276, 280, 313, 314, 324, 325, 333, or 334.

50. The ASO of any one of claims 1-49, wherein the target binding sequence comprises 15-25 nucleotides.

51. The ASO of any one of claims 1-49, wherein the target binding sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

52. The ASO of any one of claims 1-49, wherein the target binding sequence comprises 18 nucleotides.

53. A pharmaceutical composition comprising an ASO of any one of claims 1-52, and a pharmaceutically acceptable excipient.

54. A method of modifying the expression of Syntaxin binding protein 1 (STXBP1), comprising delivering an effective amount of an ASO of any one of claims 1-52 to a cell comprising the STXBP1 gene.

55. The method of claim 54, wherein the ASO binds to a pre-mRNA splicing regulatory element encoded in the STXBP1 gene.

56. The method of claim 43, wherein the splicing regulatory element is for an exon splicing enhancer (ESE), an exon splicing silencer (ESS), an intron splicing enhancer (ISE), or an intron splicing silencer (ISS).

57. The method of any one of claims 54-56, wherein delivery of the ASO to the cell results in an increase in STXBP1 production by the cell.

58. The method of claim 57, wherein delivery of the ASO to the cell results in an increase in STXBP1 production by the cell of at least 110%.

59. The method of claim 57, wherein delivery of the ASO to the cell results in an increase in STXBP1 production by the cell of at least 115%.

60. The method of claim 57, wherein delivery of the ASO to the cell results in an increase in STXBP1 production by the cell of at least 120%.

61. The method of claim 57, wherein delivery of the ASO to the cell results in an increase in STXBP1 production by the cell of at least 125%.

62. A method of treating a disease related to a STXBP1 gene mutation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising an ASO of any one of claims 1-52 or a pharmaceutical composition of claim 53.

63. The method of claim 63, wherein the disease related to a STXBP1 gene mutation is a developmental and epileptic encephalopathy (DEE).

64. The method of claim 63, wherein the disease related to a STXBP1 gene mutation is Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, Rett syndrome, or an early infantile DEE.

65. The method of any one of claims 62-64, wherein administering the composition to the subject reduces or eliminates one or more symptoms of the disease.

66. The method of claim 65, wherein administering the composition to the subject results in one or more of: (i) restores one or more cognitive abilities in the subject, (ii) reduces the number, frequency, and / or severity of seizures in the subject, (iii) improves and or restores one or more motor functions in the subject, and (iv) improves and / or restores one or more psychiatric functions in the subject.

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