Antisense oligomers for the treatment of conditions and diseases

Abstract

This invention provides a method for treating a disease or condition characterized by reduced expression or function of the NaV1.1 protein. [Solution] A method is provided for treating a disease or condition in a human subject or reducing the likelihood of developing it, comprising the step of administering a pharmaceutical composition comprising an antisense oligomer (ASO) to a human subject, wherein the ASO comprises a sequence having at least 80% sequence identity with respect to a specific sequence.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the interests of U.S. Provisional Patent Application No. 62 / 945,048, filed on 6 December 2019, and U.S. Provisional Patent Application No. 62 / 993,971, filed on 24 March 2020, respectively, which are incorporated herein by reference in their entirety. [Background technology]

[0002]

[0002] Nervous system disorders are often associated with channel diseases, which are characterized by dysfunction of ion channels that mediate neuronal excitability, neuronal interactions, and overall brain function. Mutations in the SCN1A gene, which is part of the SCN1A-SCN2A-SCN3A gene family that encodes the alpha-pore-forming subunit of neuronal voltage-gated sodium channels, are wild-type Na V 1.1 Na with reduced function compared to protein V 1.1 Protein (Na V Expression of Na (also known as "1.1") V 1.1 may result in reduced expression or both. Mutations in the SCN1A gene are associated with the incidence of diseases and conditions such as Dravet syndrome (DS) (Miller et al., 1993–2015, GeneReviews Pagon RA et al., Seattle (WA): University of Washington, Seattle, Bookshelf ID: NBK1318 and Mulley et al., 2005, Hum. Mutat. Vol. 25: pp. 535–542). [Overview of the project] [Problems that the invention aims to solve]

[0003]

[0003] Alternative splicing events in the SCN1A gene may lead to unproductive mRNA transcripts, which in turn may lead to abnormal protein expression. Therapeutic agents that can target alternative splicing events in the SCN1A gene can modulate the expression levels of functional proteins and / or inhibit abnormal protein expression in patients with Dravet syndrome. Such therapeutic agents can be used to treat conditions caused by SCN1A, SCN8A, or SCN5A protein deficiency.

[0004]

[0004] Selecting the correct dosage, formulation, administration regimen, and patient population for a drug is a critical step in drug development. For example, it is impossible for a physician to prescribe a drug to a patient without adequate information regarding dosage. For example, if a dosage or dosage range that allows for safe and predictable administration cannot be identified, the drug cannot be a medically useful or commercially viable medicine. Therefore, determining the correct drug dosage is a critical issue that needs to be addressed in clinical practice. Finding a therapeutically effective dosage and administration regimen for a drug requires balancing patient compliance, therapeutic efficacy, and side effects, which requires considerable skill. For example, suitable dosages and administration regimens may be discovered through clinical trials that form part of the approval process, require substantial investment of intellectual and financial resources from various parties, and are outside the scope of a physician's routine work. For example, patient compliance can be critical for the optimal treatment of various conditions. The higher the required dose or the more complex the treatment plan, the less likely patients are to follow it. Medical drugs have the ability to enhance a patient's quality of life (QOL), but they can only do so when used correctly. Therefore, it is clear that selecting the correct dosage, formulation, administration regimen, and patient population for a drug is complex and unpredictable. Structurally similar compounds differ significantly in their solubility, toxicity, activity, stability, and pharmacological properties. Furthermore, there are differences between animal models and human subjects. There are significant physiological differences. Therefore, translating preclinical information into clinically effective therapies is an unpredictable and challenging task. [Means for solving the problem]

[0005]

[0005] Na V 1.1 In human subjects requiring treatment of or reduction of the likelihood of developing a disease or condition characterized by reduced protein expression or function, suitable dosages, formulations, drug regimens and patient populations for treating or reducing the likelihood of developing such disease or condition are provided herein.

[0006]

[0006] In some embodiments, Na V1.1 In human subjects requiring treatment of or reduction of the likelihood of developing a disease or condition characterized by reduced protein expression or function, methods for treating or reducing the likelihood of developing such disease or condition are provided herein, the methods for treating or reducing the likelihood of developing such disease or condition in human subjects are approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 3 0, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 130, 132.5, 135, 137.5, 14 0, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 2 The step of administering a pharmaceutical composition comprising an antisense oligomer (ASO) in a first dose of 37.5, 240, 242.5, 245, 247.5, or 250 mg, wherein the ASO comprises a sequence having at least 80% sequence identity to any one of sequence numbers 21-67, 210-256, or 304-1099, thereby treating or reducing the likelihood of developing a disease or condition in a human subject.

[0007]

[0007] In some embodiments, Na V1.1 In human subjects requiring treatment or prevention of a disease or condition characterized by reduced protein expression or function, the use of antisense oligomers (ASOs) for the manufacture of a pharmacopoeia for treatment or prevention of said disease or condition is provided herein, and the pharmacopoeia are approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 9, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120 , 122.5, 125, 127.5, 130, 132.5, 135, 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 202.5, 205, 20 The first dose administered is 7.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg, and the ASO contains a sequence having at least 80% sequence identity with one of sequence numbers 21-67, 210-256, or 304-1099.

[0008]

[0008] In some embodiments, Na V 1.1 Characterized by a reduction in protein expression or function The use of an antisense oligomer (ASO) for the manufacture of a medicament for treating or preventing a disease or condition in a human subject that requires treatment or prevention of the disease or condition is provided herein, wherein the medicament is formulated to be administered at a first dose of about 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 130, 132.5, 135, 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5 or 250 mg, and the ASO comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 21-67, 210-256 or 304-1099.

[0009]

[0009] In some embodiments, Na V1.1 In a human subject requiring treatment of or reduction of the likelihood of developing a disease or condition characterized by reduced protein expression or function, a method for treating or reducing the likelihood of developing such a disease or condition is provided herein, the method comprising the step of administering to a human subject a pharmaceutical composition comprising a first dose of an antisense oligomer (ASO), the ASO comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 21-67, 210-256, or 304-1099, thereby treating or reducing the likelihood of developing such a disease or condition in a human subject, the human subject being up to 18 years of age at the time of the first dose.

[0010]

[0010] In some embodiments, Na V 1.1 In a human subject requiring treatment of or reduction of the likelihood of developing a disease or condition characterized by reduced protein expression or function, a method for treating or reducing the likelihood of developing such a disease or condition is provided herein, the method comprising the step of administering to a human subject a pharmaceutical composition comprising multiple doses of an antisense oligomer (ASO), the ASO comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 21-67, 210-256, or 304-1099, thereby treating or reducing the likelihood of developing such a disease or condition in a human subject.

[0011]

[0011] In some embodiments, the first dose is the first of several doses.

[0012] In some embodiments, human subjects are up to 18 years of age at the time of the first dose.

[0013] In some embodiments, the method involves human subjects at approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 11 7.5, 120, 122.5, 125, 127.5, 130, 132.5, 135, 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, The procedure includes administering a pharmaceutical composition containing ASO in a first dose of 237.5, 240, 242.5, 245, 247.5, or 250 mg.

[0012]

[0014] In some embodiments, the disease or condition is Dravet syndrome.

[0015] In some embodiments, subjects meet the following criteria: (i) seizure onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and triggered by high fever; (ii) no prior history of causative magnetic resonance imaging lesions; (iii) no other known etiologies of any disease or condition other than Dravet syndrome; (iv) normal development at seizure onset; (v) a pathogenic variant or variant of uncertain importance in the SCN1A gene; (vi) at least two prior treatments for epilepsy, either lacking adequate seizure management; and (vii) prior to administration. (ix) The patient has four or more seizures during a 28-day period, the seizures being one of the following selected from unilateral clonic, focal with motor signs, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic; (viii) current intervention for epilepsy, which is a ketogenic diet, a vagus nerve stimulant, or a cannabinoid or marijuana-derived product, or medication with at least one antiepileptic drug at a dose that has been stable for at least four weeks; or any combination of (ix)(i) to (viii).

[0013]

[0016] In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg and (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blockers being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d) clinical conditions other than epilepsy (i) The patient is characterized by not having any of the following conditions: (i) a medically unstable condition; (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration; (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation; (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted; (h) clinically significant abnormal clinical laboratory values ​​prior to administration; (i) aspartate aminotransferase or alanine aminotransferase >2.5 times the upper limit of normal, serum creatinine above the upper limit of normal, or platelet count below the lower limit of normal; (j) a clinically relevant abnormality in a 12-lead electrocardiogram (ECG) measured prior to administration; (k) a mental or behavioral disorder; (l) current or past four weeks of taking anticoagulants other than aspirin; or (m) one or more of any combination of (a) to (l).

[0014]

[0017] In some embodiments, human subjects are 1-18, 2-18, 3-18, 4-18, 5-18, 6-18, 7-18, 8-18, 9-18, 10-18, 11-18, 12-18, 13-18, 14-18, 15-18, 16-18, or 17-18 years old.

[0015]

[0018] In some embodiments, the human subjects are humans aged 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 years.

[0016]

[0019] In some embodiments, human subjects are under 1 year old or 1, 2, 3, 4, 5, 6, 7 They are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years old.

[0020] In some embodiments, the pharmaceutical composition is administered into the subarachnoid space of a human subject.

[0017]

[0021] In some embodiments, the pharmaceutical composition is administered into the cerebrospinal fluid of a human subject.

[0022] In some embodiments, the pharmaceutical composition is administered into the brain of a human subject.

[0023] In some embodiments, the pharmaceutical composition is administered into the cerebrospinal fluid in the brain of a human subject.

[0018]

[0024] In some embodiments, the pharmaceutical composition is administered as a bolus injection.

[0025] In some embodiments, the pharmaceutical composition is administered by infusion using a delivery pump.

[0019]

[0026] In some embodiments, the pharmaceutical composition is administered by intraventricular injection.

[0027] In some embodiments, the pharmaceutical composition is administered by intraarachnoid injection.

[0028] In some embodiments, the method reduces or improves at least one symptom of Dravet syndrome in human subjects.

[0020]

[0029] In some embodiments, the symptoms of Dravet syndrome are seizures.

[0030] In some embodiments, the administration reduces or improves the frequency, intensity, or duration of seizures.

[0021]

[0031] In some embodiments, the ASO includes a sequence having at least 83%, 88%, 94%, or 100% sequence identity with any one of sequence numbers 21-67, 210-256, or 304-1099.

[0022]

[0032] In some embodiments, the ASO consists of sequences having at least 83%, 88%, 94%, or 100% sequence identity with any one of sequence numbers 21-67, 210-256, or 304-1099.

[0023]

[0033] In some embodiments, the ASO comprises at least one modified sugar moiety.

[0034] In some embodiments, the ASO includes a T-methoxyethyl sugar moiety.

[0035] In some embodiments, the T-methoxyethyl sugar moiety is the T-2'-methoxyethyl sugar moiety.

[0024]

[0036] In some embodiments, the ASO includes a 2'-O-methoxyethyl moiety.

[0037] In some embodiments, ASO comprises a thymidine containing a 2'-O-methoxyethyl moiety.

[0025]

[0038] In some embodiments, each nucleic acid base of the ASO contains a 2'-O-methoxyethyl moiety.

[0039] In some embodiments, the ASO consists of 8 to 50 nucleic acid bases.

[0026]

[0040] In some embodiments, the ASO consists of 16 to 20 nucleic acid bases.

[0041] In some embodiments, the ASO consists of 12 to 20 nucleic acid bases.

[0042] In some embodiments, the ASO consists of 8 to 20 nucleic acid bases.

[0027]

[0043] In some embodiments, the ASO contains 5'-methylcytosine (5'-MeC).

[0044] In some embodiments, each cytosine in ASO is 5'-methylcytosine (5'-M It is eC).

[0028]

[0045] In some embodiments, the ASO includes a phosphorothioate bond.

[0046] In some embodiments, the internucleoside bonds in ASO are phosphorothioate bonds.

[0029]

[0047] In some embodiments, the ASO includes locked nucleic acid (LNA).

[0048] In some embodiments, the method further includes a step of evaluating the tolerance or efficacy of the pharmaceutical composition.

[0030]

[0049] In some embodiments, the method involves administering doses of 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, and 57 to human subjects. 5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 130, 132.5, 135, 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 2 The step further comprises administering a pharmaceutical composition containing ASO in a subsequent dose of 0.2.5, 20.5, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg.

[0031]

[0050] In some embodiments, after it is shown that an earlier dose is untolerable, a subsequent dose is lower than the earlier dose.

[0051] In some embodiments, after it has been demonstrated that the administration of a previous dose is tolerable, the subsequent dose is the same as the previous dose.

[0032]

[0052] In some embodiments, after a previous dose has been shown to be tolerable, a subsequent dose is higher than the previous dose.

[0053] In some embodiments, a subsequent dose is the same as the previous dose after it has been shown to be effective.

[0033]

[0054] In some embodiments, after a previous dose has been shown to be effective, a subsequent dose is lower than the previous dose.

[0055] In some embodiments, after it has been shown that the previous dose was ineffective, a subsequent dose is higher than the previous dose.

[0034]

[0056] In some embodiments, the subsequent dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the administration of the previous dose.

[0057] In some embodiments, the frequency of medication is maintained or reduced after a previous dose has been shown to be effective.

[0035]

[0058] In some embodiments, the frequency of administration is increased after it has been shown that the previous dose is ineffective.

[0059] In some embodiments, the method further includes the step of administering at least one additional therapeutic agent or therapy.

[0036]

[0060] In some embodiments, at least one additional therapeutic agent or therapy is administered concurrently with the dose.

[0061] In some embodiments, at least one additional therapeutic agent or therapy is administered before the dose is given.

[0037]

[0062] In some embodiments, at least one additional therapeutic agent or therapy is administered after the dose is given.

[0063] In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable excipients, carriers, or diluents.

[0038]

[0064] In some embodiments, the pharmaceutical composition is a liquid composition.

[0065] In some embodiments, the pharmaceutical composition comprises 0.1 mL to 50 mL of a diluent, in which the ASO is solubilized or diluted.

[0039]

[0066] In some embodiments, the pharmaceutical composition comprises about 0.1, 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mL of diluent.

[0040]

[0067] In some embodiments, the pharmaceutical composition comprises 1 mL to 20 mL of diluent, 2 mL to 10 mL of diluent, or 1 mL to 5 mL of diluent.

[0068] In some embodiments, the diluent comprises a cerebrospinal fluid (CSF) sample derived from the subject or an artificial cerebrospinal fluid (aCSF) solution.

[0041]

[0069] In some embodiments, the method includes the step of obtaining a cerebrospinal fluid sample from a subject.

[0070] In some embodiments, the method includes the step of solubilizing or diluting the ASO in a CSF sample from the subject.

[0042]

[0071] In some embodiments, the method includes the step of administering a pharmaceutical composition as a bolus injection.

[0072] In some embodiments, the method includes the step of administering the pharmaceutical composition as a bolus injection over a period of 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

[0043]

[0073] In some embodiments, the method includes the step of administering the pharmaceutical composition as a bolus injection using a spinal anesthetic needle.

[0074] In some embodiments, ASO is solubilized or diluted in an artificial cerebrospinal fluid (aCSF) solution.

[0044]

[0075] In some embodiments, the solution contains a cerebrospinal fluid (CSF) sample derived from the subject.

[0076] In some embodiments, the ASO is solubilized or diluted in an isotonic solution.

[0077] In some embodiments, the ASO is solubilized or diluted in a phosphate buffer solution having a pH of at least 5.8.

[0045]

[0078] In some embodiments, ASO is solubilized or diluted in a phosphate buffer solution (pH 6.6-7.6).

[0079] In some embodiments, the ASO is solubilized or diluted in a buffer containing 25–250 mM NaCl.

[0046]

[0080] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–20 mM KCl.

[0081] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM Na2HPO4.

[0047]

[0082] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM NaH2PO4.

[0083] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM CaCl2.

[0048]

[0084] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM MgCl2.

[0085] In some embodiments, ASO is solubilized or diluted in a buffer containing 25–250 mM NaCl, 0.1–20 mM KCl, 0.1–50 mM Na2HPO4, 0.1–50 mM NaH2PO4, 0.1–50 mM CaCl2, and 0.1–50 mM MgCl2.

[0049]

[0086] In some embodiments, ASO is solubilized or diluted in a buffer containing 150 mM NaCl, 3.0 mM KCl, 0.7 mM Na2HPO4, 0.3 mM NaH2PO4, 0.79 mM MgCl2, and 1.4 mM CaCl2.

[0050]

[0087] In some embodiments, the ASO is solubilized or diluted in a buffer further containing a carbohydrate. In some embodiments, the carbohydrate includes D-glucose. In some embodiments, the ASO is solubilized or diluted in a buffer further containing 1 to 100 mM D-glucose.

[0051]

[0088] In some embodiments, the ASO is solubilized or diluted in a buffer further containing 1–100 mM NaHCO3, 1–100 mM KHCO3, or a combination thereof.

[0089] In some embodiments, the ASO is solubilized or diluted in a buffer further containing an antioxidant. In some embodiments, the antioxidant is t-butylhydroxyquinoline (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin E, or any combination thereof.

[0052]

[0090] In some embodiments, the pharmaceutical formulation does not contain preservatives.

[0091] In some embodiments, ASO is present in the pharmaceutical composition at a concentration of 0.1 mg / mL to 250 mg / mL.

[0053]

[0092] In some embodiments, ASO is present in the pharmaceutical composition at concentrations of approximately 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL. In some embodiments, ASO is present in the pharmaceutical composition at concentrations of approximately 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL. In some embodiments, ASO is approximately 22.5 mg / mL, 25 mg / mL, 27.5 mg / mL, 30 mg / mL, 32.5 mg / mL, 35 mg / mL, 37.5 mg / mL, 40 mg / mL, 42.5 mg / mL, 45 mg / mL, 47.5 mg / mL, 50 mg / mL, 52.5 mg / mL, 55 mg / mL, 57.5 mg / mL, 60 mg / mL mL, 62.5mg / mL, 65mg / mL, 67.5mg / mL, 70mg / mL, 72.5mg / mL, 75mg / mL, 77.5mg / mL, 80mg / mL, 82.5mg / mL, 85mg / mL, 87 .5mg / mL, 90mg / mL, 92.5mg / mL, 95mg / mL, 97.5mg / mL, 100mg / mL, 102.5mg / mL, 105mg / mL, 107.5mg / mL, 110mg / mL, 112 .5mg / mL, 115mg / mL, 117.5mg / mL, 120mg / mL, 122.5mg / mL, 125mg / mL, 127.5mg / mL, 130mg / mL, 132.5mg / mL, 135mg / m L, 137.5mg / mL, 140mg / mL, 142.5mg / mL, 145mg / mL, 147.5mg / mL, 150mg / mL, 152.5mg / mL, 155mg / mL, 157.5mg / mL, 160 mg / mL, 162.5mg / mL, 165mg / mL, 167.5mg / mL, 170mg / mL, 172.5mg / mL, 175mg / mL, 177.5mg / mL, 180mg / mL, 182.5mg / m L, 185mg / mL, 187.5mg / mL, 190mg / mL, 192.5mg / mL, 195mg / mL, 197.5mg / mL, 200mg / mL, 202.5mg / mL, 205mg / mL, 207.5 In some embodiments, ASO is present in the pharmaceutical composition at concentrations of 11 mg / mL, 22 mg / mL, 212.5 mg / mL, 215 mg / mL, 217.5 mg / mL, 220 mg / mL, 222.5 mg / mL, 225 mg / mL, 227.5 mg / mL, 230 mg / mL, 232.5 mg / mL, 235 mg / mL, 237.5 mg / mL, 240 mg / mL, 242.5 mg / mL, 245 mg / mL, 247.5 mg / mL, or 250 mg / mL. In some embodiments, ASO is present in the pharmaceutical composition at concentrations of 11 mg / mL, 22 mg / mL, 33 mg / mL, 44 mg / mL, 55 mg / mL, 66 mg / mL, 77 mg / mL, 88 mg / mL, 99 mg / mL, or 100 mg / mL.

[0054]

[0093] In some embodiments, Na V 1.1 Reduction in protein expression or function is Na V1.1 This is associated with changes in splicing of nonsense mutation-dependent RNA decay-inducible exons (NMD exons) in premRNA that encodes proteins and contains NMD exons.

[0055]

[0094] In some embodiments, ASO facilitates the elimination of NMD exons from premRNA.

[0095] In some embodiments, the ASO contains nonsense mutation-dependent RNA decay-inducible exons (NMD exons), and Na V 1.1 It binds to the targeted region of the protein-coding premRNA.

[0056]

[0096] In some embodiments, ASO contains NMD exons, and Na V 1.1 Promotes the elimination of NMD exons from protein-coding premRNA.

[0097] In some embodiments, once ASO is introduced into the cell, the ASO becomes Na V 1.1 Increase the level of processed mRNA that codes for proteins.

[0057]

[0098] In some embodiments, once ASO is introduced into the cell, the ASO becomes Na V 1.1 Increase protein levels.

[0099] In some embodiments, the targeted region is located within an intron sequence adjacent to an NMD exon.

[0058]

[0100] In some embodiments, the targeted portion includes at least one nucleotide of the NMD exon.

[0101] In some embodiments, the targeted portion is located within an NMD exon.

[0059]

[0102] In some embodiments, the method treats a disease or condition.

[0103] In some embodiments, (a) Sequence IDs 21-67, 210-256 or 304-1 (b) an antisense oligomer (ASO) having at least 80% sequence identity with any one of 099, and (b) a pharmaceutically acceptable diluent, wherein the ASO is dissolved or suspended in a solution at a concentration of 0.1 to 200 mg / mL.

[0060]

[0104] In some embodiments, pharmaceutically acceptable diluents include artificial cerebrospinal fluid (aCSF) solutions.

[0105] In some embodiments, the solution contains a cerebrospinal fluid (CSF) sample derived from the subject.

[0061]

[0106] In some embodiments, ASO is present in the pharmaceutical composition at concentrations of approximately 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL.In some embodiments, the ASO is present in the pharmaceutical composition at a concentration of about 22.5 mg / mL, 25 mg / mL, 27.5 mg / mL, 30 mg / mL, 32.5 mg / mL, 35 mg / mL, 37.5 mg / mL, 40 mg / mL, 42.5 mg / mL, 45 mg / mL, 47.5 mg / mL, 50 mg / mL, 52.5 mg / mL, 55 mg / mL, 57.5 mg / mL, 60 mg / mL, 62.5 mg / mL, 65 mg / mL, 67.5 mg / mL, 70 mg / mL, 72.5 mg / mL, 75 mg / mL, 77.5 mg / mL, 80 mg / mL, 82.5 mg / mL, 85 mg / mL, 87.5 mg / mL, 90 mg / mL, 92.5 mg / mL, 95 mg / mL, 97.5 mg / mL, 100 mg / mL, 102.5 mg / mL, 105 mg / mL, 107.5 mg / mL, 110 mg / mL, 112.5 mg / mL, 115 mg / mL, 117.5 mg / mL, 120 mg / mL, 122.5 mg / mL, 125 mg / mL, 127.5 mg / mL, 130 mg / mL, 132.5 mg / mL, 135 mg / mL, 137.5 mg / mL, 140 mg / mL, 142.5 mg / mL, 145 mg / mL, 147.5 mg / mL, 150 mg / mL, 152.5 mg / mL, 155 mg / mL, 157.5 mg / mL, 160 mg / mL, 162.5 mg / mL, 165 mg / mL, 167.5 mg / mL, 170 mg / mL, 172.5 mg / mL, 175 mg / mL, 177.5 mg / mL, 180 mg / mL, 182.5 mg / mL, 185 mg / mL, 187.5 mg / mL, 190 mg / mL, 192.5 mg / mL, mg / mL, 197.5 mg / mL, 200 mg / mL, 202.5 mg / mL, 205 mg / mL, 207.5 mg / mL, 210 mg / mL, 212.5 mg / mL, 215 mg / mL, 217.5 mg / mL, mg / mL, 222.5 mg / mL, 225 mg / mL, 227.5 mg / mL, 230 mg / mL, 232.5 mg / mL, 235 mg / mL, 237.5 mg / mL, 240 mg / mL, 242.5 mg / mL, 245 mg / mL, 247.5 mg / mL or 250 mg / mL.

[0062]

[0107] In some embodiments, ASO is present in the pharmaceutical composition at concentrations of 11 mg / mL, 22 mg / mL, 33 mg / mL, 44 mg / mL, 55 mg / mL, 66 mg / mL, 77 mg / mL, 88 mg / mL, 99 mg / mL, or 100 mg / mL.

[0063]

[0108] In some embodiments, the ASO is solubilized or diluted in an isotonic solution.

[0109] In some embodiments, the ASO is solubilized or diluted in a phosphate buffer solution having a pH of at least 5.8.

[0064]

[0110] In some embodiments, ASO is solubilized or diluted in a phosphate buffer solution (pH 6.6-7.6).

[0111] In some embodiments, ASO is dissolved in a buffer containing 25–250 mM NaCl. It is solubilized or diluted.

[0065]

[0112] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–20 mM KCl.

[0113] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM Na2HPO4.

[0066]

[0114] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM NaH2PO4.

[0115] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM CaCl2.

[0067]

[0116] In some embodiments, the ASO is solubilized or diluted in a buffer containing 0.1–50 mM MgCl2.

[0117] In some embodiments, ASO is solubilized or diluted in a buffer containing 25–250 mM NaCl, 0.1–20 mM KCl, 0.1–50 mM Na2HPO4, 0.1–50 mM NaH2PO4, 0.1–50 mM CaCl2, and 0.1–50 mM MgCl2.

[0068]

[0118] In some embodiments, ASO is solubilized or diluted in a buffer containing 150 mM NaCl, 3.0 mM KCl, 0.7 mM Na2HPO4, 0.3 mM NaH2PO4, 0.79 mM MgCl2, and 1.4 mM CaCl2.

[0069]

[0119] In some embodiments, the ASO is solubilized or diluted in a buffer further containing a carbohydrate. In some embodiments, the carbohydrate includes D-glucose. In some embodiments, the ASO is solubilized or diluted in a buffer further containing 1 to 100 mM D-glucose.

[0070]

[0120] In some embodiments, the ASO is solubilized or diluted in a buffer further containing 1–100 mM NaHCO3, 1–100 mM KHCO3, or a combination thereof.

[0121] In some embodiments, the ASO is solubilized or diluted in a buffer further containing an antioxidant. In some embodiments, the antioxidant is t-butylhydroxyquinoline (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin E, or any combination thereof.

[0071]

[0122] In some embodiments, the pharmaceutical formulation does not contain preservatives.

[0123] In some embodiments, the pharmaceutical formulation is suitable for intraventricular or subarachnoid injection.

[0072]

[0124] In some embodiments, the pharmaceutical formulation is packaged in a single-use vial.

[0125] In some embodiments, a kit is provided herein comprising: (i) a concentrate containing an antisense oligomer (ASO), wherein the ASO contains a sequence having at least 80% sequence identity to any one of SEQ ID NOs. 21-67, 210-256, or 304-1099; (ii) a diluent, wherein the concentrate is miscible with the diluent; and (iii) instructions for use of diluting or solubilizing the ASO in the diluent.

[0073]

[0126] In some embodiments, the diluent is an artificial cerebrospinal fluid (aCSF) solution.

[0127] In some embodiments, the diluent includes an isotonic solution.

[0128] In some embodiments, the diluent includes a phosphate buffer solution having a pH of at least 5.8.

[0074]

[0129] In some embodiments, the diluent includes a phosphate buffer solution (pH 6.6-7.6).

[0130] In some embodiments, the diluent contains 25 to 250 mM NaCl.

[0075]

[0131] In some embodiments, the diluent contains 0.1 to 20 mM KCl.

[0132] In some embodiments, the diluent contains 0.1 to 50 mM Na2HPO4.

[0133] In some embodiments, the diluent contains 0.1 to 50 mM NaH2PO4.

[0076]

[0134] In some embodiments, the diluent contains 0.1 to 50 mM CaCl2.

[0135] In some embodiments, the diluent contains 0.1 to 50 mM MgCl2.

[0136] In some embodiments, the diluent includes 25-250 mM NaCl, 0.1-20 mM KCl, 0.1-50 mM Na2HPO4, 0.1-50 mM NaH2PO4, 0.1-50 mM CaCl2, and 0.1-50 mM MgCl2.

[0077]

[0137] In some embodiments, the diluent includes 150 mM NaCl, 3.0 mM KCl, 0.7 mM Na2HPO4, 0.3 mM NaH2PO4, 0.79 mM MgCl2, and 1.4 mM CaCl2.

[0078]

[0138] In some embodiments, the diluent further comprises a carbohydrate. In some embodiments, the carbohydrate comprises D-glucose. In some embodiments, the diluent further comprises 1 to 100 mM D-glucose.

[0079]

[0139] In some embodiments, the diluent further comprises 1 to 100 mM NaHCO3, 1 to 100 mM KHCO3, or a combination thereof.

[0140] In some embodiments, the diluent further comprises an antioxidant. In some embodiments, the antioxidant is t-butylhydroxyquinoline (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin E, or a combination thereof.

[0080]

[0141] In some embodiments, the diluent does not contain a preservative.

[0142] In some embodiments, instructions for diluting or solubilizing ASO in a diluent include instructions for diluting or solubilizing ASO in a diluent to a concentration of 0.1 mg / mL to 250 mg / mL.

[0081]

[0143] In some embodiments, instructions for diluting or solubilizing ASO in a diluent include instructions for diluting or solubilizing ASO in a diluent to concentrations of approximately 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL.

[0082]

[0144] In some embodiments, the instructions for diluting or solubilizing ASO in a diluent specify that ASO be added to the diluent in concentrations of approximately 22.5 mg / mL, 25 mg / mL, 27.5 mg / mL, 30 mg / mL, 32.5 mg / mL, 35 mg / mL, 37.5 mg / mL, 40 mg / mL, 42.5 mg / mL, 45 mg / mL, 47.5 mg / mL, 50 mg / mL, 52.5 mg / mL, 55 mg / mL, 57.5 mg / mL, 60 mg / mL, and 62 mg / mL. 5mg / mL, 65mg / mL, 67.5mg / mL, 70mg / mL, 72.5mg / mL, 75mg / mL, 77.5mg / mL, 80mg / mL, 82.5mg / mL, 85mg / mL, 87.5mg / mL , 90mg / mL, 92.5mg / mL, 95mg / mL, 97.5mg / mL, 100mg / mL, 102.5mg / mL, 105mg / mL, 107.5mg / mL, 110mg / mL, 112.5mg / mL , 115mg / mL, 117.5mg / mL, 120mg / mL, 122.5mg / mL, 125mg / mL, 127.5mg / mL, 130mg / mL, 132.5mg / mL, 135mg / mL, 137.5m g / mL, 140mg / mL, 142.5mg / mL, 145mg / mL, 147.5mg / mL, 150mg / mL, 152.5mg / mL, 155mg / mL, 157.5mg / mL, 160mg / mL, 16 2.5mg / mL, 165mg / mL, 167.5mg / mL, 170mg / mL, 172.5mg / mL, 175mg / mL, 177.5mg / mL, 180mg / mL, 182.5mg / mL, 185mg / m L, 187.5mg / mL, 190mg / mL, 192.5mg / mL, 195mg / mL, 197.5mg / mL, 200mg / mL, 202.5mg / mL, 205mg / mL, 207.5mg / mL, 210 Includes instructions for use on diluting or solubilizing to concentrations of mg / mL, 212.5 mg / mL, 215 mg / mL, 217.5 mg / mL, 220 mg / mL, 222.5 mg / mL, 225 mg / mL, 227.5 mg / mL, 230 mg / mL, 232.5 mg / mL, 235 mg / mL, 237.5 mg / mL, 240 mg / mL, 242.5 mg / mL, 245 mg / mL, 247.5 mg / mL, or 250 mg / mL.

[0083]

[0145] In some embodiments, instructions for diluting or solubilizing ASO in a diluent include instructions for diluting or solubilizing ASO in a diluent to concentrations of approximately 11 mg / mL, 22 mg / mL, 33 mg / mL, 44 mg / mL, 55 mg / mL, 66 mg / mL, 77 mg / mL, 88 mg / mL, 99 mg / mL, or 100 mg / mL.

[0084]

[0146] In some embodiments, instructions for diluting or solubilizing ASO in a diluent include instructions for diluting or solubilizing about 0.5 milligrams to about 500 milligrams of ASO in a diluent.

[0085]

[0147] In some embodiments, the instructions for diluting or solubilizing ASO in a diluent are approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47. 5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127. 5, 130, 132.5, 135, 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197. Includes instructions for use for diluting or solubilizing 5, 200, 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg of ASO in a diluent.

[0086]

[0148] In some embodiments, the use of antisense oligomers (ASOs) for the manufacture of pharmaceuticals for treating or preventing diseases or conditions characterized by reduced expression or function of the NaV1.1 protein in human subjects requiring treatment or prevention of such diseases or conditions. The use of a pharmaceutical product is provided herein, wherein the pharmaceutical product is administered in a first dose of approximately 0.5 milligrams to approximately 500 milligrams, and the ASO contains a sequence having at least 80% sequence identity to any one of sequence numbers 21-67, 210-256, or 304-1099. In some embodiments, the pharmaceutical product is approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 5 7.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 1 30, 132.5, 135, 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, It is administered in a first dose of 195, 197.5, 200, 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg. Inclusion of references

[0149] All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually incorporated by reference.

[0087]

[0150] The features of this disclosure are described in detail in the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description and accompanying drawings illustrating exemplary embodiments in which the principles of this disclosure are utilized. [Brief explanation of the drawing]

[0088] [Figure 1-1]

[0151] Figures 1A and 1B illustrate schematic diagrams of the therapeutic-mediated elimination of nonsense mutation-dependent mRNA degradation-inducible exons (NMD exon mRNA) from target premRNA and nonsense mutation-dependent mRNA degradation-inducible exons from premRNA to increase the expression of full-length target protein or functional RNA. Figure 1A shows a cell divided into nuclear and cytoplasmic compartments. In the nucleus, the premRNA transcript of the target gene is spliced ​​to produce processed mRNA, which is then exported to the cytoplasm and translated into the target protein. For this target gene, a portion of the processed mRNA contains nonsense mutation-dependent mRNA degradation-inducible exons (NMD exon mRNA), which are degraded in the cytoplasm, resulting in the elimination of target protein production. Figure 1B shows an example of the same cell divided into nuclear and cytoplasmic compartments. Treatment with therapeutic agents, such as antisense oligomers (ASOs), promotes the elimination of nonsense mutation-dependent mRNA decay-inducible exons from premRNA, leading to an increase in processed mRNA, which is subsequently translated into higher levels of target proteins. [Figure 1-2]

[0152] Figure 1C is a schematic diagram of therapeutic ASO-mediated elimination of nonsense mutation-dependent mRNA decay-inducible exons from premRNA, which reduces unproductive processed mRNA (e.g., those with NMD exons), increases productive mRNA (e.g., those without NMD exons), and increases the expression of full-length target proteins from productive mRNA. [Figure 1-3]

[0153] Figure 1D shows the identification of exemplary sequences in the SCN1A gene encoding a nonsense mutation-dependent mRNA decay (NMD)-inducible exon. The identification of sequences in the SCN1A gene encoding the NMD-inducible exon using comparative genomics is shown and visualized in the UCSC Genome Browser. The upper panel shows a graphical representation of the SCN1A gene for scaling. Conservation levels across 100 vertebrate species are shown as peaks. The highest peaks correspond to exons (black squares), and no peaks are observed for most introns (lines with arrowheads). A conservation peak was identified in intron 20 (NM_006920) and is shown in the middle panel. Examination of the conserved sequence identified a 64 bp exon-like sequence (lower panel, highlighted in gray) flanked by 3' and 5' splice sites (underlined sequences). Including this exon leads to a frameshift and the introduction of an immature termination codon into exon 21, making the transcript a target for NMD. [Figure 2]

[0154] Figure 2 shows the study design timeline for monitoring wild-type (WT) and Dravet syndrome (DS) mice, as well as Kaplan-Meier curves showing DS and WT littermates monitored for 14 weeks from survival. [Figure 3]

[0155] Figure 3 shows the experimental design of an EEG seizure monitoring study in DS mice and their wild littermates. [Figure 4-1]

[0156] Figures 4A–4E show the results of seizure monitoring in mice administered with ASO-22 or PBS. Figure 4A shows an exemplary ECG recording in DS mice. Figure 4B shows the number of seizures occurring in different brain regions in the two mouse groups. * indicates p<0.05. Figure 4C summarizes the total number of spontaneous seizures (generalized and focal) recorded between P22 and P46 in DS mice administered with PBS (n=21) or ASO-22 (n=21). * indicates p<0.05. Figure 4D shows the number of mice with several seizures in each group. Figure 4E shows the effect of ASO-22 on the latency to the first recorded seizure recorded between P22 and P46 in DS mice administered with PBS (n=21) or ASO-22 (n=21). [Figure 4-2]

[0156] Figures 4A-4E show the results of seizure monitoring in mice administered with ASO-22 or PBS. Figure 4A shows exemplary ECG recordings in DS mice. Figure 4B shows the number of seizures occurring in different brain regions in the two mouse groups. * indicates p<0.05. Figure 4C summarizes the total number of spontaneous seizures (generalized and focal) recorded between P22 and P46 in DS mice administered with PBS (n=21) or ASO-22 (n=21). * indicates p<0.05. Figure 4D shows the number of mice in each group that had several seizures. Figure 4E shows the effect of ASO-22 on the latency to the first recorded seizure recorded between P22 and P46 in DS mice administered with PBS (n=21) or ASO-22 (n=21). [Figure 4-3]

[0156] Figures 4A-4E show the results of seizure monitoring in mice administered with ASO-22 or PBS. Figure 4A shows exemplary ECG recordings in DS mice. Figure 4B shows the number of seizures occurring in different brain regions in the two mouse groups. * indicates p<0.05. Figure 4C summarizes the total number of spontaneous seizures (generalized and focal) recorded between P22 and P46 in DS mice administered with PBS (n=21) or ASO-22 (n=21). * indicates p<0.05. Figure 4D shows the number of mice in each group that had several seizures. Figure 4E shows the effect of ASO-22 on the latency to the first recorded seizure recorded between P22 and P46 in DS mice administered with PBS (n=21) or ASO-22 (n=21). [Figure 5-1]

[0157] Figures 5A–5G show that a single ICV injection of 20 μg of ASO-22 in P2 resulted in a reduction in SUDEP incidence and an increase in NaV1.1 protein expression in DS mice. Figure 5A is a schematic diagram of the experimental design. Figures 5B, 5C, 5D, 5E, 5F, and 5G illustrate ASO-22 exposure, Scn1a expression, and NaV1.1 expression in brain tissue at 7 or 14 weeks after a single ICV injection of ASO-22 (20 μg) or PBS in P2, respectively. [Figure 5-2]

[0157] Figures 5A-5G show that a single ICV injection of 20 μg of ASO-22 in P2 resulted in a reduction in SUDEP incidence and an increase in NaV1.1 protein expression in DS mice. Figure 5A is a schematic diagram of the experimental design. Figures 5B, 5C, 5D, 5E, 5F, and 5G illustrate ASO-22 exposure, Scn1a expression, and NaV1.1 expression in brain tissue at 7 or 14 weeks after a single ICV injection of ASO-22 (20 μg) or PBS in P2, respectively. [Figure 6]

[0158] Figures 6A-6B show the survival percentages of DS and WT mice after a single ICV injection of ASO-22 (60 μg) or PBS at P14. [Figure 7-1]

[0159] Figures 7A to 7F show ASO-22 exposure, Scn1a expression, and NaV1.1 expression in brain tissue at P35 and P90 after a single ICV injection of ASO-22 (60 μg) or PBS at P14, respectively. [Figure 7-2]

[0159] Figures 7A to 7F show ASO-22 exposure, Scn1a expression, and NaV1.1 expression in brain tissue at P35 and P90 after a single ICV injection of ASO-22 (60 μg) or PBS at P14, respectively. [Figure 8]

[0160] Figure 8 shows the experimental conditions and the number of monkeys used per group. [Figure 9]

[0161] Figures 9A-9B show the levels of ASO-22 in the brains of cynomolgus monkeys at study 3 and day 29. [Figure 10]

[0162] Figures 10A and 10B show the levels of NaV1.1 protein in the brain region of cynomolgus monkeys on day 3 and day 29. [Figure 11]

[0163] Figures 11A and 11B show the percentage of productive SCN1A genes relative to total SCN1A genes in cynomolgus monkeys on day 3 and day 29, as an assessment of target involvement. [Figure 12]

[0164] Figure 12A shows the plasma pharmacokinetics of ASO-22 in cynomolgus monkeys after intraarachnoid administration. Figure 12B shows the levels of ASO-22 in cerebrospinal fluid (CSF) of cynomolgus monkeys on study days 3 and 29. [Figure 13-1]

[0165] Figures 13A–13D illustrate the identification of alternative splicing events in SCN1A that result in NMD. Figure 13A shows SCN1A splicing isoforms in ReNcells, with or without alternative exons, as demonstrated by RT-PCR. Figure 13B shows an evaluation of alternative splicing events in the SCN1A gene in the cerebral cortex from four different sources. Figure 13C shows TBE PAGE of RT-PCR products corresponding to productive (lower band, 498 bp) and non-productive (upper band, 562 bp) Scn1a transcripts amplified from total RNA extracted from P0–P20 and 10-month-old WT C57BL / 6J mouse brains. Mouse Gapdh was used as a loading control. Figure 13D summarizes the expression of productive and non-productive Scn1a transcripts in the brain of a mouse immediately after birth, calculated using the optical density of the PCR products shown in Figure 13C. [Figure 13-2]

[0165] Figures 13A-13D illustrate the identification of alternative splicing events in SCN1A that result in NMD. Figure 13A shows SCN1A splicing isoforms in ReNcells, with or without alternative exons, as demonstrated by RT-PCR. Figure 13B shows an evaluation of alternative splicing events in the SCN1A gene in the cerebral cortex from four different sources. Figure 13C shows TBE PAGE of RT-PCR products corresponding to productive (lower band, 498 bp) and non-productive (upper band, 562 bp) Scn1a transcripts amplified from total RNA extracted from P0-P20 and 10-month-old WT C57BL / 6J mouse brains. Mouse Gapdh was used as a loading control. Figure 13D summarizes the expression of productive and non-productive Scn1a transcripts in the brain of a mouse immediately after birth, calculated using the optical density of the PCR products shown in Figure 13C. [Figure 14-1]

[0166] Figures 14A-14E illustrate that the selected ASO suppressed NMD splicing events and increased the expression of productive Scn1a mRNA in ReNcells. [Figure 14-2]

[0166] Figures 14A to 14E show that the selected ASO suppressed the NMD splicing event and increased the expression of productive Scn1a mRNA in ReNcells. [Figure 15-1]

[0167] Figures 15A-15C show the dose-dependent effects of ASO-22 on Scn1a mRNA splicing and expression in ReNcells. [Figure 15-2]

[0167] Figures 15A-15C show the dose-dependent effects of ASO-22 on splicing and expression of Scn1a mRNA in ReNcells. [Figure 16-1]

[0168] Figures 16A–16H illustrate that ASO-22 ICV injection induces a dose-dependent, persistent increase in Scn1a mRNA and NaV1.1 protein expression in mouse brains. [Figure 16-2]

[0168] Figures 16A-16H illustrate that ASO-22 ICV injection induces a dose-dependent, persistent increase in Scn1a mRNA and NaV1.1 protein expression in mouse brains. [Figure 16-3]

[0168] Figures 16A-16H illustrate that ASO-22 ICV injection induces a dose-dependent, persistent increase in Scn1a mRNA and NaV1.1 protein expression in mouse brains. [Figure 17]

[0169] Figure 17 shows the dose-dependent effect of ASO-22 on Scn1a mRNA expression in the brains of neonatal mice injected with ICV. [Figure 18]

[0170] Figure 18 shows the dose-dependent effect of ASO-22 on NaV1.1 expression in the brains of neonatal mice injected with ICV. [Figure 19]

[0171] Figure 19 shows the expression of Scn1a mRNA in mouse brains at different post-injection days. [Figure 20]

[0172] Figure 20 shows the expression of NaV1.1 in mouse brains at different post-injection days. [Figure 21]

[0173] Figure 21 shows the validation of the two anti-NaV1.1 antibodies used in the examples. The specificity of the two anti-NaV1.1 antibodies, Alomone ASC-001 and NeuroMab 75-023, was tested using total protein prepared from the brains of Scn1a- / - mice (center lane) and the brains of two wild littermates (left and right lanes). [Figure 22]

[0174] Figure 22 is a schematic diagram of the clinical findings of Dravet syndrome and its relative prevalence by age. AA: Atypical absence, AE: Acute encephalopathy, CG: Crouching gait, CPS: Complex partial seizure, DD: Developmental delay, DS: Dravet syndrome, EEG: Electroencephalogram, FSz: Complex febrile seizure, GMS: Generalized motor seizure, HS: High fever susceptibility, m: Month, MSz: Myoclonus seizure, OS: Hypostatic state, SE: Status epilepticus, SUDEP: Sudden unexpected death in epilepsy, y: Year, *60% moderate fever, mainly clonic generalized and unilateral motor seizures, **It is difficult to distinguish between AA and CPS without EEG recordings during seizures, and therefore their exact prevalence is unknown. See, for example, Gataullina and Dulac, 2007, whose full disclosure is incorporated herein by reference. [Figure 23]

[0175] Figure 23 shows TANGO (Targeted Enhancement of Nuclear Gene Output) which can be used to treat Dravet syndrome. [Figure 24]

[0176] Figure 24 illustrates the novel potential of TANGO technology in Dravet syndrome. [Figure 25]

[0177] Figure 25 shows the methods: study design. A Phase 1 / 2a open-label, two-part study conducted at approximately 20 sites across the United States. [Figure 26]

[0178] Figure 26 shows a schematic diagram of the research design. [Figure 27]

[0179] Figure 27 shows the method: patient diagram. [Figure 28]

[0180] Figure 28 shows the research evaluation. [Modes for carrying out the invention]

[0089]

[0181] Certain specific details are given in this description to provide a complete understanding of the various embodiments. However, those skilled in the art will understand that the disclosure can be carried out without these details. In other cases, well-known structures are not shown in detail and are not described in order to avoid unnecessarily obscuring the description of the embodiments.

[0090]

[0182] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but suitable methods and materials are described below.

[0091] definition

[0183] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise clearly indicated.

[0092]

[0184] It should be noted that the term “or” is generally used to mean “and / or” unless the context explicitly indicates otherwise. The terms “and / or” and “any combination thereof” and their grammatical equivalents may be used synonymously as used herein. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean “A individually, B individually, C individually, A and B, B and C, A and C, and A, B and C.” The term “or” may be used conjunctively or disjunctively unless the context specifically refers to disjunctive use.

[0093]

[0185] The terms “approximately” or “about” may mean within an acceptable margin of error of a particular value, as determined by those skilled in the art, and may vary to some extent depending on the method by which the value is measured or determined, i.e., the limits of the measurement system. For example, “approximately” may mean within one or more standard deviations per run in the art. Or, “approximately” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of the value, up to five times, more preferably up to two times. Where a particular value is described in this application and claims, unless otherwise specified, the term “approximately” should be assumed to mean within an acceptable margin of error of that particular value.

[0094]

[0186] As used herein and in the claims, the words “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “contains” and “contain”) are inclusive or unrestrictive and do not preclude any further unlisted elements or method steps. It is intended that any embodiment discussed herein can be performed with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the methods of the Disclosure can be achieved using the compositions of the Disclosure.

[0095]

[0187] References to “Embodiments,” “Some Embodiments,” “Embodiments,” “One Embodiment,” “A Certain Embodiment,” or “Other Embodiments” in this Specified Specified Characteristics, Structures, or Features described in relation to an Embodiment are included in at least some, but not all, embodiments of this Disclosure. To facilitate understanding of this Disclosure, several terms and phrases are defined below.

[0096]

[0188] The terms “oligonucleotide sequence,” “nucleic acid sequence,” “polynucleic acid sequence,” “nucleotide sequence,” and “nucleotide acid sequence” are used synonymously herein in their broadest sense and have the same meaning herein, preferably referring to DNA or RNA. A nucleic acid sequence is a polymer containing or consisting of nucleotide monomers covalently bonded to one another by phosphodiester bonds of a sugar / phosphate backbone. The term “nucleic acid sequence” also includes modified nucleic acid sequences, such as DNA or RNA that are base-modified, sugar-modified, or backbone-modified.

[0097]

[0189] In this specification, the terms “fragment” or “sequence fragment,” which have the same meaning, refer to a shorter portion of the full-length sequence of a nucleic acid molecule such as DNA or RNA, or a protein. Therefore, a fragment typically consists of sequences that are identical to the corresponding stretch within the full-length sequence. Preferred fragments of the sequence in the context of the present invention consist of entities corresponding to continuous stretches of entities in the molecule from which the fragment originates, for example, continuous stretches of nucleotides or amino acids, which correspond to at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or even 100% of the whole (i.e., full-length) molecule from which the fragment originates. For example, a “fragment” or “functional fragment” of a polynucleotide or polypeptide is a fragment of a polynucleotide or polypeptide that is shorter than the full-length immature or mature nucleotide or polypeptide and has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or even 100% or more of the activity of the full-length mature reference polynucleotide or polypeptide. The fragment of interest may be prepared by recombinant, synthetic, or digestive methods.

[0098]

[0190] The term "recombinant," when used, for example, in relation to cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified by, or are the result of, laboratory methods. Therefore, for example, the term "recombinant polynucleotide" may refer to a polynucleotide that does not exist naturally, but is synthesized or manipulated in vitro, such as a polynucleotide produced by laboratory methods. Recombinant polynucleotides can be synthesized in the laboratory and / or prepared by using recombinant DNA techniques, such as enzymatic modification of DNA, e.g., enzymatic restriction digestion, ligation, and cloning. Recombinant polypeptides can be prepared by in vitro transcription of recombinant DNA and subsequent in vitro translation of the resulting messenger RNA (mRNA). Under suitable conditions, recombinant polynucleic acids or RNA can be incorporated into cells, and recombinant polypeptides can be expressed within the cells. Recombinant proteins may contain amino acid residues not found in the protein's natural (non-recombinant) form, or they may contain modifications, such as labeled amino acid residues.

[0099]

[0191] The term “isolated” means that a polynucleotide, polypeptide, protein, or fragment thereof is separated from the cellular and other components to which it is normally associated in nature. For example, with respect to polynucleotides, an isolated polynucleotide is one that has been separated from the 5' and 3' ends that are normally associated in the naturally occurring sequence. As will be apparent to those skilled in the art, polynucleotides, polypeptides, proteins, or fragments thereof that do not exist in nature do not require “isolation” to be distinguished from their naturally occurring counterparts. Furthermore, “concentrated,” “isolated,” or “diluted” polynucleotides, polypeptides, proteins, or fragments thereof are distinguishable from their naturally occurring counterparts in that the number of molecules per unit concentration or volume is greater for “concentrated” or less for “isolated” or “diluted” than that of their naturally occurring counterparts.

[0100]

[0192] In the context of two or more nucleic acid or polypeptide sequences, the term “identical” or “identity” percentage refers to two or more sequences or subsequences that, when compared and aligned for maximum match across a comparison window, are identical or have a specified percentage of identical nucleotide or amino acid residues (i.e., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity across a specified region of the entire polypeptide sequence of the Invention or an individual domain of the polypeptide of the Invention), or a specified region as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Such sequences that are at least about 80% identical are said to be “substantially identical.” In some embodiments, two sequences are 100% identical. In some embodiments, the two sequences are 100% identical over the entire length of one of the sequences (for example, the shorter of the two sequences if the sequences are of different lengths). In various embodiments, identity may refer to the complement of the test sequences.

[0101]

[0193] In some embodiments, identity exists over regions that are at least about 2 to about 400 amino acids or nucleotides in length. In some embodiments, identity exists over regions that are at least about 2 to about 390, at least about 2 to about 380, at least about 2 to about 370, at least about 2 to about 360, at least about 2 to about 350, at least about 2 to about 340, at least about 2 to about 330, at least about 2 to about 320, at least about 2 to about 310, at least about 2 to about 300, at least about 2 to about 290, at least about 2 to about 280, at least about 2 to about 270, at least about 2 to about 260, at least about 2 to about 250, at least about 2 to about 200, at least about 2 to about 150, and at least about 2 to about 100 amino acids or nucleotides in length. In some embodiments, identity exists over regions that are at least about 2 to about 90 amino acids or nucleotides in length, at least about 2 to about 85, at least about 2 to about 80, at least about 2 to about 75, at least about 2 to about 70, at least about 2 to about 65, at least about 2 to about 60, at least about 2 to about 55, at least about 2 to about 50, at least about 2 to about 45, at least about 2 to about 40, at least about 2 to about 35, at least about 2 to about 30, at least about 2 to about 25, at least about 2 to about 20, at least about 2 to about 10, and at least about 2 to about 5.

[0102]

[0194] In some embodiments, identity is at least about 3 to about 400, about 4 to about 400, about 5 to about 400, about 6 to about 400, about 7 to about 400, about 8 to about 400, about 9 to about 400, It exists across regions that are approximately the length of amino acids or nucleotides from about 10 to 400, 11 to 400, 12 to 400, 13 to 400, 14 to 400, 15 to 400, 16 to 400, 17 to 400, 18 to 400, 19 to 400, 20 to 400, 21 to 400, 22 to 400, 23 to 400, 24 to 400, 25 to 400, 26 to 400, 27 to 400, 28 to 400, 29 to 400, 30 to 400, 31 to 400, 32 to 400, 33 to 400, 34 to 400, and 35 to 400. In some embodiments, identity is at least about 40-400, about 45-400, about 50-400, about 55-400, about 60-400, about 61-400, about 62-400, about 63-400, about 64-400, about 65-400, about 66-400, about 67-400, about 68-400, about 69-400, about 70-400, about 71- They exist over regions that are approximately 400, 72-400, 73-400, 74-400, 75-400, 80-400, 85-400, 90-400, 100-400, 150-400, 200-400, 250-400, 300-400, and 350-400 amino acids or nucleotides in length.

[0103]

[0195] In some embodiments, the identity exists over a region that is at least about 2 to about 343, about 3 to about 343, about 4 to about 343, about 7 to about 343, about 9 to about 343, about 11 to about 343, about 15 to about 343, about 16 to about 343, about 20 to about 343, about 25 to about 343, about 62 to about 343, about 2 to about 317, about 3 to about 317, about 4 to about 317, about 7 to about 317, about 9 to about 317, about 11 to about 317, about 15 to about 317, about 16 to about 317, about 20 to about 317, about 25 to about 317, about 62 to about 317, about 2 to about 300, about 3 to about 300, about 4 to about 300, about 7 to about 300, about 9 to about 300, about 11 to about 300, about 15 to about 300, about 16 to about 300, about 20 to about 300, about 25 to about 300, about 62 to about 300, about 2 to about 62, about 3 to about 62, about 4 to about 62, about 7 to about 62, about 9 to about 62, about 11 to about 62, about 15 to about 62, about 16 to about 62, about 20 to about 62, about 25 to about 62 amino acids or nucleotides in length.

[0104]

[0196] The term "genetically modified" means, in turn, containing and / or expressing a foreign gene or nucleic acid sequence that modifies the genotype or phenotype of a cell or its progeny. In other words, it refers to any addition, deletion or disruption of the endogenous nucleotides of a cell.

[0105]

[0197] The term "operatively linked" may refer to a functional relationship between two or more nucleic acid sequences, for example, a functional relationship between a transcriptional regulatory or signal sequence and the transcribed sequence. For example, a target motif or nucleic acid encoding a target motif is operatively linked to a coding sequence when it is expressed as a preprotein involved in targeting the polypeptide encoded by the coding sequence to the cell membrane, intracellular or extracellular compartment. For example, a signal peptide or nucleic acid encoding a signal peptide is operatively linked to a coding sequence when it is expressed as a preprotein involved in the secretion of the polypeptide encoded by the coding sequence. For example, a promoter is operatively linked when it stimulates or modulates the transcription of a coding sequence.

[0106]

[0198] The terms "subject" or "patient" include vertebrates or mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees and other apes and monkey species, livestock, such as cows, horses, sheep, goats, pigs, companion animals, such as rabbits, dogs and cats, rodents, such as rats, mice and guinea pigs, and experimental animals, etc. In one aspect, the mammal is a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, the "non-human animal" is a mammal, such as a rodent , such as a rat or a mouse. In one embodiment, the non-human animal is a mouse.

[0107]

[0199] A "control" is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative". Treatment method

[0200] In some aspects, Na V1.1 A method for treating or preventing a disease or condition characterized by reduced protein expression or function in a human subject requiring treatment or prevention of the disease or condition, wherein the human subject is given approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 13 A method is provided herein that includes the step of administering a pharmaceutical composition comprising an antisense oligomer (ASO) in a first dose of 5, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg, wherein the ASO comprises a sequence having at least 80% sequence identity to any one of sequence numbers 21-67, 210-256, or 304-1099, thereby treating or preventing a disease or condition in a human subject. In some embodiments, the ASO comprises a sequence having at least 80% sequence identity to any one of sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b, thereby treating or preventing a disease or condition in a human subject.

[0108]

[0201] In some embodiments, Na V1.1 A method for treating or preventing a disease or condition characterized by reduced protein expression or function in a human subject requiring treatment or prevention, comprising the step of administering to the human subject a pharmaceutical composition comprising a first dose of an antisense oligomer (ASO), wherein the ASO comprises a sequence having at least 80% sequence identity to any one of sequence numbers 21-67, 210-256, or 304-1099, thereby treating or preventing the disease or condition in a human subject, the human subject being up to 18 years of age, is provided herein. In some embodiments, the ASO comprises a sequence having at least 80% sequence identity to any one of sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b, thereby treating or preventing the disease or condition in a human subject, the human subject being up to 18 years of age.

[0109]

[0202] In some embodiments, Na V 1.1 A method for treating or preventing a disease or condition characterized by reduced protein expression or function in a human subject requiring treatment or prevention of such disease or condition is provided herein, comprising the step of administering to the human subject a pharmaceutical composition comprising a single dose of an antisense oligomer (ASO), wherein the ASO comprises a sequence having at least 80% sequence identity to any one of sequence numbers 21-67, 210-256, or 304-1099, thereby treating or preventing the disease or condition in a human subject. In some embodiments, the ASO comprises a sequence having at least 80% sequence identity to any one of sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b, thereby treating or preventing the disease or condition in a human subject.

[0110]

[0203] In some embodiments, the pharmaceutical composition is administered into the subarachnoid space of a human subject. In some embodiments, the pharmaceutical composition is administered into the cerebrospinal fluid of a human subject. In some embodiments, the pharmaceutical composition is administered into the brain of a human subject. In some embodiments, the pharmaceutical composition is administered into the cerebrospinal fluid in the brain of a human subject.

[0111]

[0204] In some embodiments, the pharmaceutical composition is administered as a bolus injection. In terms of administration methods, the pharmaceutical composition is administered by infusion using a delivery pump. In some embodiments, the pharmaceutical composition is administered by intracerebral injection. In some embodiments, the pharmaceutical composition is administered by subarachnoid injection.

[0112] therapeutic dose

[0205] In some embodiments, the first dose is a single dose. In some embodiments, the method further includes a step of evaluating the tolerability or efficacy of the pharmaceutical composition.

[0113]

[0206] In some embodiments, the methods described herein include administering to a human subject a pharmaceutical composition comprising an ASO described herein in a first dose of about 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg.

[0114]

[0207] In some embodiments, the methods described herein apply to human subjects in doses of approximately 0.1 to 1000 mg, approximately 0.2 to 1000 mg, approximately 0.3 to 1000 mg, approximately 0.4 to 1000 mg, approximately 0.5 to 1000 mg, approximately 0.6 to 1000 mg, approximately 0.7 to 1000 mg, approximately 0.8 to 1000 mg, approximately 0.9 to 1000 mg, 1 to 1000 mg, approximately 2 to 1000 mg, approximately 3 to 1000 mg, and approximately 4 to 1 000mg, about 5 to about 1000mg, about 6 to about 1000mg, about 7 to about 1000mg, about 8 to about 1000mg, about 9 to about 1000mg, about 10 to about 1000mg, about 15 to about 1000mg, about 20 to about 10 00mg, about 25 to about 1000mg, about 30 to about 1000mg, about 35 to about 1000mg, about 40 to about 1000mg, about 45 to about 1000mg, about 50 to about 1000mg, about 55 to about 1000mg, about 60 ~1000mg, 65~1000mg, 70~1000mg, 75~1000mg, 80~1000mg, 85~1000mg, 90~1000mg, 95~1000mg , about 100 to about 1000 mg, about 150 to about 1000 mg, about 200 to about 1000 mg, about 250 to about 1000 mg, about 300 to about 1000 mg, about 350 to about 1000 mg, about 400 to about 1000 mg, The procedure includes administering a pharmaceutical composition containing an ASO as described herein in a first dose of approximately 450-1000 mg, approximately 500-1000 mg, approximately 550-1000 mg, approximately 600-1000 mg, approximately 650-1000 mg, approximately 700-1000 mg, approximately 750-1000 mg, approximately 800-1000 mg, approximately 850-1000 mg, approximately 900-1000 mg, or approximately 950-1000 mg.

[0115]

[0208] In some embodiments, the methods described herein apply to human subjects in doses of 0.1-1000 mg, 0.2-1000 mg, 0.3-1000 mg, 0.4-1000 mg, 0.5-1000 mg, 0.6-1000 mg, 0.7-1000 mg, 0.8-1000 mg, 0.9-1000 mg, 1-1000 mg, 2-1000 mg, 3-1000 mg, 4-1000 mg, 5-1000 mg, 6-1000 mg, 7-1000 mg, 8-1000 mg, 9-1000 mg, 10-1000 mg, 15-1000 mg, 20-1000 mg, 25-1000 mg, 30-1000 mg, 35-1000 mg, and 40-1000 mg. g, 45~1000mg, 50~1000mg, 55~1000mg, 60~1000mg, 65~1000mg, 70~1000mg, 75~1000 mg, 80~1000mg, 85~1000mg, 90~1000mg, 95~1000mg, 100~1000mg, 150~1000mg, 200~1 000mg, 250~1000mg, 300~1000mg, 350~1000mg, 400~1000mg, 450~1000mg, 500~1000 mg, 550~1000mg, 600~1000mg, 650~1000mg, 700~1000mg, 750~1000mg, 800~1000mg, 8 The procedure includes administering a pharmaceutical composition containing the ASO described herein in a first dose of 50-1000 mg, 900-1000 mg, or 950-1000 mg.

[0116]

[0209] In some embodiments, the methods described herein apply to human subjects in doses of approximately 0.1 to 950 mg, approximately 0.1 to 900 mg, approximately 0.1 to 850 mg, approximately 0.1 to 800 mg, approximately 0.1 to 750 mg, approximately 0.1 to 700 mg, approximately 0.1 to 650 mg, approximately 0.1 to 600 mg, approximately 0.1 to 550 mg, approximately 0.1 to 500 mg, approximately 0.1 to 450 mg, and approximately 0.1 ~400mg, approximately 0.1~350mg, approximately 0.1~300mg, approximately 0.1~250mg, approximately 0.1~200mg, approximately 0.1~150mg, approximately 0.1~100mg, approximately 0. 1 to about 95 mg, about 0.1 to about 90 mg, about 0.1 to about 85 mg, about 0.1 to about 80 mg, about 0.1 to about 75 mg, about 0.1 to about 70 mg, about 0.1 to about 65 mg, about 0.1 to about 60 mg , about 0.1 to about 55 mg, about 0.1 to about 50 mg, about 0.1 to about 45 mg, about 0.1 to about 40 mg, about 0.1 to about 35 mg, about 0.1 to about 30 mg, about 0.1 to about mg, about 0.1 to about 2 5 mg, about 0.1 to about 20 mg, about 0.1 to about 10 mg, about 0.1 to about 9 mg, about 0.1 to about 8 mg, about 0.1 to about 7 mg, about 0.1 to about 6 mg, about 0.1 to about 5 mg, about 0.1 to about 4 The procedure includes administering a pharmaceutical composition containing an ASO as described herein in a first dose of mg, approximately 0.1 to approximately 3, approximately 0.1 to approximately 2 mg, approximately 0.1 to approximately 1 mg, approximately 0.1 to approximately 0.9 mg, approximately 0.1 to approximately 0.8 mg, approximately 0.1 to approximately 0.7 mg, approximately 0.1 to approximately 0.6 mg, approximately 0.1 to approximately 0.5 mg, approximately 0.1 to approximately 0.4 mg, approximately 0.1 to approximately 0.3 mg, or approximately 0.1 to approximately 0.2 mg.

[0117]

[0210] In some embodiments, the methods described herein apply to human subjects in doses of 0.1-950 mg, 0.1-900 mg, 0.1-850 mg, 0.1-800 mg, 0.1-750 mg, 0.1-700 mg, 0.1-650 mg, 0.1-600 mg, 0.1-550 mg, 0.1-500 mg, 0.1-450 mg, 0.1-400 mg, 0.1-350 mg, 0.1-300 mg, 0.1~250mg, 0.1~200mg, 0.1~150mg, 0.1~100mg, 0.1~95mg, 0.1~90mg, 0.1~85mg, 0.1~80mg, 0.1~75mg, 0.1~70mg, 0.1~65mg, 0.1~60mg, 0.1~55mg, 0.1~50mg, 0.1~45mg, 0.1~40mg, 0.1~35mg, 0.1~30mg, 0.1~ The procedure includes administering a pharmaceutical composition containing an ASO as described herein in a first dose of mg, 0.1-25 mg, 0.1-20 mg, 0.1-10 mg, 0.1-9 mg, 0.1-8 mg, 0.1-7 mg, 0.1-6 mg, 0.1-5 mg, 0.1-4 mg, 0.1-3 mg, 0.1-2 mg, 0.1-1 mg, 0.1-0.9 mg, 0.1-0.8 mg, 0.1-0.7 mg, 0.1-0.6 mg, 0.1-0.5 mg, 0.1-0.4 mg, 0.1-0.3 mg, or 0.1-0.2 mg.

[0118]

[0211] In some embodiments, the methods described herein apply to human subjects in doses of approximately 1 to 400 mg, approximately 2 to 400 mg, approximately 3 to 400 mg, approximately 4 to 400 mg, approximately 5 to 400 mg, approximately 6 to 400 mg, approximately 7 to 400 mg, approximately 8 to 400 mg, approximately 9 to 400 mg, approximately 10 to 400 mg, approximately 20 to 400 mg, approximately 30 to 400 mg, approximately 40 to 400 mg, approximately 50 to 400 mg, approximately 60 to 400 mg, approximately 70 to 400 mg, approximately 80 to 400 mg, approximately 90 to 400 mg, approximately 100 to 400 mg, approximately 110 to 400 mg, approximately 120 to 400 mg, and approximately 13 0-400mg, 140-400mg, 150-400mg, 160-400mg, 170-400mg, 180-400mg, 190-400mg, 200-400mg, 210-400mg, 220-400mg, 230-400mg, 240-400mg, 250-400mg, 260-400mg, 270-400mg, 280-400mg, 290-400mg, 300-400mg, 310-400mg, 320-400mg, 330-400mg, 340-400mg The procedure includes administering a pharmaceutical composition containing the ASO described herein in a first dose of mg, approximately 350-400 mg, approximately 360-400 mg, approximately 370-400 mg, approximately 380-400 mg, or approximately 390-400 mg.

[0119]

[0212] In some embodiments, the methods described herein apply to human subjects in doses of 1-400 mg, 2-400 mg, 3-400 mg, 4-400 mg, 5-400 mg, 6-400 mg, 7-400 mg, 8-400 mg, 9-400 mg, 10-400 mg, 20-400 mg, 30-400 mg, 40-400 mg, 50-400 mg, 60-400 mg, 70-400 mg, 80-400 mg, 90-400 mg, 100-400 mg, 110-400 mg, 120-400 mg, 130-400 mg, approximately 140-400 mg, 150-400 mg, approximately 160-400 mg, and 170-400 mg. The procedure includes administering a pharmaceutical composition containing the ASO described herein in a first dose of g, 180-400 mg, 190-400 mg, 200-400 mg, 210-400 mg, 220-400 mg, 230-400 mg, 240-400 mg, 250-400 mg, 260-400 mg, 270-400 mg, 280-400 mg, 290-400 mg, 300-400 mg, 310-400 mg, 320-400 mg, 330-400 mg, 340-400 mg, 350-400 mg, 360-400 mg, 370-400 mg, 380-400 mg, or 390-400 mg.

[0120]

[0213] In some embodiments, the methods described herein apply to human subjects in doses of approximately 10-390 mg, approximately 10-380 mg, approximately 10-370 mg, approximately 10-360 mg, approximately 10-350 mg, approximately 10-340 mg, approximately 10-330 mg, approximately 10-320 mg, approximately 10-310 mg, approximately 10-300 mg, approximately 10-290 mg, approximately 10-280 mg, approximately 10-270 mg, approximately 10-260 mg, approximately 10-250 mg, approximately 10-240 mg, approximately 10-230 mg, approximately 10-220 mg, and approximately 10-21 mg. The procedure includes administering a pharmaceutical composition containing an ASO as described herein in a first dose of 0 mg, approximately 10 to approximately 200 mg, approximately 10 to approximately 190 mg, approximately 10 to approximately 180 mg, approximately 10 to approximately 170 mg, approximately 10 to approximately 160 mg, approximately 10 to approximately 150 mg, approximately 10 to approximately 140 mg, approximately 10 to approximately 130 mg, approximately 10 to approximately 120 mg, approximately 10 to approximately 110 mg, approximately 10 to approximately 90 mg, approximately 10 to approximately 80 mg, approximately 10 to approximately 70 mg, approximately 10 to approximately 60 mg, approximately 10 to approximately 50 mg, approximately 10 to approximately 40 mg, approximately 10 to approximately 30 mg, or approximately 10 to approximately 20 mg.

[0121]

[0214] In some embodiments, the methods described herein apply to human subjects in doses of 10-390 mg, 10-380 mg, 10-370 mg, 10-360 mg, 10-350 mg, 10-340 mg, 10-330 mg, 10-320 mg, 10-310 mg, 10-300 mg, 10-290 mg, 10-280 mg, 10-270 mg, 10-260 mg, 10-250 mg, 10-240 mg, 10-230 mg, 10-220 mg, and 10-210 mg. The procedure includes administering a pharmaceutical composition containing an ASO as described herein in a first dose of mg, 10-200 mg, 10-190 mg, 10-180 mg, 10-170 mg, 10-160 mg, 10-150 mg, 10-140 mg, 10-130 mg, 10-120 mg, 10-110 mg, 10-90 mg, 10-80 mg, 10-70 mg, 10-60 mg, 10-50 mg, 10-40 mg, 10-30 mg, or 10-20 mg.

[0122]

[0215] For certain rights, the methods described herein apply to human subjects in doses of approximately 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, and 36 mg. Approximately 37mg, approximately 38mg, approximately 39mg, approximately 40mg, approximately 41mg, approximately 42mg, approximately 43mg, approximately 44mg, approximately 45mg, approximately 46mg, approximately 47mg, approximately 48mg, approximately 49mg, approximately 50mg, approximately 51mg, approximately 52mg, approximately 53mg, approximately 54mg, approximately 55mg, approximately 56mg, approximately 57mg, approximately 58mg, approximately 59mg, approximately 60mg, approximately 61mg, approximately 62mg, approximately 63mg, approximately 64mg, approximately 65mg, approximately 66mg, approximately 67mg, approximately 68mg, approximately 69mg, approximately 70mg, approximately 71mg, approximately 72mg, approximately 73mg, approximately 74mg, approximately 75mg, approximately 76mg, approximately 77mg, approximately 78mg, approximately 79mg, approximately 80mg, approximately 81mg, approximately 82mg, approximately 83mg, approximately 84mg, approximately 85mg, approximately 86mg, approximately 87mg, approximately 88mg, approximately 89mg, approximately 90mg, approximately 91mg, approximately 92mg, approximately 93mg, approximately 94mg, approximately 95mg, approximately 96mg, approximately 97mg, approximately 98mg, approximately 99mg, approximately 100mg, approximately 101mg, approximately 102mg, approximately 103mg, approximately 104mg, approximately 105mg, approximately 106mg, approximately 107mg, approximately 108mg, approximately 109mg, approximately 110mg, approximately 111mg, approximately 112mg, approximately 113mg, approximately 114mg, approximately 115mg, approximately 116mg Approximately 117mg, approximately 118mg, approximately 119mg, approximately 120mg, approximately 121mg, approximately 122mg, approximately 123mg, approximately 124mg, approximately 125mg, approximately 126mg, approximately 127mg, approximately 128mg, approximately 129mg, approximately 130mg, approximately 131mg, approximately 132mg, approximately 133mg, approximately 134mg, approximately 135mg, approximately 136mg, approximately 137mg, approximately 138mg, approximately 139mg, approximately 140mg, approximately 141mg, approximately 142mg, approximately 143mg, approximately 144mg, approximately 145mg, approximately 146mg, approximately 147mg, approximately 148mg, approximately 149mg, approximately 150mg, approximately 151mg, approximately 15 2mg, approximately 153mg, approximately 154mg, approximately 155mg, approximately 156mg, approximately 157mg, approximately 158mg, approximately 159mg, approximately 160mg, approximately 161mg, approximately 162mg, approximately 163mg, approximately 164mg, approximately 165mg, approximately 166mg, approximately 167mg, approximately 168mg, approximately 169mg, approximately 170mg, approximately 171mg, approximately 172mg, approximately 173mg, approximately 174mg, approximately 175mg, approximately 176mg, approximately 177mg, approximately 178mg, approximately 179mg, approximately 180mg, approximately 181mg, approximately 182mg, approximately 183mg, approximately 184mg, approximately 185mg, approximately 186mg, approximately 187mg,Approximately 188mg, approximately 189mg, approximately 190mg, approximately 191mg, approximately 192mg, approximately 193mg, approximately 194mg, approximately 195mg, approximately 196mg, approximately 197mg, approximately 198mg, approximately 199mg, approximately 200mg, approximately 201mg, approximately 202mg, approximately 203mg, approximately 204mg, approximately 205mg, approximately 206mg, approximately 207mg, approximately 208mg, approximately 209mg, approximately 210mg, approximately 211mg, approximately 212mg, approximately 213mg, approximately 214mg, approximately 215mg, approximately 216mg, approximately 217mg, approximately 218mg, approximately 219mg, approximately 220mg, approximately 221mg, approximately 222mg, approximately 223mg g, approximately 224mg, approximately 225mg, approximately 226mg, approximately 227mg, approximately 228mg, approximately 229mg, approximately 230mg, approximately 231mg, approximately 232mg, approximately 233mg, approximately 234mg, approximately 235mg, approximately 236mg, approximately 237mg, approximately 238mg, approximately 239mg, approximately 240mg, approximately 241mg, approximately 242mg, approximately 243mg, approximately 244mg, approximately 245mg, approximately 246mg, approximately 247mg, approximately 248mg, approximately 249mg, approximately 250mg, approximately 251mg, approximately 252mg, approximately 253mg, approximately 254mg, approximately 255mg, approximately 256mg, approximately 257mg, approximately 258mg, approximately 25 9mg, approximately 260mg, approximately 261mg, approximately 262mg, approximately 263mg, approximately 264mg, approximately 265mg, approximately 266mg, approximately 267mg, approximately 268mg, approximately 269mg, approximately 270mg, approximately 271mg, approximately 272mg, approximately 273mg, approximately 274mg, approximately 275mg, approximately 276mg, approximately 277mg, approximately 278mg, approximately 279mg, approximately 280mg, approximately 281mg, approximately 282mg, approximately 283mg, approximately 284mg, approximately 285mg, approximately 286mg, approximately 287mg, approximately 288mg, approximately 289mg, approximately 290mg, approximately 291mg, approximately 292mg, approximately 293mg, approximately 294mg, approximately 295mg, approximately 296mg, approximately 297mg, approximately 298mg, approximately 299mg, approximately 300mg, approximately 301mg, approximately 302mg, approximately 303mg, approximately 304mg, approximately 305mg, approximately 306mg, approximately 307mg, approximately 308mg, approximately 309mg, approximately 310mg, approximately 311mg, approximately 312mg, approximately 313mg, approximately 314mg, approximately 315mg, approximately 316mg, approximately 317mg, approximately 318mg, approximately 319mg, approximately 320mg, approximately 321mg, approximately 322mg, approximately 323mg, approximately 324mg, approximately 325mg, approximately 326mg, approximately 327mg, approximately 328mg, approximately 329mg, approximately 330mgApproximately 331, mg, approx. 332 mg, approx. 333 mg, approx. 334 mg, approx. 335 mg, approx. 336 mg, approx. 337 mg, approx. 338 mg, approx. 339 mg, approx. 340 mg, approx. 341 mg, approx. 342 mg, approx. 50mg, approx. 351mg, approx. 352mg, approx. 353mg, approx. 354mg, approx. 355mg, approx. 356mg, approx. 357mg, approx. 358mg, approx. 359mg, approx. 360mg, approx. The procedure includes administering a pharmaceutical composition containing the ASO described herein in a first dose of 369 mg, approximately 370 mg, approximately 371 mg, approximately 372 mg, approximately 373 mg, approximately 374 mg, approximately 375 mg, approximately 376 mg, approximately 377 mg, approximately 378 mg, approximately 379 mg, approximately 380 mg, approximately 381 mg, approximately 382 mg, approximately 383 mg, approximately 384 mg, approximately 385 mg, approximately 386 mg, approximately 387 mg, approximately 388 mg, approximately 389 mg, approximately 390 mg, approximately 391 mg, approximately 392 mg, approximately 393 mg, approximately 394 mg, approximately 395 mg, approximately 396 mg, approximately 397 mg, approximately 398 mg, approximately 399 mg, or 400 mg.

[0123]

[0216] In some embodiments, the methods described herein apply to human subjects with doses of 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg. g, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71 mg, 72mg, 73mg, 74mg, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96 mg, 97mg, 98mg, 99mg, 100mg, 101mg, 102mg, 103mg, 104mg, 105mg, 106mg, 107mg, 108mg, 109mg, 110mg, 111mg, 112mg, 113mg, 114mg, 115mg, 116mg, 117m g, 118mg, 119mg, 120mg, 121mg, 122mg, 123mg, 124mg, 125mg, 126mg, 127mg, 128mg, 129mg, 130mg, 131mg, 132mg, 133mg, 134mg, 135mg, 136mg, 137mg, 138 mg, 139mg, 140mg, 141mg, 142mg, 143mg, 144mg, 145mg, 146mg, 147mg, 148mg, 149mg, 150mg, 151mg, 152mg, 153mg, 154mg, 155mg, 156mg, 157mg, 158mg, 1 59mg, 160mg, 161mg, 162mg, 163mg, 164mg, 165mg, 166mg, 167mg, 168mg, 169mg, 170mg, 171mg, 172mg, 173mg, 174mg, 175mg, 176mg, 177mg, 178mg, 179mg,180mg、181mg、182mg、183mg、184mg、185mg、186mg、187mg、188mg、189mg、190mg、191mg、192mg、193mg、194mg、195mg、196mg、197mg、198mg、199mg、200mg、201mg、202mg、203mg、204mg、205mg、206mg、207mg、208mg、209mg、210mg、211mg、212mg、213mg、214mg、215mg、216mg、217mg、218mg、219mg、220mg、221mg、222mg、223mg、224mg、225mg、226mg、227mg、228mg、229mg、230mg、231mg、232mg、233mg、234mg、235mg、236mg、237mg、238mg、239mg、240mg、241mg、242mg、243mg、244mg、245mg、246m、 g, 247mg, 248mg, 249mg, 250mg, 251mg, 252mg, 253mg, 254mg, 255mg, 256mg, 257mg, 258mg, 259mg, 260mg, 261mg, 262mg, 263mg, 264mg, 265mg, 266mg , 267mg, 268mg, 269mg, 270mg, 271mg, 272mg, 273mg, 274mg, 275mg, 276mg, 277mg, 278mg, 279mg, 280mg, 281mg, 282mg, 283mg, 284mg, 285mg, 286mg, 287mg, 288mg, 289mg, 290mg, 291mg, 292mg, 293mg, 294mg, 295mg, 296mg, 297mg, 298mg, 299mg, 300mg, 301mg, 302mg, 303mg, 304mg, 305mg, 306mg, 3 07mg, 308mg, 309mg, 310mg, 311mg, 312mg, 313mg, 314mg, 315mg, 316mg, 317mg, 318mg, 319mg, 320mg, 321mg, 322mg, 323mg, 324mg, 325mg, 326mg, 32 7mg, 328mg, 329mg, 330mg, 331mg, 332mg, 333mg, 334mg, 335mg, 336mg, 337mg, 338mg, 339mg, 340mg, 341mg, 342mg, 343mg, 344mg, 345mg, 346mg, 347 mg, 348mg, 349mg, 350mg, 351mg, 352mg, 353mg, 354mg, 355mg, 356mg, 357mg, 358mg, 359mg, 360mg, 361mg, 362mg, 363mg, 364mg, 365mg, 366mg, 367m The procedure includes administering a pharmaceutical composition containing the ASO described herein in a first dose of g, 368 mg, 369 mg, 370 mg, 371 mg, 372 mg, 373 mg, 374 mg, 375 mg, 376 mg, 377 mg, 378 mg, 379 mg, 380 mg, 381 mg, 382 mg, 383 mg, 384 mg, 385 mg, 386 mg, 387 mg, 388 mg, 389 mg, 390 mg, 391 mg, 392 mg, 393 mg, 394 mg, 395 mg, 396 mg, 397 mg, 398 mg, 399 mg, or 400 mg.

[0124] Treatment target population

[0217] In some embodiments, the human subject is at most 18 years old. In some embodiments, the human subject is 1 - 18, 2 - 18, 3 - 18, 4 - 18, 5 - 18, 6 - 18, 7 - 18, 8 - 18, 9 - 18, 10 - 18, 11 - 18, 12 - 18, 13 - 18, 14 - 18, 15 - 18, 16 - 18 or 17 - 18 years old. In some embodiments, the human subject is a human of 1 - 17, 1 - 16, 1 - 15, 1 - 14, 1 - 13, 1 - 12, 1 - 11, 1 - 10, 1 - 9, 1 - 8, 1 - 7, 1 - 6, 1 - 5, 1 - 4, 1 - 3 or 1 - 2 years old. In some embodiments, the human subject is less than 1 year old or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 years old.

[0125]

[0218] In some embodiments, the human subject is at most 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 year old.

[0126]

[0219] In some embodiments, the human subject is less than 1 year old or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 years old.

[0127]

[0220] In some embodiments, the human subject is 1 - 35, 2 - 35, 3 - 35, 4 - 35, 5 - 35, 6 - 3,5, 7 - 35, 8 - 35, 9 - 35, 10 - 35, 11 - 35, 12 - 35, 13 - 35, 14 - 35, 15 - 35, 16 - 35, 17 - 35, 18 - 35, 19 - 35, 20 - 35, 21 - 35, 22 - 35, 23 - 35, 24 - 35, 25 - 35 , 26 - 35, 27 - 35, 28 - 35, 29 - 35, 30 - 35, 31 - 35, 32 - 35, 33 - 35 or 34 - 35 years old.

[0128]

[0221] In some embodiments, the human subjects are humans aged 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 years.

[0129]

[0222] In some embodiments, the human subjects are humans aged 2-35, 2-34, 2-33, 2-32, 2-31, 2-30, 2-29, 2-28, 2-27, 2-26, 2-25, 2-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2x-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 years. In some embodiments, the human subjects are humans aged 3-35, 3-34, 3-33, 3-32, 3-31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 years.

[0130]

[0223] In some embodiments, the human subjects are individuals aged 4-35, 4-34, 4-33, 4-32, 4-31, 4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 years.

[0131]

[0224] In some embodiments, the human subjects are humans aged 5-35, 5-34, 5-33, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, or 5-6 years.

[0132]

[0225] In some embodiments, the human subjects are humans aged 6-35, 6-34, 6-33, 6-32, 6-31, 6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, or 6-7 years.

[0133]

[0226] In some embodiments, the human subjects are individuals aged 7-35, 7-34, 7-33, 7-32, 7-31, 7-30, 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, and 7-8 years.

[0134]

[0227] In some embodiments, the human subjects are humans aged 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, or 8-9 years.

[0135]

[0228] In some embodiments, subjects meet the following criteria: (i) seizure onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and triggered by high fever; (ii) no prior history of causative magnetic resonance imaging lesions; (iii) no other known etiologies of any disease or condition other than Dravet syndrome; (iv) normal development at seizure onset; (v) a pathogenic variant or variant of uncertain importance in the SCN1A gene; (vi) at least two prior treatments for epilepsy in which either lacked adequate seizure management; and (vii) prior to administration. (ix) The patient has four or more seizures during a 28-day period, the seizures being one of the following selected from unilateral clonic, focal with motor signs, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic; (viii) current intervention for epilepsy, which is a ketogenic diet, a vagus nerve stimulant, or a cannabinoid or marijuana-derived product, or medication with at least one antiepileptic drug at a dose that has been stable for at least four weeks; or any combination of (ix)(i) to (viii).

[0136]

[0229] In some embodiments, subjects are (i) seizure onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and triggered by high fever, (ii) no prior history of causative magnetic resonance imaging lesions, (iii) no other known etiologies of any disease or condition other than Dravet syndrome, (iv) normal development at seizure onset, (v) pathogenic variant or variant of uncertain importance in the SCN1A gene, (vi) at least two prior treatments for epilepsy in which either lacked adequate seizure management, (v (ii) Four or more seizures during the 28 days prior to administration, which are selected from unilateral clonic, focal, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic; (viii) at least one or more of the following: ketogenic diet therapy, vagus nerve stimulants or cannabinoids or marijuana-derived products, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks.In some embodiments, subjects are (i) seizure onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and triggered by high fever, (ii) no prior history of causative magnetic resonance imaging lesions, (iii) no other known etiologies of any disease or condition other than Dravet syndrome, (iv) normal development at seizure onset, (v) pathogenic variant or variant of uncertain importance in the SCN1A gene, (vi) at least two prior treatments for epilepsy in which either lacked adequate seizure management, (v (ii) Four or more seizures during the 28 days prior to administration, which are selected from unilateral clonic, focal, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic; (viii) at least two or more of the following: ketogenic diet therapy, vagus nerve stimulants or cannabinoids or marijuana-derived products, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks. In some embodiments, subjects are (i) seizure-onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and triggered by high fever; (ii) no prior history of causative magnetic resonance imaging lesions; (iii) no other known etiologies of any disease or condition other than Dravet syndrome; (iv) normal development at seizure onset; (v) a pathogenic variant or variant of uncertain importance in the SCN1A gene; (vi) at least two prior treatments for epilepsy in which either lacked adequate seizure management; and (vii) four or more seizures in the 28 days prior to administration, selected from unilateral clonic, focal with motor signs, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic. (viii) seizures, (viii) a current intervention for epilepsy, which is a ketogenic diet, a vagus nerve stimulant, or a cannabinoid or marijuana-derived product, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks. In some embodiments, the subject is characterized by (i) seizure onset before 12 months of age with recurrent focal motor or hemipteric or generalized tonic-clonic seizures that are often prolonged and triggered by high fever, (ii) no prior history of causative magnetic resonance imaging lesions, (iii) no other known etiology of any disease or condition other than Dravet syndrome, (iv) normal development at seizure onset, (v) a pathogenic variant or variant of uncertain importance in the SCN1A gene, (vi) at least two prior treatments for epilepsy in which any of the following lacked adequate seizure control, (v (ii) Four or more seizures during the 28 days prior to administration, which are selected from unilateral clonic seizures, focal seizures with motor signs, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic seizures, tonic or atonic (falling seizures), and clonic seizures; (viii) At least four or more of the following: ketogenic diet therapy, vagus nerve stimulants or cannabinoids or marijuana-derived products, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks.In some embodiments, subjects are (i) seizure onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and triggered by high fever, (ii) no prior history of causative magnetic resonance imaging lesions, (iii) no other known etiologies of any disease or condition other than Dravet syndrome, (iv) normal development at seizure onset, (v) pathogenic variant or variant of uncertain importance in the SCN1A gene, (vi) at least two prior treatments for epilepsy in which either lacked adequate seizure management, (v (ii) Four or more seizures during the 28 days prior to administration, which are selected from unilateral clonic, focal, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic; (viii) at least five or more of the following: ketogenic diet therapy, vagus nerve stimulants or cannabinoids or marijuana-derived products, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks.In some embodiments, subjects are (i) seizure onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and triggered by high fever, (ii) no prior history of causative magnetic resonance imaging lesions, (iii) no other known etiologies of any disease or condition other than Dravet syndrome, (iv) normal development at seizure onset, (v) pathogenic variant or variant of uncertain importance in the SCN1A gene, (vi) at least two prior treatments for epilepsy in which either lacked adequate seizure management, (v (ii) Four or more seizures during the 28 days prior to administration, which are selected from unilateral clonic, focal, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic; (viii) at least six or more of the following: ketogenic diet therapy, vagus nerve stimulants or cannabinoids or marijuana-derived products, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks. In some embodiments, the subjects are (i) seizure-onset before 12 months of age with recurrent focal motor, unilateral convulsive, or generalized tonic-clonic seizures that are often prolonged and caused by high fever; (ii) no prior history of causative magnetic resonance imaging lesions; (iii) no other known etiologies of any disease or condition other than Dravet syndrome; (iv) normal development at the time of seizure onset; (v) a pathogenic variant or variant of uncertain importance in the SCN1A gene; (vi) at least two prior treatments for epilepsy in which either lacked adequate seizure management; and (vii) four or more seizures in the 28 days prior to administration, which are unilateral clonic, focal, focal to bilateral tonic-clonic, or generalized tonic-clonic seizures with motor signs. (i) seizures that are selected from tonic, tonic or nonatonic (falling seizures) and clonic; (viii) current interventions for epilepsy, such as ketogenic diet therapy, vagus nerve stimulants or cannabinoids or marijuana-derived products; or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks. In some embodiments, the subject is characterized by (i) seizure onset before 12 months of age with recurrent focal motor or hemipteric or generalized tonic-clonic seizures that are often prolonged and triggered by high fever; (ii) no prior history of causative magnetic resonance imaging lesions; (iii) no other known etiologies of any disease or condition except Dravet syndrome; (iv) normal development at seizure onset; (v) pathogenic variant or variant of uncertain importance in the SCN1A gene; or (vi) at least two prior treatments for epilepsy in which any of the following lacked adequate seizure control. (vii) Four or more seizures during the 28 days prior to administration, which are selected from unilateral clonic, focal, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic, tonic or atonic (falling seizures), and clonic seizures, and (viii) all eight of the following: ketogenic diet therapy, vagus nerve stimulants or cannabinoids or marijuana-derived products, current interventions for epilepsy, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks.

[0137]

[0230] In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg and A (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blockers being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d) clinical conditions other than epilepsy (i) The patient is characterized by not having any of the following conditions: (i) a clinically relevant medical condition other than epilepsy, (ii) a clinically relevant symptom or clinically significant illness in the four weeks prior to administration, (ii) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (iii) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or has a CSF drainage shunt implanted, (iv) clinically significant abnormal clinical laboratory values ​​prior to administration, (iii) aspartate aminotransferase or alanine aminotransferase greater than 2.5 times the upper limit of normal, serum creatinine greater than the upper limit of normal, or platelet count less than the lower limit of normal, (iv) a clinically relevant abnormality in a 12-lead electrocardiogram (ECG) measured prior to administration, (iv) a mental or behavioral disorder, (v) current or past four weeks of taking an anticoagulant other than aspirin, or any one or more of any combination of (iv)(a) to (v).In some embodiments, the target is the following: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg (b) a known pathogenic mutation in another gene causing epilepsy, which is homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blockers being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant being not aspirin, (d) (i) a clinically significant medical condition other than epilepsy, (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase more than 2.5 times the upper limit of normal, serum creatinine more than the upper limit of normal, or platelet count less than the lower limit of normal, (j) a clinically relevant abnormality in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) a mental or behavioral disorder, and (l) one or more of the following medications currently or in the past four weeks:In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Ar (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blocker being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d (i) a clinically significant medical condition other than epilepsy, (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase 2.5 times the upper limit of normal, serum creatinine above the upper limit of normal, or platelet count below the lower limit of normal, (j) a clinically relevant abnormality in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) a mental or behavioral disorder, and (l) taking anticoagulants other than aspirin, either currently or in the past four weeks.In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, G (b) a known pathogenic mutation in another epilepsy-causing gene, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blockers being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant (i) Aspirin is not prescribed if (i) you have any of the following conditions: (d) any clinically significant medical condition other than epilepsy; (e) any clinically relevant symptoms or clinically significant illness other than epilepsy in the four weeks prior to administration; (f) any history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or any history of bacterial meningitis or brain malformation; (g) any spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or has a CSF drainage shunt implanted; (h) any clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase levels greater than 2.5 times the upper limit of normal, serum creatinine levels greater than the upper limit of normal, or platelet counts below the lower limit of normal; (j) any clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured prior to administration; (k) any mental or behavioral disorders; or (l) any current or past four-week history of aspirin. It is characterized by not having three or more of the following anticoagulant medications. In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Ar (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blocker being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d (i) a clinically significant medical condition other than epilepsy, (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase 2.5 times the upper limit of normal, serum creatinine above the upper limit of normal, or platelet count below the lower limit of normal, (j) a clinically relevant abnormality in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) a mental or behavioral disorder, and (l) taking anticoagulants other than aspirin, either currently or in the past four weeks.In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Ar (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blocker being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d (i) a clinically significant medical condition other than epilepsy, (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase 2.5 times the upper limit of normal, serum creatinine above the upper limit of normal, or platelet count below the lower limit of normal, (j) a clinically relevant abnormality in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) a mental or behavioral disorder, and (l) taking anticoagulants other than aspirin, either currently or in the past four weeks.In some embodiments, the subjects are further: (a) one of the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg, and Asp1866Tyr. b) Known pathogenic mutations in another gene causing epilepsy, homozygous in the case of known recessive genetic diseases, (c) sodium channel blockers as maintenance treatment and The patient is currently being treated with anticoagulants, and the sodium channel blockers are phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide, or rufinamide, and the anticoagulant is not aspirin; (d) a clinically significant medical condition other than epilepsy; (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration; (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation; (g) spinal deformity or alteration of the free flow of cerebrospinal fluid (CSF). (i) The patient is characterized by not having any of the following conditions: (h) a CSF drainage shunt implanted; (i) clinically significant abnormal clinical laboratory values ​​prior to administration; (i) aspartate aminotransferase or alanine aminotransferase levels exceeding 2.5 times the upper limit of normal; serum creatinine levels exceeding the upper limit of normal; or platelet counts below the lower limit of normal; (j) clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured prior to administration; (k) mental or behavioral disorders; and (l) taking six or more non-aspirin anticoagulants currently or in the past four weeks.In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Ar (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blocker being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d (i) clinically significant medical conditions other than epilepsy, (e) clinically relevant symptoms or clinically significant illnesses other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformities or other conditions that alter the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase more than 2.5 times the upper limit of normal, serum creatinine more than the upper limit of normal, or platelet count below the lower limit of normal, (j) clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) mental or behavioral disorders, and (l) not having taken seven or more non-aspirin anticoagulants currently or in the past four weeks.In some embodiments, the subjects are further: (a) one of the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg, and Asp1866Tyr; (b) a known pathogenic mutation in another epilepsy-causing gene that is homozygous in the case of a known recessive genetic disorder; (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blocker being phenyto (d) a clinically significant medical condition other than epilepsy, (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome or a history of bacterial meningitis or brain malformation, (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or has a CSF drainage shunt implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase more than 2.5 times the upper limit of normal, or serum creatine more than the upper limit of normal. (i) platelet count below the lower limit of normal, (j) clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured before administration, (k) psychiatric or behavioral disorders, and (l) no more than eight of the following: current or past four weeks of taking non-aspirin anticoagulants. In some embodiments, subjects are further characterized by: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Ar (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blocker being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d (i) clinically significant medical conditions other than epilepsy, (e) clinically relevant symptoms or clinically significant illnesses other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformities or other conditions that alter the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase 2.5 times the upper limit of normal, serum creatinine above the upper limit of normal, or platelet count below the lower limit of normal, (j) clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) mental or behavioral disorders, and (l) taking nine or more non-aspirin anticoagulants currently or in the past four weeks.In some embodiments, the subjects are further: (a) the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Ar (b) a known pathogenic mutation in another gene causing epilepsy, homozygous in the case of a known recessive genetic disorder, (c) currently treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blocker being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant not being aspirin, (d (i) a clinically significant medical condition other than epilepsy, (e) a clinically relevant symptom or clinically significant illness other than epilepsy in the four weeks prior to administration, (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase 2.5 times the upper limit of normal, serum creatinine above the upper limit of normal, or platelet count below the lower limit of normal, (j) a clinically relevant abnormality in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) a mental or behavioral disorder, and (l) not having 10 or more of the following medications other than aspirin currently or in the past four weeks.In some embodiments, the subjects are further: (a) one of the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg, and Asp1866Tyr; and (b) homozygous in the case of a known recessive genetic disease. (c) Known pathogenic mutations in another gene causing epilepsy, (d) Currently being treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blockers being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant being not aspirin, (e) Clinically significant medical conditions other than epilepsy, (f) Clinically relevant symptoms or clinically significant illnesses other than epilepsy in the four weeks prior to administration, (g) A history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformations, (g) The patient is characterized by not having (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase 2.5 times the upper limit of normal, serum creatinine above the upper limit of normal, or platelet count below the lower limit of normal, (j) clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) mental or behavioral disorders, and (l) 11 or more non-aspirin anticoagulant medications currently or in the past 4 weeks.In some embodiments, the subjects are further: (a) one of the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg, and Asp1866Tyr; (b) in the case of a known recessive genetic disease. (c) homozygous known pathogenic mutations in another gene causing epilepsy, (d) currently being treated with sodium channel blockers and anticoagulants as maintenance treatment, the sodium channel blockers being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide or rufinamide, and the anticoagulant being not aspirin, (e) clinically significant medical conditions other than epilepsy, (c) clinically relevant symptoms or clinically significant illnesses other than epilepsy in the four weeks prior to administration. (f) a history of brain or spinal cord disease other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformation, (g) spinal deformity or other condition that alters the free flow of cerebrospinal fluid (CSF) or in which a CSF drainage shunt is implanted, (h) clinically significant abnormal clinical laboratory values ​​prior to administration, (i) aspartate aminotransferase or alanine aminotransferase greater than 2.5 times the upper limit of normal, serum creatinine greater than the upper limit of normal, or platelet count less than the lower limit of normal, (j) clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured prior to administration, (k) mental or behavioral disorders, and (l) no current or past four-week use of any non-aspirin anticoagulant.

[0138]

[0231] In some embodiments, the subject is further characterized by not having a known pathogenic mutation in another gene that causes epilepsy. In some embodiments, the subject is further characterized by not having any other clinically relevant symptoms or clinically significant illnesses in the past four weeks. In some embodiments, the subject is further characterized by not having a specific mutation in the SCN1A gene that has been demonstrated to cause gain-of-function. In some embodiments, the subject is further characterized by not currently being treated with antiepileptic drugs that act primarily as sodium channel blockers. In some embodiments, the subject is further characterized by not having any other clinically significant medical condition(s) that is critically unstable.

[0139]

[0232] In some embodiments, the subjects have childhood epilepsy, epileptic encephalopathy, refractory myoclonic epilepsy, or severe myoclonic epilepsy in infancy. In some embodiments, the subjects have myoclonic epilepsy, generalized epilepsy, epilepsy, or brain diseases. , having a central nervous system disorder, a neurological disorder or an epileptic syndrome. In some embodiments, the treatment methods described herein include methods for treating a disease or condition or reducing the likelihood of developing it, where the disease or condition is childhood epilepsy, epileptic encephalopathy, refractory myoclonus epilepsy or severe myoclonus epilepsy in infancy. In some embodiments, the treatment methods described herein include methods for treating a disease or condition or reducing the likelihood of developing it, where the disease or condition is myoclonus epilepsy, generalized epilepsy, epilepsy, brain disorder, central nervous system disorder, neurological disorder or an epileptic syndrome. In some embodiments, the subject has seizures that are not controlled by the current antiepileptic drug (AED) regimen. In some embodiments, the AED regimen includes clobazam, cannabidiol, levetiracetam, stiripentol or valproic acid / valproate.

[0140]

[0233] As used herein, the term “magnetic resonance imaging lesion” refers to any damage or abnormal change in the tissue of a living organism caused by magnetic resonance imaging. As used herein, the term “magnetic resonance imaging” refers to a form of medical imaging that measures the response of atomic nuclei in body tissue to high-frequency radio waves when placed in a strong magnetic field to produce images of internal organs.

[0141]

[0234] As used herein, the term "ketogenic diet" refers to a high-fat, moderate-protein, low-carbohydrate diet used, for example, in medication to treat refractory epilepsy in children. This diet causes the body to burn fat rather than carbohydrates.

[0142]

[0235] The terms “vagus nerve stimulant” or “vagus nerve stimulation (VNS)” as used herein refer to a medical procedure that includes the step of delivering electrical impulses to the vagus nerve. For example, it is used as an adjunct treatment for certain types of refractory epilepsy and treatment-resistant depression.

[0143]

[0236] As used herein, the term "cannabinoid" refers to the chemical substances found in cannabis. Exemplary cannabinoids include, but are not limited to, the phytocannabinoids tetrahydrocannabinol (THC) (Delta9-THC or Delta8-THC) and cannabidiol (CBD). As used herein, cannabinoids may be natural substances or synthetic chemical substances.

[0144]

[0237] The terms “marijuana” or “cannabis,” as used herein, refer to psychoactive substances derived from the cannabis plant, primarily used for medical or recreational purposes. The exemplary primary psychoactive component of cannabis is tetrahydrocannabinol (THC).

[0145]

[0238] As used herein, the term "sodium channel blocker" refers to drugs that impair the conduction of sodium ions (Na+) through sodium channels. Examples of sodium channel blockers, but not limited to them, include alkaloids (e.g., saxitoxin, neosaxitoxin, tetrodotoxin), local anesthetics (e.g., lidocaine), anticonvulsants (e.g., phenytoin, oxycarbazepine (a derivative of carbamazepine)), and antiarrhythmic agents of class Ia (e.g., quinidine, procainamide, and disopyramide), class Ib (e.g., lidocaine, mexiletine, tokainide, and phenytoin), and class Ic (e.g., encainide, flecainide, moriscidine, and propafenone).

[0146]

[0239] The term "cerebrospinal fluid (CSF)" as used herein refers to the brain and spine. This refers to the clear, colorless fluid found in the spinal cord. CSF acts as a cushion or buffer, for example, providing basic mechanical and immunological protection to the brain inside the skull, and plays a crucial role in the brain's autoregulation of cerebral blood flow. The term "artificial cerebrospinal fluid (aCSF)," as used herein, refers to a biological buffer solution commonly used as a vehicle solution for the administration of drugs to the central nervous system (CNS). aCSF, for example, closely matches the electrolyte concentration and physiological compatibility of endogenous CSF, enabling a life-sustaining environment for nerve tissue by maintaining homeostasis, osmotic pressure, and pH at physiological levels.

[0147]

[0240] As used herein, the term "CSF drainage shunt" refers to a system that, as part of the circulatory process, drains excess fluid from the brain to another part of the body from which the fluid is absorbed. CSF shunts are used, for example, to treat hydrocephalus.

[0148]

[0241] The term "electrocardiogram (EKG or ECG)," as used herein, refers to a test that measures the electrical activity of the heart, for example, by producing a graph of voltage versus time of the heart's electrical activity. With each heartbeat, an electrical impulse (or "wave") travels through the heart.

[0149]

[0242] "Aspartate transaminase (AST)," also known as aspartate aminotransferase, AspAT / ASAT / AAT, or (serum) glutamate oxaloacetate transaminase (GOT, SGOT), refers, as used herein, to pyridoxal phosphate (PLP)-dependent transaminase enzyme (EC 2.6.1.1). AST includes either recombinant or naturally occurring forms of the AST protein or its variants or homologs that maintain AST activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to AST). Exemplary AST activity includes, but is not limited to, catalyzing the reversible transfer of an α-amino group between aspartate and glutamate, and playing a role in amino acid metabolism as itself. In some embodiments, the variant or homolog has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous amino acid segment of 50, 100, 150, or 200 amino acids) compared to the naturally occurring AST protein. In some embodiments, the AST protein is substantially identical to the protein identified by UniProt reference number P17174 or a variant or homolog that is substantially identical to it. In some embodiments, the AST protein is substantially identical to the protein identified by UniProt reference number P00505 or a variant or homolog that is substantially identical to it.

[0150]

[0243] "Alanine transaminase (ALT)," also known as alanine aminotransferase (ALAT), serum glutamate-pyruvate transaminase (SGPT), or serum glutamate-pyruvate transaminase (SGPT), refers, as used herein, to the transaminase enzyme (EC 2.6.1.2). ALT includes either recombinant or naturally occurring forms of the ALT protein or its variants or homologs that maintain ALT activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to ALT). Exemplary ALT activity includes, but is not limited to, catalyzing two parts of the alanine cycle. In some embodiments, the variant or homolog has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous amino acid segment of 50, 100, 150, or 200 amino acids) compared to the naturally occurring ALT protein. In some embodiments, the ALT protein is the protein identified by UniProt reference number P24298 or a variant that is substantially identical to it. Alternatively, they are substantially identical to homologs.

[0151]

[0244] In some embodiments, serum AST levels, serum ALT levels, and their ratio (AST / ALT ratio) are clinically measured as biomarkers of liver health.

[0245] The term “laboratory vale” as used herein refers to a value obtained by a clinical test or measurement. Exemplary, but not limited to, clinical tests or measurements may relate to hematology, blood coagulation, clinical chemistry, plasma, urinalysis, serum, serum or urine pregnancy, urine or cerebrospinal fluid.

[0152] Treatment schedule

[0246] In some embodiments, the first dose is a single dose. In some embodiments, the first dose is the first of multiple doses. In some embodiments, the method further includes a step of evaluating the tolerability or efficacy of the pharmaceutical composition.

[0153]

[0247] In some embodiments, the methods described herein further include administering to a human subject a pharmaceutical composition comprising an ASO described herein in a subsequent dose of 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg.

[0154]

[0248] In some embodiments, the methods described herein apply to human subjects in doses of approximately 0.1 to 1000 mg, approximately 0.2 to 1000 mg, approximately 0.3 to 1000 mg, approximately 0.4 to 1000 mg, approximately 0.5 to 1000 mg, approximately 0.6 to 1000 mg, approximately 0.7 to 1000 mg, approximately 0.8 to 1000 mg, approximately 0.9 to 1000 mg, 1 to 1000 mg, approximately 2 to 1000 mg, approximately 3 to 1000 mg, and approximately 4 to 10 00mg, about 5 to about 1000mg, about 6 to about 1000mg, about 7 to about 1000mg, about 8 to about 1000mg, about 9 to about 1000mg, about 10 to about 1000mg, about 15 to about 1000mg, about 20 to about 100 0mg, about 25 to about 1000mg, about 30 to about 1000mg, about 35 to about 1000mg, about 40 to about 1000mg, about 45 to about 1000mg, about 50 to about 1000mg, about 55 to about 1000mg, about 60 to about 1000mg, about 65 to about 1000mg, about 70 to about 1000mg, about 75 to about 1000mg, about 80 to about 1000mg, about 85 to about 1000mg, about 90 to about 1000mg, about 95 to about 1000mg, about 100 to about 1000mg, about 150 to about 1000mg, about 200 to about 1000mg, about 250 to about 1000mg, about 300 to about 1000mg, about 350 to about 1000mg, about 400 to about 1000mg, about 45 The procedure further includes administering a pharmaceutical composition containing the ASO described herein in subsequent doses of 0 to approximately 1000 mg, approximately 500 to approximately 1000 mg, approximately 550 to approximately 1000 mg, approximately 600 to approximately 1000 mg, approximately 650 to approximately 1000 mg, approximately 700 to approximately 1000 mg, approximately 750 to approximately 1000 mg, approximately 800 to approximately 1000 mg, approximately 850 to approximately 1000 mg, approximately 900 to approximately 1000 mg, or approximately 950 to approximately 1000 mg.

[0155]

[0249] In some embodiments, the methods described herein apply to human subjects in doses of 0.1-1000 mg, 0.2-1000 mg, 0.3-1000 mg, 0.4-1000 mg, 0.5-1000 mg, 0.6-1000 mg, 0.7-1000 mg, 0.8-1000 mg, 0.9-1000 mg, 1-1000 mg, 2-1000 mg, 3-1000 mg, 4- 1000mg, 5~1000mg, 6~1000mg, 7~1000mg, 8~1000mg, 9~1000mg, 10~1000mg, 15~1000mg, 20~1000 mg, 25~1000mg, 30~1000mg, 35~1000mg, 40~1000mg, 45~1000mg, 50~1000mg, 55~1000mg, 60~100 0mg, 65~1000mg, 70~1000mg, 75~1000mg, 80~1000mg, 85~1000mg, 90~1000mg, 95~1000mg, 100~1 000mg, 150~1000mg, 200~1000mg, 250~1000mg, 300~1000mg, 350~1000mg, 400~1000mg, 450~100 The procedure further comprises administering a pharmaceutical composition containing the ASO described herein in subsequent doses of 0 mg, 500-1000 mg, 550-1000 mg, 600-1000 mg, 650-1000 mg, 700-1000 mg, 750-1000 mg, 800-1000 mg, 850-1000 mg, 900-1000 mg, or 950-1000 mg.

[0156]

[0250] In some embodiments, the methods described herein apply to human subjects in doses of approximately 0.1 to 950 mg, approximately 0.1 to 900 mg, approximately 0.1 to 850 mg, approximately 0.1 to 800 mg, approximately 0.1 to 750 mg, approximately 0.1 to 700 mg, approximately 0.1 to 650 mg, approximately 0.1 to 600 mg, approximately 0.1 to 550 mg, approximately 0.1 to 500 mg, approximately 0.1 to 450 mg, approximately 0.1 to Approximately 400 mg, approximately 0.1 to approximately 350 mg, approximately 0.1 to approximately 300 mg, approximately 0.1 to approximately 250 mg, approximately 0.1 to approximately 200 mg, approximately 0.1 to approximately 150 mg, approximately 0.1 to approximately 100 mg, approximately 0.1 ~about 95mg, about 0.1 to about 90mg, about 0.1 to about 85mg, about 0.1 to about 80mg, about 0.1 to about 75mg, about 0.1 to about 70mg, about 0.1 to about 65mg, about 0.1 to about 60mg, About 0.1 to about 55 mg, about 0.1 to about 50 mg, about 0.1 to about 45 mg, about 0.1 to about 40 mg, about 0.1 to about 35 mg, about 0.1 to about 30 mg, about 0.1 to about mg, about 0.1 to about 25 mg, about 0.1 to about 20 mg, about 0.1 to about 10 mg, about 0.1 to about 9 mg, about 0.1 to about 8 mg, about 0.1 to about 7 mg, about 0.1 to about 6 mg, about 0.1 to about 5 mg, about 0.1 to about 4 mg The procedure further comprises administering a pharmaceutical composition containing an ASO as described herein in subsequent doses of approximately 0.1 to 0.3 mg, approximately 0.1 to 0.2 mg, approximately 0.1 to 0.1 mg, approximately 0.1 to 0.9 mg, approximately 0.1 to 0.8 mg, approximately 0.1 to 0.7 mg, approximately 0.1 to 0.6 mg, approximately 0.1 to 0.5 mg, approximately 0.1 to 0.4 mg, approximately 0.1 to 0.3 mg, or approximately 0.1 to 0.2 mg.

[0157]

[0251] In some embodiments, the methods described herein apply to human subjects in doses of 0.1-950 mg, 0.1-900 mg, 0.1-850 mg, 0.1-800 mg, 0.1-750 mg, 0.1-700 mg, 0.1-650 mg, 0.1-600 mg, 0.1-550 mg, 0.1-500 mg, 0.1-450 mg, 0.1-400 mg, 0.1-350 mg, 0.1-300 mg, 0.1~250mg, 0.1~200mg, 0.1~150mg, 0.1~100mg, 0.1~95mg, 0.1~90mg, 0.1~85mg, 0.1~80mg, 0.1~75mg, 0.1~70mg, 0.1~65mg, 0.1~60mg, 0.1~55mg, 0.1~50mg, 0.1~45mg, 0.1~40mg, 0.1~35mg, 0.1~30mg, 0.1~ The procedure further includes administering a pharmaceutical composition containing an ASO as described herein in subsequent doses of mg, 0.1-25 mg, 0.1-20 mg, 0.1-10 mg, 0.1-9 mg, 0.1-8 mg, 0.1-7 mg, 0.1-6 mg, 0.1-5 mg, 0.1-4 mg, 0.1-3 mg, 0.1-2 mg, 0.1-1 mg, 0.1-0.9 mg, 0.1-0.8 mg, 0.1-0.7 mg, 0.1-0.6 mg, 0.1-0.5 mg, 0.1-0.4 mg, 0.1-0.3 mg, or 0.1-0.2 mg.

[0158]

[0252] In some embodiments, the methods described herein apply to human subjects in doses of approximately 1 to 400 mg, approximately 2 to 400 mg, approximately 3 to 400 mg, approximately 4 to 400 mg, approximately 5 to 400 mg, approximately 6 to 400 mg, approximately 7 to 400 mg, approximately 8 to 400 mg, approximately 9 to 400 mg, approximately 10 to 400 mg, approximately 20 to 400 mg, approximately 30 to 400 mg, approximately 40 to 400 mg, approximately 50 to 400 mg, approximately 60 to 400 mg, approximately 70 to 400 mg, approximately 80 to 400 mg, approximately 90 to 400 mg, approximately 100 to 400 mg, approximately 110 to 400 mg, approximately 120 to 400 mg, approximately 130 to 400 mg, and approximately 140 to 400 mg. mg, about 150 to about 400 mg, about 160 to about 400 mg, about 170 to about 400 mg, about 180 to about 400 mg, about 190 to about 400 mg, about 200 to about 400 mg, about 210 to about 400 mg, About 220 to about 400 mg, about 230 to about 400 mg, about 240 to about 400 mg, about 250 to about 400 mg, about 260 to about 400 mg, about 270 to about 400 mg, about 280 to about 400 mg, about 29 The procedure further includes administering a pharmaceutical composition containing the ASO described herein in subsequent doses of 0 to approximately 400 mg, approximately 300 to approximately 400 mg, approximately 310 to approximately 400 mg, approximately 320 to approximately 400 mg, approximately 330 to approximately 400 mg, approximately 340 to approximately 400 mg, approximately 350 to approximately 400 mg, approximately 360 to approximately 400 mg, approximately 370 to approximately 400 mg, approximately 380 to approximately 400 mg, or approximately 390 to approximately 400 mg.

[0159]

[0253] In some embodiments, the methods described herein apply to human subjects in doses of 1-400 mg, 2-400 mg, 3-400 mg, 4-400 mg, 5-400 mg, 6-400 mg, 7-400 mg, 8-400 mg, 9-400 mg, 10-400 mg, 20-400 mg, 30-400 mg, 40-400 mg, 50-400 mg, 60-400 mg, 70-400 mg, 80-400 mg, 90-400 mg, 100-400 mg, 110-400 mg, 120-400 mg, 130-400 mg, approximately 140-400 mg, 150-400 mg, approximately 160-400 mg, 170-400 mg, The procedure further comprises administering a pharmaceutical composition containing the ASO described herein in a subsequent dose of 180-400 mg, 190-400 mg, 200-400 mg, 210-400 mg, 220-400 mg, 230-400 mg, 240-400 mg, 250-400 mg, 260-400 mg, 270-400 mg, 280-400 mg, 290-400 mg, 300-400 mg, 310-400 mg, 320-400 mg, 330-400 mg, 340-400 mg, 350-400 mg, 360-400 mg, 370-400 mg, 380-400 mg, or 390-400 mg.

[0160]

[0254] In some embodiments, the methods described herein apply to human subjects in doses of approximately 10-390 mg, approximately 10-380 mg, approximately 10-370 mg, approximately 10-360 mg, approximately 10-350 mg, approximately 10-340 mg, approximately 10-330 mg, approximately 10-320 mg, approximately 10-310 mg, approximately 10-300 mg, approximately 10-290 mg, approximately 10-280 mg, approximately 10-270 mg, approximately 10-260 mg, approximately 10-250 mg, approximately 10-240 mg, approximately 10-230 mg, approximately 10-220 mg, and approximately 10-210 mg. The procedure further comprises administering a pharmaceutical composition containing an ASO as described herein in a subsequent dose of approximately 10-200 mg, approximately 10-190 mg, approximately 10-180 mg, approximately 10-170 mg, approximately 10-160 mg, approximately 10-150 mg, approximately 10-140 mg, approximately 10-130 mg, approximately 10-120 mg, approximately 10-110 mg, approximately 10-90 mg, approximately 10-80 mg, approximately 10-70 mg, approximately 10-60 mg, approximately 10-50 mg, approximately 10-40 mg, approximately 10-30 mg, or approximately 10-20 mg.

[0161]

[0255] In some embodiments, the methods described herein apply to human subjects in doses of 10-390 mg, 10-380 mg, 10-370 mg, 10-360 mg, 10-350 mg, 10-340 mg, 10-330 mg, 10-320 mg, 10-310 mg, 10-300 mg, 10-290 mg, 10-280 mg, 10-270 mg, 10-260 mg, 10-250 mg, 10-240 mg, 10-230 mg, 10-220 mg, and 10-210 mg. The procedure further comprises administering a pharmaceutical composition containing an ASO as described herein in a subsequent dose of 10-200 mg, 10-190 mg, 10-180 mg, 10-170 mg, 10-160 mg, 10-150 mg, 10-140 mg, 10-130 mg, 10-120 mg, 10-110 mg, 10-90 mg, 10-80 mg, 10-70 mg, 10-60 mg, 10-50 mg, 10-40 mg, 10-30 mg, or 10-20 mg.

[0162]

[0256] For certain rights, the methods described herein apply to human subjects approximately 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, approx. 39mg, approximately 40mg, approximately 41mg, approximately 42mg, approximately 43mg, approximately 44mg, approximately 45mg, approximately 46mg, approximately 47mg, approximately 48mg, approximately 49mg, approximately 50mg, approximately 51mg, approximately 52mg, approximately 53mg, approximately 54mg, approximately 55mg, approximately 56mg, approximately 57mg, approximately 58mg, approximately 59mg, approximately 60mg, approximately 61mg, approximately 62mg, approximately 63mg, approximately 64mg, approximately 65mg, approximately 66mg, approximately 67mg, approximately 68mg, approximately 69mg, approximately 70mg, approximately 71mg, approximately 72mg, approximately 73mg, approximately 74mg, approximately 75mg, approximately 76mg, approximately 77mg, approximately 78mg, approximately 79mg, approximately 80mg Approximately 81mg, approximately 82mg, approximately 83mg, approximately 84mg, approximately 85mg, approximately 86mg, approximately 87mg, approximately 88mg, approximately 89mg, approximately 90mg, approximately 91mg, approximately 92mg, approximately 93mg, approximately 94mg, approximately 95mg, approximately 96mg, approximately 97mg, approximately 98mg, approximately 99mg, approximately 100mg, approximately 101mg, approximately 102mg, approximately 103mg, approximately 104mg, approximately 105mg, approximately 106mg, approximately 107mg, approximately 108mg, approximately 109mg, approximately 110mg, approximately 111mg, approximately 112mg, approximately 113mg, approximately 114mg, approximately 115mg, approximately 116mg, approximately 117mg, approximately 118mg, approximately 1 19mg, approximately 120mg, approximately 121mg, approximately 122mg, approximately 123mg, approximately 124mg, approximately 125mg, approximately 126mg, approximately 127mg, approximately 128mg, approximately 129mg, approximately 130mg, approximately 131mg, approximately 132mg, approximately 133mg, approximately 134mg, approximately 135mg, approximately 136mg, approximately 137mg, approximately 138mg, approximately 139mg, approximately 140mg, approximately 141mg, approximately 142mg, approximately 143mg, approximately 144mg, approximately 145mg, approximately 146mg, approximately 147mg, approximately 148mg, approximately 149mg, approximately 150mg, approximately 151mg, approximately 152mg, approximately 153mg, approximately 154mg,Approximately 155mg, approximately 156mg, approximately 157mg, approximately 158mg, approximately 159mg, approximately 160mg, approximately 161mg, approximately 162mg, approximately 163mg, approximately 164mg, approximately 165mg, approximately 166mg, approximately 167mg, approximately 168mg, approximately 169mg, approximately 170mg, approximately 171mg, approximately 172mg, approximately 173mg, approximately 174mg, approximately 175mg, approximately 176mg, approximately 177mg, approximately 178mg, approximately 179mg, approximately 180mg, approximately 181mg, approximately 182mg, approximately 183mg, approximately 184mg, approximately 185mg, approximately 186mg, approximately 187mg, approximately 188mg, approximately 189mg, approximately 190mg mg, approximately 191mg, approximately 192mg, approximately 193mg, approximately 194mg, approximately 195mg, approximately 196mg, approximately 197mg, approximately 198mg, approximately 199mg, approximately 200mg, approximately 201mg, approximately 202mg, approximately 203mg, approximately 204mg, approximately 205mg, approximately 206mg, approximately 207mg, approximately 208mg, approximately 209mg, approximately 210mg, approximately 211mg, approximately 212mg, approximately 213mg, approximately 214mg, approximately 215mg, approximately 216mg, approximately 217mg, approximately 218mg, approximately 219mg, approximately 220mg, approximately 221mg, approximately 222mg, approximately 223mg, approximately 224mg, approximately 225mg, approximately 226mg, approximately 227mg, approximately 228mg, approximately 229mg, approximately 230mg, approximately 231mg, approximately 232mg, approximately 233mg, approximately 234mg, approximately 235mg, approximately 236mg, approximately 237mg, approximately 238mg, approximately 239mg, approximately 240mg, approximately 241mg, approximately 242mg, approximately 243mg, approximately 244mg, approximately 245mg, approximately 246mg, approximately 247mg, approximately 248mg, approximately 249mg, approximately 250mg, approximately 251mg, approximately 252mg, approximately 253mg, approximately 254mg, approximately 255mg, approximately 256mg, approximately 257mg, approximately 258mg, approximately 259mg, approximately 260mg, approximately 261mg g, approximately 262mg, approximately 263mg, approximately 264mg, approximately 265mg, approximately 266mg, approximately 267mg, approximately 268mg, approximately 269mg, approximately 270mg, approximately 271mg, approximately 272mg, approximately 273mg, approximately 274mg, approximately 275mg, approximately 276mg, approximately 277mg, approximately 278mg, approximately 279mg, approximately 280mg, approximately 281mg, approximately 282mg, approximately 283mg, approximately 284mg, approximately 285mg, approximately 286mg, approximately 287mg, approximately 288mg, approximately 289mg, approximately 290mg, approximately 291mg, approximately 292mg, approximately 293mg, approximately 294mg, approximately 295mg, approximately 296mg, approximately 2, 97mg, about 298mg, about 299mg, about 300mg, about 301mg, about 302mg, about 303mg, about 304mg, about 305mg, about 306mg, about 307mg, about 308mg, about 309mg, about 310mg, Approximately 311mg, approximately 312mg, approximately 313mg, approximately 314mg, approximately 315mg, approximately 316mg, approximately 317mg, approximately 318mg, approximately 319mg, approximately 320mg, approximately 321mg, approximately 322mg, approximately 323mg, approximately 324m g, about 325 mg, about 326 mg, about 327 mg, about 328 mg, about 329 mg, about 330 mg, about 331 mg, about 332 mg, about 333 mg, about 334 mg, about 335 mg, about 336 mg, about 337 mg, about 33 8mg, about 339mg, about 340mg, about 341mg, about 342mg, about 343mg, about 344mg, about 345mg, about 346mg, about 347mg, about 348mg, about 349mg, about 350mg, about 351mg, about 3 52mg, about 353mg, about 354mg, about 355mg, about 356mg, about 357mg, about 358mg, about 359mg, about 360mg, about 361mg, about 362mg, about 363mg, about 364mg, about 365mg, Approximately 366mg, approximately 367mg, approximately 368mg, approximately 369mg, approximately 370mg, approximately 371mg, approximately 372mg, approximately 373mg, approximately 374mg, approximately 375mg, approximately 376mg, approximately 377mg, approximately 378mg, approximately 379m The procedure further includes administering a pharmaceutical composition containing the ASO described herein in a subsequent dose of approximately 380 mg, approximately 381 mg, approximately 382 mg, approximately 383 mg, approximately 384 mg, approximately 385 mg, approximately 386 mg, approximately 387 mg, approximately 388 mg, approximately 389 mg, approximately 390 mg, approximately 391 mg, approximately 392 mg, approximately 393 mg, approximately 394 mg, approximately 395 mg, approximately 396 mg, approximately 397 mg, approximately 398 mg, approximately 399 mg, or 400 mg.

[0163]

[0257] In some embodiments, the methods described herein apply to human subjects with doses of 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg. g, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71 mg, 72mg, 73mg, 74mg, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96 mg, 97mg, 98mg, 99mg, 100mg, 101mg, 102mg, 103mg, 104mg, 105mg, 106mg, 107mg, 108mg, 109mg, 110mg, 111mg, 112mg, 113mg, 114mg, 115mg, 116mg, 117m g, 118mg, 119mg, 120mg, 121mg, 122mg, 123mg, 124mg, 125mg, 126mg, 127mg, 128mg, 129mg, 130mg, 131mg, 132mg, 133mg, 134mg, 135mg, 136mg, 137mg, 138 mg, 139mg, 140mg, 141mg, 142mg, 143mg, 144mg, 145mg, 146mg, 147mg, 148mg, 149mg, 150mg, 151mg, 152mg, 153mg, 154mg, 155mg, 156mg, 157mg, 158mg, 1 59mg, 160mg, 161mg, 162mg, 163mg, 164mg, 165mg, 166mg, 167mg, 168mg, 169mg, 170mg, 171mg, 172mg, 173mg, 174mg, 175mg, 176mg, 177mg, 178mg, 179mg,180mg、181mg、182mg、183mg、184mg、185mg、186mg、187mg、188mg、189mg、190mg、191mg、192mg、193mg、194mg、195mg、196mg、197mg、198mg、199mg、200mg、201mg、202mg、203mg、204mg、205mg、206m、 g、207mg、208mg、209mg、210mg、211mg、212mg、213mg、214mg、215mg、216mg、217mg、218mg、219mg、220mg、221mg、222mg、223mg、224mg、225mg、226mg、227mg、228mg、229mg、230mg、231mg、232mg、233mg、234mg、235mg、236mg、237mg、238mg、239mg、240mg、241mg、242mg、243mg、244mg、245mg、246mg、247mg、248mg、249mg、250mg、251mg、252mg、253mg、254mg、255mg、256mg、257mg、258mg、259mg、260mg、261mg、262mg、263mg、264mg、265mg、266mg、267mg、268mg、269mg、270mg、271mg、272mg、273mg、274mg、275mg、276mg、277mg、278mg、279mg、280mg、281mg、282mg、283mg、284mg、285mg、286mg、287mg、288mg、289mg、290mg、291mg、292mg、293mg、294mg、295mg、296mg、297mg、298mg、299mg、300mg、301mg、302mg、303mg、304mg、305mg、306mg、307mg、308mg、309mg、310mg、311mg、312mg、313mg、314mg、315mg、316mg、317mg、318mg、319mg、320mg、321mg、322mg、323mg、324mg、325mg、326mg、327mg、328mg、329mg、330mg、331mg、332mg、333mg、334mg、335mg、336mg、337mg、338mg、339mg、340mg、341mg、342mg、343mg、344mg、345mg、346mg、347mg、348mg、349mg、350mg、351mg、352mg、353mg、354mg、355mg、356mg、357mg、358mg、359mg、360mg、361mg、362mg、363mg、364mg、365mg、366mg、367mg、368mg、369mg、370mg、371mg、372mg、The procedure further includes administering a pharmaceutical composition containing the ASO described herein in a subsequent dose of 373 mg, 374 mg, 375 mg, 376 mg, 377 mg, 378 mg, 379 mg, 380 mg, 381 mg, 382 mg, 383 mg, 384 mg, 385 mg, 386 mg, 387 mg, 388 mg, 389 mg, 390 mg, 391 mg, 392 mg, 393 mg, 394 mg, 395 mg, 396 mg, 397 mg, 398 mg, 399 mg, or 400 mg.

[0164]

[0258] In some embodiments, the methods described herein further include a step of evaluating the tolerability or efficacy of the pharmaceutical composition. In some embodiments, after it has been shown that an earlier dose was intolerable, a subsequent dose is lower than the earlier dose. In some embodiments, after it has been shown that an earlier dose was effective, a subsequent dose is the same as the earlier dose. In some embodiments, after it has been shown that an earlier dose was effective, a subsequent dose is lower than the earlier dose. In some embodiments, after it has been shown that an earlier dose was ineffective, a subsequent dose is higher than the earlier dose.

[0165]

[0259] In some embodiments, the subsequent dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the administration of the previous dose.

[0260] In some embodiments, the subsequent dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the administration of the previous dose. In some embodiments, the subsequent dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the administration of the previous dose. In some embodiments, the subsequent dose is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 of the dose of the previous dose. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 weeks after the previous dose. In some embodiments, the subsequent dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the previous dose. In some embodiments, the subsequent dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after the previous dose.

[0166]

[0261] In some embodiments, subsequent doses are administered at the same interval. For example, each subsequent dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the previous dose. In some embodiments, each subsequent dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the previous dose. In some embodiments, each subsequent dose is administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 weeks after the previous dose. In some embodiments, each subsequent dose is administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the previous dose.

[0167]

[0262] In some embodiments, subsequent doses are administered at different intervals.

[0263] In some embodiments, the frequency of administration is maintained or reduced after the previous dose has been shown to be effective. In some embodiments, the frequency of administration is increased after the previous dose has been shown to be ineffective. In some embodiments, the method further includes the step of administering at least one additional therapeutic agent or therapy. In some embodiments, at least one additional therapeutic agent or therapy is administered concurrently with the dose. In some embodiments, at least one additional therapeutic agent or therapy is administered before the dose. In some embodiments, at least one additional therapeutic agent or therapy is administered after the dose.

[0168] Dravet syndrome and other related diseases

[0264] The terms “condition,” “disease,” and “disorder” are used synonymously herein in their broadest sense and include susceptibility. In some embodiments, the disease or condition is Dravet syndrome. In some embodiments, the method reduces or improves at least one symptom of Dravet syndrome in a human subject. In some embodiments, the symptom of Dravet syndrome is seizures. In some embodiments, the administration reduces or improves seizure frequency, seizure intensity, or seizure duration.

[0169]

[0265] Dravet syndrome (DS), also known as severe myoclonus epilepsy in infancy (SMEI), is an epileptic encephalopathy that presents within the first year of life. Dravet syndrome is an increasingly recognized epileptic encephalopathy, and its clinical diagnosis is supported by the presence of sodium channel gene mutations in approximately 70–80% of patients. DS is a severe, progressive developmental, and epileptic encephalopathy characterized by high seizure frequency and severity, intellectual disability, and a high risk of sudden, unexpected death in epilepsy. Mutations in ion channel genes play a significant role in the pathogenesis of various epilepsy syndromes, and as a result, some epilepsies are considered channel diseases. Voltage-gated sodium channels (VGSCs) play an essential role in neuronal excitability and are therefore associated with DS. It is not surprising that numerous mutations have been identified in the genes encoding the VGSC subunit. The disease is described, for example, in Mulley et al., 2005 and OMIM number 607208 (Online Mendelian Inheritance). The disease is described in the Man, Johns Hopkins University, 1966–2015, and both are incorporated herein by reference. Born.

[0170]

[0266] Between 70% and 80% of patients carry abnormalities in the sodium channel al subunit gene (SCN1A), with truncated mutations accounting for approximately 40%, and significantly correlated with an earlier age of seizure onset. Sequencing mutations were found in approximately 70% of cases, including truncated (40%) and missense mutations (40%), with the remainder being splice site changes. Most mutations are de novo, but familial mutations occur in 5–10% of cases and are usually essentially missense. The remaining SCN1A mutations include splice site and missense mutations, most of which fall within the pore-forming region of the sodium channel. Currently, more than 500 mutations have been associated with DS, randomly distributed along the gene (Mulley et al., Neurol. 2006, Vol. 67, pp. 1094–1095).

[0171]

[0267] The SCN1A gene is located in a cluster of sodium channel genes on human chromosome 2q24, and is a neuronal voltage-gated sodium channel. V It encodes the α-pore-forming subunit known as 1.1. The SCN1A gene spans approximately 100kb of genomic DNA and contains 26 exons. V1.1 The protein consists of four domains, each having six transmembrane segments. Two splice variants have been identified, resulting in long and short isoforms that differ in the presence or absence of 11 amino acids in the cytoplasmic loop between domains 1 and 2 in exon 11 (the full disclosure thereof is incorporated herein by reference, Miller et al., 1993–2015 and Mulley et al., 2005, Vol. 25, pp. 535–542). In some embodiments, the variants or homologs have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive nucleotides) compared to the naturally occurring SCN1A gene. In some embodiments, the SCN1A gene is substantially identical to the gene identified by Ensembl reference number ENSG00000144285, or to a variant or homolog that is substantially identical thereto.

[0172]

[0268] Alternative splicing events in the SCN1A gene can lead to unproductive mRNA transcripts, which in turn can lead to abnormal protein expression. Therapies that can target alternative splicing events in the SCN1A gene can modulate the expression levels of functional proteins and / or inhibit abnormal protein expression in DS patients. Such therapies include Na V 1.1 It can be used to treat conditions caused by protein deficiency.

[0173]

[0269] One alternative splicing event that can lead to unproductive mRNA transcripts is the inclusion of extra exons in the mRNA transcript, which can induce nonsense mutation-dependent mRNA decay. This disclosure relates to mature protein-coding mRNA, and consequently, translated functional Na V1.1 The present invention provides compositions and methods for modulating alternative splicing of SCN1A to increase protein production. These compositions and methods include antisense oligomers (ASOs) that can promote constitutive splicing, which can induce exon skipping of SCN1A premRNA. In various embodiments, functional Na V 1.1 Proteins are increased using the methods disclosed herein, Na V 1.1 Conditions caused by protein deficiency can be treated.

[0174]

[0270] In some cases, the disease or condition is SMEB (Small-Symptom-Effective Biology).

[0271] In some cases, the disease or condition is GEFS+.

[0272] In some cases, the disease or condition is a febrile seizure (e.g., febrile seizure, familial, 3A).

[0175]

[0273] In some cases, the disorder or condition is autism (also known as autism spectrum disorder or ASD).

[0274] In some cases, the disease or condition is migraine (e.g., migraine, familial hemiplegia, 3).

[0176]

[0275] In some cases, the disease or condition is Alzheimer's disease.

[0276] In some embodiments, the disease or condition is SMEB. In some embodiments, the disease or condition is GEFS+. In some embodiments, the disease or condition is a febrile seizure (e.g., febrile seizure, familial, 3A). In some embodiments, the disease or condition is autism (also known as autism spectrum disorder or ASD). In some embodiments, the disease or condition is a migraine (e.g., migraine, familial hemiplegic, 3). In some embodiments, the disease or condition is Alzheimer's disease. In some embodiments, the disease or condition is SCN2A encephalopathy. In some embodiments, the disease or condition is SCN8A encephalopathy. In some embodiments, the disease or condition is SCN5A arrhythmia.

[0177]

[0277] In some embodiments, the disease or condition is Na V 1.1 It is induced by mutations in the protein encoded by the SCN1A gene. V 1.1" is also known as sodium channel, voltage-gated, type I, alpha subunit (SCN1A), and as used herein, refers to the protein encoded by the SCN1A gene in humans. V 1.1 is Na V 1.1 Activity (e.g., Na V 1. Maintaining activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to 1.1, Na V 1.1 This includes either recombinant or naturally occurring forms of proteins or their variants or homologs. In some embodiments, the variant or homolog is a naturally occurring Na V 1.1 Compared to a protein, it has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a continuous amino acid portion of 50, 100, 150, or 200 amino acids). In some embodiments, Na V1.1 The protein is substantially identical to the protein identified by UniProt reference number P35498, or to a variant or homolog that is substantially identical to it.

[0178]

[0278] In some cases, the mutation occurs in Na V 1.1 This is a loss-of-function mutation. In some cases, Na V 1.1 Loss-of-function mutations in wild-type Na V For the function of 1.1, Na V 1.1 contains one or more mutations that reduce or impair the function of (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some cases, Na V A loss-of-function mutation in 1.1 includes one or more mutations that result in a disease phenotype. Exemplary loss-of-function mutations, but not limited to, include R859C, T875M, V1353L, I1656M, R1657C, A1685V, M1841T, and R1916G.

[0179]

[0279] In other examples, the mutation was Na V 1.1 This is a gain-of-function mutation. In such cases, the gain-of-function mutation is wild-type Na V For the function of 1.1, Na V This includes one or more mutations that prolong the activation of 1.1 (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In such cases, Na V A gain-of-function mutation in 1.1 includes one or more mutations that result in a disease phenotype. Exemplary gain-of-function mutations, but not limited to, include D188V, W1204R, R1648H, and D1866Y.

[0180]

[0280] In some embodiments, the disease or condition is encephalopathy. In some cases, encephalopathy is Na V It is induced by loss-of-function mutations in 1.1.

[0281] In some embodiments, the encephalopathy is an epileptic encephalopathy. Exemplary epileptic encephalopathy includes, but is not limited to, Dravet syndrome (DS) (also known as severe myoclonic epilepsy in infancy or SMEI), severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB), febrile seizures (FS), generalized febrile seizures plus epilepsy (GEFS+), epileptic encephalopathy, early infancy, 13, generalized epilepsy of unknown cause, focal epilepsy of unknown cause, and These include oclonic-apoptotic epilepsy, Lennox-Gastaut syndrome, West syndrome, idiopathic convulsions, early myoclonic encephalopathy, progressive myoclonic epilepsy, alternating hemiplegia in children, unclassified epileptic encephalopathy, sudden unexpected death in epilepsy (SUDEP), early infantile SCN1A encephalopathy, early infantile epileptic encephalopathy (EIEE), autism, infantile epilepsy with migratory focal seizures, or sick sinus syndrome¹. In some embodiments, the disease or condition is, at the discretion of the court, an epileptic encephalopathy selected from Dravet syndrome (DS) (also known as severe myoclonic epilepsy in infancy or SMEI), severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB), febrile seizures (FS), generalized febrile seizures plus epilepsy (GEFS+), epileptic encephalopathy, early infantile, 13, generalized epilepsy of unknown cause, focal epilepsy of unknown cause, myoclonic-astatic epilepsy, Lennox-Gastaut syndrome, West syndrome, idiopathic convulsions, early myoclonic encephalopathy, progressive myoclonic epilepsy, alternating hemiplegia in children, unclassified epileptic encephalopathy, sudden unexpected death in epilepsy (SUDEP), and sick sinus syndrome 1.

[0181]

[0282] In some cases, GEFS+ is type 2 epilepsy with generalized febrile seizures.

[0283] In some cases, the febrile seizures are classified as febrile seizures, familial, and 3A.

[0182]

[0284] In some cases, SMEB is classified as SMEB without generalized spike waves (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more features of SMEI (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0183]

[0285] In some embodiments, GEFS+ is epilepsy type 2 with generalized febrile seizures. In some embodiments, febrile seizures are familial, type 3A. In some embodiments, SMEB is SMEB without generalized spike waves (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking two or more features of SMEI (SMEB-O), or refractory childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).

[0184]

[0286] In some embodiments, Na V 1.1 Diseases or conditions induced by loss-of-function mutations include, but are not limited to, Dravet syndrome (DS) (also known as SMEI), severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB), febrile seizures (FS), generalized febrile seizures plus epilepsy (GEFS+), epileptic encephalopathy, early infancy, 13, generalized epilepsy of unknown cause, focal epilepsy of unknown cause, myoclonic-astatic epilepsy, Lennox-Gastaut syndrome, West syndrome, idiopathic convulsions, early myoclonic encephalopathy, progressive myoclonic epilepsy, alternating hemiplegia in children, unclassified epileptic encephalopathy, sudden unexpected death in epilepsy (SUDEP), sick sinus syndrome Symptoms include Syndrome 1, early infant SCN1A encephalopathy, early infant epileptic encephalopathy (EIEE), autism, or infantile epilepsy with migratory focal seizures.

[0185]

[0287] In the relevant embodiments, the method involves using an ASO to reduce the expression of a protein or functional RNA. In some embodiments, the ASO is Na V1.1 In target cells having NMD-inducible exons (NIEs) containing protein-coding premRNA, Na V 1.1 Used to reduce protein expression. In some embodiments, the target is Na V 1.1 The cell contains a gain-of-function mutation, e.g., migraine. In some embodiments, the ASO is Na in the target cell. V 1.1 Used to reduce protein expression, the target is Na V 1.1 contains gain-of-function mutations, e.g., migraine, familial hemiplegia, 3.

[0186]

[0288] In some embodiments, Na V 1.1 The level of protein-coding mRNA is measured in control cells, e.g., those not treated with antisense oligomers or those treated with antisense oligomers that do not bind to the targeted region of SCN1A NIE containing premRNA. V 1.1 Compared to the amount of mRNA encoding the protein, the amount decreases by 1.1 to 10 times.

[0187]

[0289] In some embodiments, the disease or condition is Na V 1.1 Genetic epilepsy. Na V 1.1 Genetic epilepsy is Na V 1.1 Even if it includes loss-of-function mutations, Na V 1.1 may also include gain-of-function mutations. In some cases, Na V 1.1 Genetic epilepsy involves one or more hereditary mutations. In other cases, Na V 1.1 Genetic epilepsy involves one or more de novo mutations. In some cases, Na V1.1 Genetic epilepsy includes Dravet syndrome (DS) (also known as severe myoclonic epilepsy in infancy or SMEI), severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB), febrile seizures (FS), generalized febrile seizures plus epilepsy (GEFS+), epileptic encephalopathy, early infant, 13, generalized epilepsy of unknown cause, focal epilepsy of unknown cause, myoclonic-astatic epilepsy, Lennox-Gastaut syndrome, West syndrome, idiopathic convulsions, early myoclonic encephalopathy, progressive myoclonic epilepsy, alternating hemiplegia in children, unclassified epileptic encephalopathy, early infant SCN1A encephalopathy, early infant epileptic encephalopathy (EIEE), and infant epilepsy with sudden unexpected death in epilepsy (SUDEP) or migratory focal seizures. In some cases, Na V 1.1 Loss-of-function mutations in Na and related Na V 1.1 Genetic epilepsy includes Dravet syndrome (DS) (also known as severe myoclonic epilepsy in infancy or SMEI), severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB), febrile seizures (FS), generalized febrile seizures plus epilepsy (GEFS+), epileptic encephalopathy, early infant, 13, generalized epilepsy of unknown cause, focal epilepsy of unknown cause, myoclonic-astatic epilepsy, Lennox-Gastaut syndrome, West syndrome, idiopathic convulsions, early myoclonic encephalopathy, progressive myoclonic epilepsy, alternating hemiplegia in children, unclassified epileptic encephalopathy, early infant SCN1A encephalopathy, early infant epileptic encephalopathy (EIEE), sudden unexpected death in epilepsy (SUDEP), and infantile epilepsy with migratory focal seizures.

[0188]

[0290] In some embodiments, the disease or condition is associated with SCN1A gene haploinsufficiency. Exemplary diseases or conditions associated with SCN1A gene haploinsufficiency include, but are not limited to, Dravet syndrome (DS) (also known as SMEI), severe myoclonic epilepsy in infancy (SMEI-borderline SMEB), febrile seizures (FS), generalized febrile seizures plus epilepsy (GEFS+), epileptic encephalopathy, early infantile, 13, generalized epilepsy of unknown cause, focal epilepsy of unknown cause, myoclonic-astatic epilepsy, Lennox-Gastaut syndrome, West syndrome, idiopathic convulsions, early myoclonic encephalopathy, progressive myoclonic epilepsy, alternating hemiplegia in children, unclassified epileptic encephalopathy, and epilepsy. These include sudden unexpected death in epilepsy (SUDEP), sick sinus syndrome 1, early infant SCN1A encephalopathy, early infant epileptic encephalopathy (EIEE), or infantile epilepsy with migratory focal seizures. In some cases, the disease or condition is Dravet syndrome (DS) (also known as SMEI), severe myoclonic epilepsy in infancy (SMEI)-borderline (SMEB), febrile seizures (FS), generalized febrile seizures plus epilepsy (GEFS+), epileptic encephalopathy, early infant, 13, generalized epilepsy of unknown cause, focal epilepsy of unknown cause, myoclonic-astatic epilepsy, Lennox-Gastaut syndrome, West syndrome, idiopathic convulsions, early myoclonic encephalopathy, progressive myoclonic epilepsy, alternating hemiplegia in children, unclassified epileptic encephalopathy, sudden unexpected death in epilepsy (SUDEP), sick sinus syndrome 1, early infant SCN1A encephalopathy, early infant epileptic encephalopathy (EIEE), or infantile epilepsy with migratory focal seizures.

[0189]

[0291] In some cases, the disease or condition is Dravet syndrome (DS).

[0292] As used herein, the term “epilepsy” refers to a group of neurological disorders characterized by recurrent epileptic seizures. As used herein, “epileptic seizure” refers to an episode that may vary from short, nearly undetectable periods to prolonged, violent tremors. Exemplary types of seizures include, but are not limited to, convulsive, nonconvulsive, focal, and generalized seizures. Exemplary types of generalized seizures include, but are not limited to, tonic-clonic, tonic, clonic, myoclonic, absence, and astonic seizures.

[0190]

[0293] In some embodiments, the disease or condition is Na V It is induced by gain-of-function mutations in 1.1. Na V 1.1 Examples of diseases or conditions associated with gain-of-function mutations include, but are not limited to, migraines. In some cases, Na V 1.1 The disease or condition induced by a gain-of-function mutation is migraine. In some embodiments, migraine is migraine, familial hemiplegia, 3.

[0191]

[0294] In some embodiments, the method is Na V 1.1 Na by target cells with NIE containing protein-coding premRNA V 1.1 A method to reduce protein expression, targeting Na V 1.1 It has a gain-of-function mutation. In such embodiments, the subject is Na V 1.1 Alleles or cells in which Na is produced in increased amounts of protein V It has an allele encoding the mutant SCN1A that induces increased activity of 1.1. In some embodiments, Na V The increased activity of 1.1 is due to mutant Na V1.1 The invention is characterized by a long-term or nearly persistent sodium current mediated by the channel, rapid deactivation deceleration, a positive shift in steady-state inactivation, higher channel availability between repeated stimuli, increased depolarization-induced persistent sodium current without inactivation, delayed entry into inactivation, accelerated rapid recovery of inactivation, and / or rescue of folding defects by incubation at low temperatures or co-expression of interacting proteins. In any of these embodiments, the antisense oligomer binds to a targeted portion of the NIE containing premRNA transcribed from a second allele, thereby inhibiting or blocking exon skipping of pseudoexons from the premRNA and functional Na in the target cell. V 1.1 Decreased levels of mature mRNA encoding proteins and Na V 1.1 Causes a decrease in protein expression.

[0192] composition

[0295] In some embodiments, the ASO includes a sequence having at least 83%, 88%, 94%, or 100% sequence identity with any one of sequence numbers 21-67, 210-256, or 304-1099. In some embodiments, the ASO consists of a sequence having at least 83%, 88%, 94%, or 100% sequence identity with any one of sequence numbers 21-67, 210-256, or 304-1099. Morphologically, an ASO comprises a sequence having at least 83%, 88%, 94%, or 100% sequence identity with any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b. In some embodiments, the ASO consists of a sequence having at least 83%, 88%, 94%, or 100% sequence identity with any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b.

[0193]

[0296] In some embodiments, the ASO includes sequences having at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 884%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% sequence identity with respect to any one of sequence numbers 21-67, 210-256, or 304-1099. In some embodiments, the ASO consists of sequences having at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 884%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% sequence identity with respect to any one of sequence numbers 21-67, 210-256, or 304-1099. In some embodiments, the ASO includes a sequence having at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 884%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% sequence identity with respect to any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b.In some embodiments, the ASO consists of sequences having at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 884%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% sequence identity with respect to any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b.

[0194]

[0297] In some embodiments, the ASO described herein comprises at least one modified sugar moiety.

[0298] In some embodiments, the ASO described herein includes a T-methoxyethyl sugar moiety. In some embodiments, the T-methoxyethyl sugar moiety is a T-2'-methoxyethyl sugar moiety. In some embodiments, the ASO described herein includes a 2'-O-methoxyethyl moiety. In some embodiments, the ASO described herein includes a thymidine containing a 2'-O-methoxyethyl moiety. In some embodiments, each nucleic acid base of the ASO described herein includes a 2'-O-methoxyethyl moiety.

[0195]

[0299] In some embodiments, the ASO described herein consists of 8 to 50 nucleic acid bases. In some embodiments, the ASO described herein consists of 16 to 20 nucleic acid bases. In some embodiments, the ASO described herein consists of 12 to 20 nucleic acid bases. In some embodiments, the ASO described herein consists of 8 to 20 nucleic acid bases.

[0196]

[0300] In some embodiments, the ASO described herein is 5-100, 6-100 It consists of 7-100, 8-100, 9-100, 10-100, 11-100, 12-100, 13-100, 14-100, 15-100, 16-100, 17-100, 18-100, 19-100, 20-100, 21-100, 22-100, 23-100, 24-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 85-100, or 90-100 nucleic acid bases. In some embodiments, the ASO described herein consists of 5-100, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, or 5-10 nucleic acid bases. In some embodiments, the ASO described herein consists of 8-50, 8-45, 8-40, 8-35, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, or 8-16 nucleic acid bases. In some embodiments, the ASO described herein consists of 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, or 18-20 nucleic acid bases. In some embodiments, the ASO described herein consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleic acid bases.

[0197]

[0301] In some embodiments, the ASOs described herein include 5'-methylcytosine (5'-MeC). In some embodiments, each cytosine in the ASOs described herein is 5'-methylcytosine (5'-MeC).

[0198]

[0302] In some embodiments, the ASOs described herein include phosphorothioate bonds. In some embodiments, each nucleoside bond in the ASOs described herein is a phosphorothioate bond.

[0199]

[0303] In some embodiments, the ASO described herein includes locked nucleic acid (LNA).

[0304] In some embodiments, the ASO described herein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 LNAs. In some embodiments, the ASO described herein includes 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 LNAs. In some embodiments, the ASO described herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 LNAs.

[0200]

[0305] In some embodiments, the 5' terminal nucleotide of the ASO described herein is LNA. In some embodiments, the 3' terminal nucleotide of the ASO described herein is LNA. In some embodiments, the 5' and 3' terminal nucleotides of the ASO described herein are LNA.

[0201] ASO (Antisense Oligomer)

[0306] This specification provides compositions comprising antisense oligomers that induce exon skipping by binding to a targeted portion of a premRNA-containing SCN1A NIE. As used herein, the terms “ASO” and “antisense oligomer” are used interchangeably and refer to compositions comprising an antisense oligomer that induces exon skipping by Watson-Crick base pairing or fluctuation base pairing (GU) of a targeted nucleic acid (e.g., a premRNA-containing SCN1A NIE). ASOs refer to oligomers, such as polynucleotides, that contain nucleic acid bases that hybridize to a target sequence. ASOs can have an exact sequence complementary to the target sequence, or nearly complementary (e.g., sufficient complementarity to bind to the target sequence and enhance splicing at the splice site). ASOs are designed to bind (hybridize) to the target nucleic acid (e.g., the targeted portion of a premRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not the target nucleic acid (several sites other than the target nucleic acid). The design of ASOs can take into account the occurrence of sufficiently similar nucleic acid sequences at other locations in the genome or cell's premRNA or transcriptome, so as to limit the possibility of the ASO binding to other sites and causing “off-target” effects. Any antisense oligomer known in the art, for example, the one described herein in PCT application PCT / US2014 / 054151, published as International Publication No. 2015 / 035091, entitled “Reducing Nonsense-Mediated mRNA Decay,” which is incorporated herein by reference, can be used to carry out the method described herein.

[0202]

[0307] In some embodiments, the ASO "specifically hybridizes" or is "specific" to the targeted portion of the NIE containing the target nucleic acid or premRNA. Typically, such hybridization occurs at temperatures substantially greater than 37°C. m This hybridization preferably occurs at at least 50°C, typically 60°C to about 90°C. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, T m This is the temperature at which 50% of the target sequence hybridizes to complementary oligonucleotides.

[0203]

[0308] Oligomers, such as oligonucleotides, are “complementary” to each other if hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration. A double-stranded polynucleotide can be “complementary” to another polynucleotide if hybridization can occur between one of the first polynucleotide chains and one of the second polynucleotide chains. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion (e.g., percentage) of bases in opposing chains that are expected to form hydrogen bonds with each other according to generally accepted base-pairing rules. The sequence of an antisense oligomer (ASO) does not need to be 100% complementary to the sequence of the target nucleic acid to hybridize. In certain embodiments, ASOs may have sequence complementarity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with respect to the targeting region in the target nucleic acid sequence to which they are targeted. For example, 18 of the 20 nucleic acid bases of an oligomeric compound are complementary to the target region, and therefore, the specifically hybridizing ASO represents 90 percent complementarity. In this example, the remaining non-complementary nucleic acid bases may cluster with each other or be scattered with complementary nucleic acid bases, and do not need to be adjacent to each other or to complementary nucleic acid bases. The percentage of complementarity between the ASO and the region of the target nucleic acid can be routinely determined using the BLAST program (a basic local alignment search tool) and the PowerBLAST program, which is publicly known in the art (the entire content of which is incorporated herein by reference: Altschul et al., J.Mol.Biol., 1990, Vol. 215, pp. 403-410; Zhang and Madden, Genome Res., 1997, Vol. 7, pp. 649-656).

[0204]

[0309] ASO does not need to hybridize to all nucleic acid bases in the target sequence; the nucleic acid bases that hybridize may be adjacent or not. ASO can hybridize on one or more segments of a premRNA transcript, thereby preventing intervening or adjacent segments from participating in the hybridization event (e.g., forming loop or hairpin structures). In certain embodiments, ASO hybridizes to non-adjacent nucleic acid bases in the target premRNA transcript. For example, ASO can hybridize to nucleic acid bases in a premRNA transcript separated by one or more nucleic acid bases that ASO does not hybridize to.

[0205]

[0310] The ASOs described herein include nucleic acid bases that are complementary to the nucleic acid bases present in the targeted region of the NIE containing premRNA. The term “ASO” includes nucleic acid bases that can hybridize to complementary nucleic acid bases on the target mRNA, but do not contain sugar moieties such as peptide nucleic acids (PNAs), and any other oligomeric molecules. An ASO may include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides having modified or substituted sugar groups and / or a modified skeleton. In some embodiments, all nucleotides in the ASO are modified nucleotides. Chemical modifications of ASO or components of ASO that are suitable for the methods and compositions described herein will be obvious to those skilled in the art, and can be found, for example, in U.S. Patent Nos. 8,258,109, 5,656,612, U.S. Patent Application Publication No. 2012 / 0190728, which are incorporated herein by whole reference, and in Dias and Stein, Mol. Cancer Ther. 2002, pp. 347-355.

[0206]

[0311] One or more nucleic acid bases of the ASO may be naturally occurring unmodified nucleic acid bases such as adenine, guanine, cytosine, thymine, and uracil, or any synthetic or modified nucleic acid base that is sufficiently similar to the unmodified nucleic acid bases so as to be able to hydrogen bond with the nucleic acid bases present on the target premRNA. Examples of modified nucleic acid bases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine (5-hydroxymethoylcytosine).

[0207]

[0312] The ASOs described herein also include a skeletal structure that connects the components of the oligomer. The terms “skeletal structure” and “oligomer linkage” can be used interchangeably and refer to the linkage between monomers in the ASO. In naturally occurring oligonucleotides, the skeletal structure includes a 3'-5' phosphodiester linkage that connects the sugar portions of the oligomer. The skeletal structures or oligomer links of the ASOs described herein may include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, phosphoramidates, and the like. For example, see LaPlanche et al., Nucleic Acids Res. Vol. 14: p. 9081 (1986); Stec et al., J.Am.Chem.Soc. Vol. 106: p. 6077 (1984); Stein et al., Nucleic Acids Res. Vol. 16: p. 3209 (1988); Zon et al., Anti-Cancer Drug Design Vol. 6: p. 539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews Vol. 90: p. 543 (1990), the contents of which are incorporated herein by reference. In some embodiments, the ASO skeletal structure does not contain phosphorus, but rather peptide bonds, for example, The peptide nucleic acid (PNA) contains peptide bonds, or binding groups including carbamates, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the skeletal modification is a phosphothioate bond. In some embodiments, the skeletal modification is a phosphoramidate bond.

[0208]

[0313] In embodiments, the stereochemistry of each phosphonucleotide bond in the ASO skeleton is random. In embodiments, the stereochemistry of each phosphonucleotide bond in the ASO skeleton is controlled and not random. For example, U.S. Patent Application Publication 2014 / 0194610, “Methods for the Synthesis of Functionalized Nucleic Acids,” incorporated herein by reference, describes a method for independently selecting the chirality of each phosphonucleotide in a nucleic acid oligomer. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs listed in Tables 5 and 6, and includes ASOs having non-random phosphonucleotide bonds. In embodiments, the composition used in the method of the present invention includes a pure diastereomer ASO. In embodiments, the composition used in the method of the present invention comprises an ASO having a diastereomer purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0209]

[0314] In embodiments, the ASO has a non-random mixture of Rp and Sp configurations in its phosphonucleotide interbonding. For example, it has been suggested that a mixture of Rp and Sp is necessary in antisense oligonucleotides or antisense oligomers to achieve a balance between good activity and nuclease stability (see Wan et al., 2014, "Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate Linkages, Nucleic Acids Research, Vol. 42 (No. 22): pp. 13456-13468. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in SEQ ID NOs. 21-114, and comprises about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder being Sp or comprising about 100% Rp. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in SEQ ID NOs. 21 to 114, with a range of approximately 10% to approximately 100% Rp, approximately 15% to approximately 100% Rp, approximately 20% to approximately 100% Rp, approximately 25% to approximately 100% Rp, approximately 30% to approximately 100% Rp, approximately 35% to approximately 100% Rp, approximately 40% to approximately 100% Rp, approximately 45% to approximately 100% Rp, and approximately 50% %~approximately 100% Rp, approximately 55%~approximately 100% Rp, approximately 60%~approximately 100% Rp, approximately 65%~approximately 100% Rp, approximately 70%~approximately 100% Rp, approximately 75%~approximately 100% Rp, approximately 80%~approximately 100% Rp, approximately 85%~approximately 100% Rp, approximately 90%~approximately 100% Rp, or approximately 95%~approximately 100% Rp, approximately 20%~approximately 80% Rp, approximately 25%~approximately 75% Rp, approximately 30%~approximately 70% Rp, approximately 40%~approximately 60% Rp, also It contains approximately 45% to 55% Rp, with the remainder being Sp.

[0210]

[0315] In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in Sequence Numbers 21 to 114, and comprises about 5 to 100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder being Rp or comprising about 100% Sp. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in Sequence ID Nos. 21 to 114, with concentrations of approximately 10% to approximately 100% Sp, approximately 15% to approximately 100% Sp, approximately 20% to approximately 100% Sp, approximately 25% to approximately 100% Sp, approximately 30% to approximately 100% Sp, approximately 35% to approximately 100% Sp, approximately 40% to approximately 100% Sp, approximately 45% to approximately 100% Sp, approximately 50% to approximately 100% Sp, and approximately 5 This includes 5% to approximately 100% Sp, approximately 60% to approximately 100% Sp, approximately 65% ​​to approximately 100% Sp, approximately 70% to approximately 100% Sp, approximately 75% to approximately 100% Sp, approximately 80% to approximately 100% Sp, approximately 85% to approximately 100% Sp, approximately 90% to approximately 100% Sp, or approximately 95% to approximately 100% Sp, approximately 20% to approximately 80% Sp, approximately 25% to approximately 75% Sp, approximately 30% to approximately 70% Sp, approximately 40% to approximately 60% Sp, or approximately 45% to approximately 55% Sp, with the remainder being Rp.

[0211]

[0316] In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in SEQ ID NOs. 21-67, 210-256, or 304-1099, and comprises about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder being Sp or comprising about 100% Rp. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in SEQ ID NOs. 21-67, 210-256, or 304-1099, in amounts of about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, and about 50% to about This includes 100% Rp, approximately 55% to 100% Rp, approximately 60% to 100% Rp, approximately 65% ​​to 100% Rp, approximately 70% to 100% Rp, approximately 75% to 100% Rp, approximately 80% to 100% Rp, approximately 85% to 100% Rp, approximately 90% to 100% Rp, or approximately 95% to 100% Rp, approximately 20% to 80% Rp, approximately 25% to 75% Rp, approximately 30% to 70% Rp, approximately 40% to 60% Rp, or approximately 45% to 55% Rp, with the remainder being Sp.

[0212]

[0317] In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in Sequence IDs 21-67, 210-256 or 304-1099, with a content of about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, and at least about 7% It comprises 5% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder being Rp or comprising about 100% Sp. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in SEQ ID NOs. 21-67, 210-256 or 304-1099, and comprises about 10%-about 100% Sp, about 15%-about 100% Sp, about 20%-about 100% Sp, about 25%-about 100% Sp, about 30%-about 100% Sp, about 35%-about 100% Sp, about 40%-about 100% Sp, about 45%-about 100% Sp, and about 50%-about This includes 100% Sp, approximately 55% to 100% Sp, approximately 60% to 100% Sp, approximately 65% ​​to 100% Sp, approximately 70% to 100% Sp, approximately 75% to 100% Sp, approximately 80% to 100% Sp, approximately 85% to 100% Sp, approximately 90% to 100% Sp, or approximately 95% to 100% Sp, approximately 20% to 80% Sp, approximately 25% to 75% Sp, approximately 30% to 70% Sp, approximately 40% to 60% Sp, or approximately 45% to 55% Sp, with the remainder being Rp.

[0213]

[0318] In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b, and includes about 5 to 100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder being Sp or about 100% Rp. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b, with a range of approximately 10% to approximately 100% Rp, approximately 15% to approximately 100% Rp, approximately 20% to approximately 100% Rp, approximately 25% to approximately 100% Rp, approximately 30% to approximately 100% Rp, approximately 35% to approximately 100% Rp, approximately 40% to approximately 100% Rp, and approximately 45% to approximately 100% This includes Rp, approximately 50% to 100% Rp, approximately 55% to 100% Rp, approximately 60% to 100% Rp, approximately 65% ​​to 100% Rp, approximately 70% to 100% Rp, approximately 75% to 100% Rp, approximately 80% to 100% Rp, approximately 85% to 100% Rp, approximately 90% to 100% Rp, or approximately 95% to 100% Rp, approximately 20% to 80% Rp, approximately 25% to 75% Rp, approximately 30% to 70% Rp, approximately 40% to 60% Rp, or approximately 45% to 55% Rp, with the remainder being Sp.

[0214]

[0319] In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b, and includes about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder being Rp or about 100% Sp. In embodiments, the ASO used in the method of the present invention includes, but is not limited to, any of the ASOs described herein in any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b, in amounts of about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, and about This includes 35% to approximately 100% Sp, approximately 40% to approximately 100% Sp, approximately 45% to approximately 100% Sp, approximately 50% to approximately 100% Sp, approximately 55% to approximately 100% Sp, approximately 60% to approximately 100% Sp, approximately 65% ​​to approximately 100% Sp, approximately 70% to approximately 100% Sp, approximately 75% to approximately 100% Sp, approximately 80% to approximately 100% Sp, approximately 85% to approximately 100% Sp, approximately 90% to approximately 100% Sp, or approximately 95% to approximately 100% Sp, approximately 20% to approximately 80% Sp, approximately 25% to approximately 75% Sp, approximately 30% to approximately 70% Sp, approximately 40% to approximately 60% Sp, or approximately 45% to approximately 55% Sp, with the remainder being Rp.

[0215]

[0320] Any of the ASOs described herein may include a sugar moiety containing ribose or deoxyribose, as found in naturally occurring nucleotides, or a modified sugar moiety or sugar analog containing a morpholine ring. Non-limiting examples of modified sugar moieties include 2' substitutions, e.g., 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;N3'->P5' phosphoramidate, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. In some embodiments, the sugar modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar modification is an extra crosslink, such as in locked nucleic acids (LNAs). In some embodiments, the sugar analog contains a morpholine ring, for example, phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety includes ribofuranyl or 2'-deoxyribofuranyl modification. In some embodiments, the sugar moiety includes 2',4'-restricted 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety includes cEt2',4'-restricted 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety includes tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety includes ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety includes MCE modification. The modifications are publicly known in the art and are described in the literature, for example, Jarver et al., 2014, Nucleic Acid Therapeutics, Vol. 24 (No. 1): pp. 37-47, incorporated by reference for the purposes of this specification, and "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics, Vol. 24 (No. 1): pp. 37-47, incorporated by reference for the purposes of this specification.

[0216]

[0321] In some embodiments, each monomer of ASO is modified in the same manner, for example, each bond in the ASO skeleton contains a phosphorothioate bond, or each ribose sugar moiety contains a 2'O-methyl modification. Such modifications present in each monomer component of ASO are called “homogeneous modifications.” In some examples, combinations of different modifications may be desired. For example, ASO may contain a combination of a phosphorodiamidate bond and a sugar moiety (morpholinose) containing a morpholine ring. Combinations of different modifications to ASO are called “mixed modifications” or “mixed chemistry.”

[0217]

[0322] In some embodiments, the ASO comprises one or more skeletal modifications. In some embodiments, the ASO comprises one or more partial sugar modifications. In some embodiments, the ASO comprises one or more skeletal modifications and one or more partial sugar modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate skeleton. In some embodiments, the ASO comprises phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of an ASO (e.g., nucleic acid bases, sugar moieties, skeletons), may be modified to achieve desired properties or activities of the ASO, or to reduce undesirable properties or activities of the ASO. For example, modifying one or more components of an ASO or any ASO By modifying the ASO, it is possible to enhance its binding affinity to target sequences on premRNA transcripts; reduce binding to any non-target sequences; reduce degradation by cellular nucleases (i.e., RNase H); improve the uptake of ASO into cells and / or the nucleus; alter the pharmacokinetics or pharmacodynamics of ASO; and / or modulate the half-life of ASO.

[0218]

[0323] In some embodiments, the ASO is composed of a 2'-O-(2-methoxyethyl)(MOE) phosphorothioate modified nucleotide. ASOs composed of such nucleotides are particularly well suited to the methods disclosed herein, and oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation, increased bioavailability, and, for example, suitability for oral delivery in some embodiments described herein. For example, see Geary et al., J Pharmacol Exp Ther. 2001; vol. 296 (no. 3): pp. 890-897; Geary et al., J Pharmacol Exp Ther. 2001; vol. 296 (no. 3): pp. 898-904, incorporated herein by reference.

[0219]

[0324] Methods for synthesizing ASO are known to those skilled in the art. Alternatively, or in addition to, ASO can be obtained from commercial sources.

[0325] Unless otherwise specified, the left end of a single-stranded nucleic acid sequence (e.g., pre-mRNA transcripts, oligonucleotides, ASOs, etc.) is called the 5' end, and the left end of a single-stranded or double-stranded nucleic acid sequence is called the 5' direction. Similarly, the right end or direction of a nucleic acid sequence (single-stranded or double-stranded) is called the 3' end or 3' direction. Generally, the region or sequence that is 5' relative to the reference point in a nucleic acid is called "upstream," and the region or sequence that is 3' relative to the reference point in a nucleic acid is called "downstream." Generally, the 5' direction or 5' end of mRNA is where the start codon or start codon is located, while the 3' end or 3' direction is where the stop codon is located. In some embodiments, nucleotides upstream of the reference point in a nucleic acid can be denoted by a negative number, while nucleotides downstream of the reference point can be denoted by a positive number. For example, a reference point (e.g., an exon-exon junction in mRNA) can be designated as a "zero" site, and nucleotides directly adjacent to the reference point and upstream of it can be designated as "minus 1", for example "-1", while nucleotides directly adjacent to the reference point and downstream of it can be designated as "plus 1", for example "+1".

[0220]

[0326] In some embodiments, the ASO is complementary to (and binds to) the targeted region of the SCN1A NIE containing the premRNA, which is downstream (3' direction) of the 5' splice site (or 3' end of the NIE) of the exon containing the premRNA (e.g., the direction indicated by a positive number relative to the 5' splice site). In some embodiments, the ASO is complementary to the targeted region of the SCN1A NIE containing the premRNA, which lies in the region approximately +1 to approximately +500 relative to the 5' splice site (or 3' end) of the exon containing the premRNA. In some embodiments, the ASO may be complementary to the targeted region of the SCN1A NIE containing the premRNA, which lies in the region between nucleotides +6 and +496 relative to the 5' splice site (or 3' end) of the exon containing the premRNA. In some embodiments, the ASO is approximately +1 to +500, approximately +1 to +490, approximately +1 to +480, approximately +1 to +470, approximately +1 to +460, approximately +1 to +450, approximately +1 to +440, approximately +1 to +430, approximately +1 to +420, approximately +1 to +410, approximately +1 to +400, approximately +1 to +390, approximately +1 to +380, and approximately +1 ~approximately +370, approximately +1~approximately +360, approximately +1~approximately +350, approximately +1~approximately +340, approximately +1~approximately +330, approximately +1~approximately +320, approximately +1~approximately +310, approximately +1~approximately +300, approximately +1~approximately +290, approximately +1~approximately +280, approximately +1~approximately +270, approximately +1~approximately +260, approximately +1~approximately +250, approximately +1~approximately +240, approximately +1~approximately +230, approximately +1~approximately +220, approximately +1~approximately +210, approximately +1~approximately +200, It is complementary to the targeted portion located within the range of approximately +1 to +190, approximately +1 to +180, approximately +1 to +170, approximately +1 to +160, approximately +1 to +150, approximately +1 to +140, approximately +1 to +130, approximately +1 to +120, approximately +1 to +110, approximately +1 to +100, approximately +1 to +90, approximately +1 to +80, approximately +1 to +70, approximately +1 to +60, approximately +1 to +50, approximately +1 to +40, approximately +1 to +30, or approximately +1 to +20. In some embodiments, the ASO is complementary to the targeted portion, which is located within a region of approximately +1 to +100, approximately +100 to +200, approximately +200 to +300, approximately +300 to +400, or approximately +400 to +500 relative to the 5' splice site (or 3' end) of the exon containing it.

[0221]

[0327] In some embodiments, the ASO is complementary to (and binds to) the targeted region of the SCN1A NIE containing the premRNA, which is upstream (5' direction) of the 5' splice site (or 3' end) of the exon containing the premRNA (e.g., the direction indicated by a negative number relative to the 5' splice site). In some embodiments, the ASO is complementary to the targeted region of the SCN1A NIE containing the premRNA, which is located in the region approximately -4 to approximately -270 relative to the 5' splice site (or 3' end) of the exon containing the premRNA. In some embodiments, the ASO may be complementary to the targeted region of the SCN1A NIE containing the premRNA, which is located in the region between nucleotides -1 and -264 relative to the 5' splice site (or 3' end) of the exon containing the premRNA. In some embodiments, the ASO is approximately -1 to approximately -270, approximately -1 to approximately -260, approximately -1 to approximately -250, approximately -1 to approximately -240, approximately -1 to approximately -230, approximately -1 to approximately -220, approximately -1 to approximately -210, approximately -1 to approximately -200, approximately -1 to approximately -190, approximately -1 to approximately -180, approximately -1 to approximately -170, approximately -1 to approximately - In some embodiments, the ASO is complementary to the targeted portion located in the regions of approximately -1 to -150, approximately -1 to -140, approximately -1 to -130, approximately -1 to -120, approximately -1 to -110, approximately -1 to -100, approximately -1 to -90, approximately -1 to -80, approximately -1 to -70, approximately -1 to -60, approximately -1 to -50, approximately -1 to -40, approximately -1 to -30, or approximately -1 to -20 relative to the 5' splice site (or 3' end) of the exon containing the ASO.

[0222]

[0328] In some embodiments, the ASO is complementary to the targeting region of the SCN1A NIE containing the premRNA, which is upstream (5' direction) (e.g., direction indicated by a negative number) of the 3' splice site (or 5' end) of the exon containing the premRNA in the SCN1A NIE containing the premRNA. In some embodiments, the ASO is complementary to the targeted region of the SCN1A NIE containing the premRNA, which lies in the region approximately -1 to approximately -500 relative to the 3' splice site (or 5' end) of the exon containing the premRNA. In some embodiments, the ASO is complementary to the targeted region of the SCN1A NIE containing the premRNA, which lies in the region approximately -1 to -496 relative to the 3' splice site of the exon containing the premRNA. In some embodiments, the ASO is approximately -1 to approximately -500, approximately -1 to approximately -490, approximately -1 to approximately -480, approximately -1 to approximately -470, approximately -1 to approximately -460, approximately -1 to approximately -450, approximately -1 to approximately -440, approximately -1 to approximately -430, approximately -1 to approximately -420, approximately -1 to approximately -410, approximately -1 to approximately -400, approximately -1 to approximately -390, approximately -1 to approximately -380, approximately -1 to approximately -370, approximately -1 to approximately -360, approximately -1 to approximately -350, approximately -1 to approximately - 340, approx. -1 to approx. -330, approx. -1 to approx. -320, approx. -1 to approx. -310, approx. -1 to approx. -300, approx. -1 to approx. -290, approx. -1 to approx. -280, approx. -1 to approx. -270, approx. -1 to approx. -260, approx. -1 to approx. -250, approx. -1 to approx. -240, approx. -1 to approx. -230, approx. -1 to approx. -220, approx. -1 to approx. -210, approx. -1 to approx. -200, approx. -1 to approx. -190, approx. -1 to approx. -180, approx. -1 to approx. -170, approx. -1 to approx. -160, approx. -1 to approx. -150, approx. -1 to approx. -1 In some embodiments, the ASO is complementary to the targeted portion located in the regions of approximately -1 to -130, approximately -1 to -120, approximately -1 to -110, approximately -1 to -100, approximately -1 to -90, approximately -1 to -80, approximately -1 to -70, approximately -1 to -60, approximately -1 to -50, approximately -1 to -40, or approximately -1 to -30 relative to the 3' splice site of the exon containing it.

[0223]

[0329] In some embodiments, the ASO is complementary to the targeting region of the premRNA-containing SCN1A NIE that is downstream (3' direction) (e.g., direction indicated by a positive number) of the 3' splice site (5' end) of the exon containing the premRNA in the SCN1A NIE containing the premRNA. In some embodiments, the ASO is complementary to the targeted portion of the premRNA-containing SCN1A NIE that lies in a region approximately +1 to approximately +100 relative to the 3' splice site of the exon containing the premRNA. In some embodiments, the ASO is complementary to the targeted portion that lies in a region approximately +1 to approximately +90, approximately +1 to approximately +80, approximately +1 to approximately +70, approximately +1 to approximately +60, approximately +1 to approximately +50, approximately +1 to approximately +40, approximately +1 to approximately +30, approximately +1 to approximately +20, or approximately +1 to approximately +10 relative to the 3' splice site of the exon containing the premRNA.

[0224]

[0330] In some embodiments, the targeted region of the SCN1A NIE containing premRNA is located within a region from +100 to -100 relative to the 5' splice site (3' end) of the exon containing the premRNA. In some embodiments, the targeted region of the SCN1A NIE containing premRNA is located within the NIE. In some embodiments, the targeted region of the SCN1A NIE containing premRNA includes a pseudoexon and an intron boundary.

[0225]

[0331] The ASO can be of any length suitable for effective enhancement of specific binding and splicing. In some embodiments, the ASO consists of 8 to 50 nucleic acid bases. For example, the ASO may have a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleic acid bases. In some embodiments, the ASO consists of more than 50 nucleic acid bases. In some embodiments, the ASO has lengths of 8-50 nucleic acid bases, 8-40 nucleic acid bases, 8-35 nucleic acid bases, 8-30 nucleic acid bases, 8-25 nucleic acid bases, 8-20 nucleic acid bases, 8-15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 nucleic acid bases, 10-40 nucleic acid bases, 10-35 nucleic acid bases, 10-30 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 1 2-30 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, 12-15 nucleic acid bases, 13-50 nucleic acid bases, 13-40 nucleic acid bases, 13-35 nucleic acid bases, 13-30 nucleic acid bases, 13-25 nucleic acid bases, 13-20 nucleic acid bases, 14-50 nucleic acid bases, 14-40 nucleic acid bases, 14-35 nucleic acid bases, 14-30 nucleic acid bases, 14-25 nucleic acid bases, 14-20 nucleic acid salts The ASO consists of 15-50 nucleic acid bases, 15-40 nucleic acid bases, 15-35 nucleic acid bases, 15-30 nucleic acid bases, 15-25 nucleic acid bases, 15-20 nucleic acid bases, 20-50 nucleic acid bases, 20-40 nucleic acid bases, 20-35 nucleic acid bases, 20-30 nucleic acid bases, 20-25 nucleic acid bases, 25-50 nucleic acid bases, 25-40 nucleic acid bases, 25-35 nucleic acid bases, or 25-30 nucleic acid bases. In some embodiments, the ASO has a length of 18 nucleotides. In some embodiments, the ASO has a length of 15 nucleotides. In some embodiments, the ASO has a length of 25 nucleotides.

[0226]

[0332] In some embodiments, two or more ASOs are used that have different chemistry but are complementary to the same targeted region of the NIE containing premRNA. In some embodiments, two or more ASOs are used that are complementary to different targeted regions of the NIE containing premRNA.

[0227]

[0333] In embodiments, the antisense oligonucleotide or antisense oligomer of the present invention is chemically linked to one or more moieties or conjugates, for example, a targeted moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, e.g., cholesterol moieties, cholesteryl moieties, aliphatic chains, e.g., dodecanediol or undecyl residues, polyamine or polyethylene glycol chains, or adamantane acetate. Oligonucleotides containing lipophilic moieties and methods for their preparation are described in published literature. In embodiments, the antisense oligonucleotide or antisense oligomer is conjugated with, but are not limited to, a moiety containing debasalized nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, e.g., N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. A conjugate can be linked to one or more nucleotides, including antisense oligonucleotides or antisense oligomers, at any of several positions on a sugar, base, or phosphate group, for example, using a linker, as understood in the art and described in the literature. The linker can be a divalent or trivalent branched linker. In embodiments, the conjugate is linked to the 3' end of the antisense oligonucleotide or antisense oligomer. A method for preparing oligonucleotide conjugates is, for example, U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery," which is incorporated herein by reference. This is described in "agents for oligonucleotides".

[0228]

[0334] In some embodiments, the nucleic acid targeted by ASO is an SCN1A NIE containing premRNA expressed in cells such as eukaryotic cells. In some embodiments, the term “cells” may refer to a population of cells. In some embodiments, the cells are within the subject. In some embodiments, the cells are isolated from the subject. In some embodiments, the cells are ex vivo. In some embodiments, the cells are cells or cell lines associated with a condition or disease. In some embodiments, the cells are in vitro (e.g., cell culture).

[0229]

[0335] In some embodiments, ASO is a salt of a nucleotide. In some embodiments, ASO is a salt of a nucleotide, a fully phosphorothioate-bonded oligonucleotide. In some embodiments, ASO is a salt of a nucleotide in which the salt is bonded to a phosphate bond. In some embodiments, ASO is a salt of a nucleotide, a fully phosphorothioate-bonded oligonucleotide in which the salt is bonded to a phosphate bond. In some embodiments, ASO is a sodium salt of a nucleotide. In some embodiments, ASO is a sodium salt of a nucleotide, a fully phosphorothioate-bonded oligonucleotide. In some embodiments, ASO is a sodium salt of a nucleotide in which the sodium salt is bonded to a phosphate bond. In some embodiments, ASO is a sodium salt of a nucleotide, a fully phosphorothioate-bonded oligonucleotide in which the sodium salt is bonded to a phosphate bond. In some embodiments, ASO is a potassium salt of a nucleotide. In some embodiments, ASO is a potassium salt of a nucleotide, a fully phosphorothioate-bonded oligonucleotide. In some embodiments, ASO is a potassium salt of a nucleotide in which the potassium salt is bonded to a phosphate bond. In some embodiments, ASO is a potassium salt of a nucleotide, a fully phosphorothioate-bonded oligonucleotide in which the potassium salt is bonded to a phosphate bond. It is a lium salt.

[0230]

[0336] In some embodiments, ASO is the monosodium salt of a 2-nucleotide (2-mer). In some embodiments, ASO is the disodium salt of a 3-nucleotide (3-mer). In some embodiments, ASO is the trisodium salt of a 4-nucleotide (4-mer). In some embodiments, ASO is the tetrasodium salt of a 5-nucleotide (5-mer). In some embodiments, ASO is the pentasodium salt of a 6-nucleotide (6-mer). In some embodiments, ASO is the hexasodium salt of a 7-nucleotide (7-mer). In some embodiments, ASO is the heptasodium salt of an 8-nucleotide (8-mer). In some embodiments, ASO is the octasodium salt of a 9-nucleotide (9-mer). In some embodiments, ASO is the nonasodium salt of a 10-nucleotide (10-mer). In some embodiments, ASO is the decanasodium salt of an 11-nucleotide (11-mer). In some embodiments, ASO is the undecasodium salt of a 12-nucleotide (12-mer). In some embodiments, ASO is the dodecasodium salt of a 13-nucleotide (13-mer). In some embodiments, ASO is the tripedacodium salt of a 14-nucleotide (14-mer). In some embodiments, ASO is the tetradecasodium salt of a 15-nucleotide (15-mer). In some embodiments, ASO is the pentadecanodium salt of a 16-nucleotide (16-mer). In some embodiments, ASO is the hexadecanodium salt of a 17-nucleotide (17-mer). In some embodiments, ASO is the heptadecanodium salt of an 18-nucleotide (18-mer). In some embodiments, ASO is the octadecanodium salt of a 19-nucleotide (19-mer). In some embodiments, ASO is the nonadecanodium salt of a 20-nucleotide (20-mer). In some embodiments, ASO is the eicosanate salt of a 21-nucleotide (21-mer). In some embodiments, ASO is the henicosa sodium salt of a 22-nucleotide (22-mer).In some embodiments, ASO is a docosodium salt of 23-nucleotides (23-mer). In some embodiments, ASO is a tricosodium salt of 24-nucleotides (24-mer). In some embodiments, ASO is a tetracosodium salt of 25-nucleotides (25-mer). In some embodiments, ASO is a pentacosodium salt of 26-nucleotides (26-mer). In some embodiments, ASO is a hexacosaodium salt of 27-nucleotides (27-mer). In some embodiments, ASO is a heptacosaodium salt of 28-nucleotides (28-mer). In some embodiments, ASO is an octacosodium salt of 29-nucleotides (29-mer). In some embodiments, ASO is a nonacosaodium salt of 30-nucleotides (30-mer). In some embodiments, ASO is a triacontasodium salt of 31-nucleotides (31-mer). In some embodiments, ASO is the hentoriaconta sodium salt of 32-nucleotides (32-mer). In some embodiments, ASO is the dotoriaconta sodium salt of 33-nucleotides (33-mer). In some embodiments, ASO is the tritoriaconta sodium salt of 34-nucleotides (34-mer). In some embodiments, ASO is the tetratriaconta sodium salt of 35-nucleotides (35-mer). In some embodiments, ASO is the pentariaconta sodium salt of 36-nucleotides (36-mer). In some embodiments, ASO is the hexariaconta sodium salt of 37-nucleotides (37-mer). In some embodiments, ASO is the heptatriaconta sodium salt of 38-nucleotides (38-mer). In some embodiments, ASO is the octariaconta sodium salt of 39-nucleotides (39-mer). In some embodiments, ASO is the nonariaconta sodium salt of 40-nucleotides (40-mer). In some embodiments, ASO is the tetraconta sodium salt of 41-nucleotide (41-mer). In some embodiments, ASO is 42. - It is the hentetraconta sodium salt of nucleotide (42-mer). In some embodiments, ASO is the dotetraconta sodium salt of nucleotide (43-mer). In some embodiments, ASO is the tritetraconta sodium salt of nucleotide (44-mer). In some embodiments, ASO is the tetratetraconta sodium salt of nucleotide (45-mer). In some embodiments, ASO is the pentatetraconta sodium salt of nucleotide (46-mer). In some embodiments, ASO is the hexatetraconta sodium salt of nucleotide (47-mer). In some embodiments, ASO is the heptatetraconta sodium salt of nucleotide (48-mer). In some embodiments, ASO is the octatetraconta sodium salt of nucleotide (49-mer). In some embodiments, ASO is the nonatetraconta sodium salt of nucleotide (50-mer). In some embodiments, ASO is the pentaconta sodium salt of a 51-nucleotide (51-mer).

[0231]

[0337] In some embodiments, ASO is the monosodium salt of a fully phosphorothioate-linked oligonucleotide (2-mer). In some embodiments, ASO is the disodium salt of a fully phosphorothioate-linked oligonucleotide (3-mer). In some embodiments, ASO is the trisodium salt of a fully phosphorothioate-linked oligonucleotide (4-mer). In some embodiments, ASO is the tetrasodium salt of a fully phosphorothioate-linked oligonucleotide (5-mer). In some embodiments, ASO is the pentasodium salt of a fully phosphorothioate-linked oligonucleotide (6-mer). In some embodiments, ASO is the hexasodium salt of a fully phosphorothioate-linked oligonucleotide (7-mer). In some embodiments, ASO is the heptasodium salt of an 8-nucleotide (8-mer). In some embodiments, ASO is the octa-sodium salt of a fully phosphorothioate-linked oligonucleotide of 9-nucleotides (9-mer). In some embodiments, ASO is the nona-sodium salt of a fully phosphorothioate-linked oligonucleotide of 10-nucleotides (10-mer). In some embodiments, ASO is the deca-sodium salt of a fully phosphorothioate-linked oligonucleotide of 11-nucleotides (11-mer). In some embodiments, ASO is the undeca-sodium salt of a fully phosphorothioate-linked oligonucleotide of 12-nucleotides (12-mer). In some embodiments, ASO is the dodeca-sodium salt of a fully phosphorothioate-linked oligonucleotide of 13-nucleotides (13-mer). In some embodiments, ASO is the trideca-sodium salt of a fully phosphorothioate-linked oligonucleotide of 14-nucleotides (14-mer). In some embodiments, ASO is the tetradeca-sodium salt of a fully phosphorothioate-linked oligonucleotide of 15-nucleotides (15-mer).In some embodiments, ASO is the pentadeca sodium salt of a fully phosphorothioate-linked oligonucleotide of 16-nucleotides (16-mers). In some embodiments, ASO is the hexadeca sodium salt of a fully phosphorothioate-linked oligonucleotide of 17-nucleotides (17-mers). In some embodiments, ASO is the heptadeca sodium salt of a fully phosphorothioate-linked oligonucleotide of 18-nucleotides (18-mers). In some embodiments, ASO is the octadeca sodium salt of a fully phosphorothioate-linked oligonucleotide of 19-nucleotides (19-mers). In some embodiments, ASO is the nonadeca sodium salt of a fully phosphorothioate-linked oligonucleotide of 20-nucleotides (20-mers). In some embodiments, ASO is the eicosa sodium salt of a fully phosphorothioate-linked oligonucleotide of 21-nucleotides (21-mers). Some embodiments. In some embodiments, ASO is the henicosa sodium salt of a fully phosphorothioate-linked oligonucleotide (22-mer) of 22 nucleotides. In some embodiments, ASO is the docosana sodium salt of a fully phosphorothioate-linked oligonucleotide (23-mer) of 23 nucleotides. In some embodiments, ASO is the tricosana sodium salt of a fully phosphorothioate-linked oligonucleotide (24-mer) of 24 nucleotides. In some embodiments, ASO is the tetracosana sodium salt of a fully phosphorothioate-linked oligonucleotide (25-mer) of 25 nucleotides. In some embodiments, ASO is the pentacosa sodium salt of a fully phosphorothioate-linked oligonucleotide (26-mer) of 26 nucleotides. In some embodiments, ASO is the hexacosa sodium salt of a fully phosphorothioate-linked oligonucleotide (27-mer) of 27 nucleotides. In some embodiments, ASO is the heptacosa sodium salt of a fully phosphorothioate-linked oligonucleotide of 28-nucleotides (28-mer). In some embodiments, ASO is the octacosa sodium salt of a fully phosphorothioate-linked oligonucleotide of 29-nucleotides (29-mer). In some embodiments, ASO is the nonacosa sodium salt of a fully phosphorothioate-linked oligonucleotide of 30-nucleotides (30-mer). In some embodiments, ASO is the triaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 31-nucleotides (31-mer). In some embodiments, ASO is the hentriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 32-nucleotides (32-mer). In some embodiments, ASO is the dotriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 33-nucleotides (33-mer). In some embodiments, ASO is the tritriaconta sodium salt of a fully phosphorothioate-bonded oligonucleotide of a 34-nucleotide (34-mer).In some embodiments, ASO is the tetratriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 35-nucleotides (35-mer). In some embodiments, ASO is the pentatriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 36-nucleotides (36-mer). In some embodiments, ASO is the hexatriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 37-nucleotides (37-mer). In some embodiments, ASO is the heptatriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 38-nucleotides (38-mer). In some embodiments, ASO is the octatriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 39-nucleotides (39-mer). In some embodiments, ASO is the nonatriaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 40-nucleotides (40-mer). In some embodiments, ASO is the tetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 41-nucleotide (41-mer). In some embodiments, ASO is the hentetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 42-nucleotide (42-mer). In some embodiments, ASO is the dotetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 43-nucleotide (43-mer). In some embodiments, ASO is the tritetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 44-nucleotide (44-mer). In some embodiments, ASO is the tetratetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 45-nucleotide (45-mer). In some embodiments, ASO is the pentatetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 46-nucleotide (46-mer).In some embodiments, ASO is the hexatetraconta sodium salt of a fully phosphorothioate-bonded oligonucleotide of 47-nucleotides (47-mers). In some embodiments, ASO is of 48-nucleotides (48-mers). ASO is the heptatetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide. In some embodiments, ASO is the octatetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 49-nucleotides (49-mer). In some embodiments, ASO is the nonatetraconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 50-nucleotides (50-mer). In some embodiments, ASO is the pentaconta sodium salt of a fully phosphorothioate-linked oligonucleotide of 51-nucleotides (51-mer).

[0232]

[0338] In some embodiments, ASO is a monopotassium salt of 2-nucleotides (2-mer). In some embodiments, ASO is a dipotassium salt of 3-nucleotides (3-mer). In some embodiments, ASO is a tripotassium salt of 4-nucleotides (4-mer). In some embodiments, ASO is a tetrapotassium salt of 5-nucleotides (5-mer). In some embodiments, ASO is a pentapotassium salt of 6-nucleotides (6-mer). In some embodiments, ASO is a hexapotassium salt of 7-nucleotides (7-mer). In some embodiments, ASO is a heptapotassium salt of 8-nucleotides (8-mer). In some embodiments, ASO is an octapotassium salt of 9-nucleotides (9-mer). In some embodiments, ASO is a nonapotassium salt of 10-nucleotides (10-mer). In some embodiments, ASO is a decapotassium salt of 11-nucleotides (11-mer). In some embodiments, ASO is an undecapotassium salt of 12-nucleotides (12-mer). In some embodiments, ASO is the dodecapotassium salt of 13-nucleotides (13-mer). In some embodiments, ASO is the tripecapotassium salt of 14-nucleotides (14-mer). In some embodiments, ASO is the tetradecapotassium salt of 15-nucleotides (15-mer). In some embodiments, ASO is the pentadecapotassium salt of 16-nucleotides (16-mer). In some embodiments, ASO is the hexadecapotassium salt of 17-nucleotides (17-mer). In some embodiments, ASO is the heptadecapotassium salt of 18-nucleotides (18-mer). In some embodiments, ASO is the octadecapotassium salt of 19-nucleotides (19-mer). In some embodiments, ASO is the nonadecapotassium salt of 20-nucleotides (20-mer). In some embodiments, ASO is the eicosapotassium salt of 21-nucleotides (21-mer). In some embodiments, ASO is a henicosapotassium salt of a 22-nucleotide (22-mer). In some embodiments, ASO is a docosapotassium salt of a 23-nucleotide (23-mer).In some embodiments, ASO is a tricosapotassium salt of 24-nucleotides (24-mer). In some embodiments, ASO is a tetracosapotassium salt of 25-nucleotides (25-mer). In some embodiments, ASO is a pentacosapotassium salt of 26-nucleotides (26-mer). In some embodiments, ASO is a hexacosapotassium salt of 27-nucleotides (27-mer). In some embodiments, ASO is a heptacosapotassium salt of 28-nucleotides (28-mer). In some embodiments, ASO is an octacosapotassium salt of 29-nucleotides (29-mer). In some embodiments, ASO is a nonacosapotassium salt of 30-nucleotides (30-mer). In some embodiments, ASO is a triacontapotassium salt of 31-nucleotides (31-mer). In some embodiments, ASO is a hentriacontapotassium salt of 32-nucleotides (32-mer). In some embodiments, ASO is a dotriaconta potassium salt of 33-nucleotides (33-mer). In some embodiments, ASO is a tritriaconta potassium salt of 34-nucleotides (34-mer). In some embodiments, ASO is a tetratriaconta potassium salt of 35-nucleotides (35-mer). In some embodiments, ASO is a pentatriaconta potassium salt of 36-nucleotides (36-mer). In some embodiments, ASO is a hexatriaconta potassium salt of 37-nucleotides (37-mer). In some embodiments, ASO is 38. - It is the heptatriaconta potassium salt of nucleotide (38-mer). In some embodiments, ASO is the octatriconta potassium salt of nucleotide (39-mer). In some embodiments, ASO is the nonatriaconta potassium salt of nucleotide (40-mer). In some embodiments, ASO is the tetraconta potassium salt of nucleotide (41-mer). In some embodiments, ASO is the hentetraconta potassium salt of nucleotide (42-mer). In some embodiments, ASO is the dotetraconta potassium salt of nucleotide (43-mer). In some embodiments, ASO is the tritetraconta potassium salt of nucleotide (44-mer). In some embodiments, ASO is the tetratetraconta potassium salt of nucleotide (45-mer). In some embodiments, ASO is the pentatetraconta potassium salt of nucleotide (46-mer). In some embodiments, ASO is the hexatetraconta potassium salt of 47-nucleotides (47-mer). In some embodiments, ASO is the heptatetraconta potassium salt of 48-nucleotides (48-mer). In some embodiments, ASO is the octatetraconta potassium salt of 49-nucleotides (49-mer). In some embodiments, ASO is the nonatetraconta potassium salt of 50-nucleotides (50-mer). In some embodiments, ASO is the pentaconta potassium salt of 51-nucleotides (51-mer).

[0233]

[0339] In some embodiments, ASO is the monopotassium salt of a fully phosphorothioate-linked oligonucleotide (2-mer). In some embodiments, ASO is the dipotassium salt of a fully phosphorothioate-linked oligonucleotide (3-mer). In some embodiments, ASO is the tripotassium salt of a fully phosphorothioate-linked oligonucleotide (4-mer). In some embodiments, ASO is the tetrapotassium salt of a fully phosphorothioate-linked oligonucleotide (5-mer). In some embodiments, ASO is the pentapotassium salt of a fully phosphorothioate-linked oligonucleotide (6-mer). In some embodiments, ASO is the hexapotassium salt of a fully phosphorothioate-linked oligonucleotide (7-mer). In some embodiments, ASO is the heptapotassium salt of an 8-nucleotide (8-mer). In some embodiments, ASO is the octapotassium salt of a fully phosphorothioate-linked oligonucleotide of 9-nucleotides (9-mer). In some embodiments, ASO is the nonapotassium salt of a fully phosphorothioate-linked oligonucleotide of 10-nucleotides (10-mer). In some embodiments, ASO is the decapotassium salt of a fully phosphorothioate-linked oligonucleotide of 11-nucleotides (11-mer). In some embodiments, ASO is the undecapotassium salt of a fully phosphorothioate-linked oligonucleotide of 12-nucleotides (12-mer). In some embodiments, ASO is the dodecapotassium salt of a fully phosphorothioate-linked oligonucleotide of 13-nucleotides (13-mer). In some embodiments, ASO is the tridecapotassium salt of a fully phosphorothioate-linked oligonucleotide of 14-nucleotides (14-mer). In some embodiments, ASO is the tetradecapotassium salt of a fully phosphorothioate-linked oligonucleotide of 15-nucleotides (15-mer).In some embodiments, ASO is the pentadeca potassium salt of a fully phosphorothioate-linked oligonucleotide of 16-nucleotides (16-mers). In some embodiments, ASO is the hexadeca potassium salt of a fully phosphorothioate-linked oligonucleotide of 17-nucleotides (17-mers). In some embodiments, ASO is the heptadeca potassium salt of a fully phosphorothioate-linked oligonucleotide of 18-nucleotides (18-mers). In some embodiments, ASO is the fully phosphorothioate-linked oligonucleotide of 19-nucleotides (19-mers). ASO is the octadeca potassium salt of an Oate-linked oligonucleotide. In some embodiments, ASO is the nonadeca potassium salt of a fully phosphorothioate-linked oligonucleotide of 20-nucleotides (20-mers). In some embodiments, ASO is the eicosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 21-nucleotides (21-mers). In some embodiments, ASO is the henicosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 22-nucleotides (22-mers). In some embodiments, ASO is the docosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 23-nucleotides (23-mers). In some embodiments, ASO is the tricosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 24-nucleotides (24-mers). In some embodiments, ASO is the tetracosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 25-nucleotides (25-mers). In some embodiments, ASO is the pentacosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 26-nucleotides (26-mer). In some embodiments, ASO is the hexacosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 27-nucleotides (27-mer). In some embodiments, ASO is the heptacosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 28-nucleotides (28-mer). In some embodiments, ASO is the octacosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 29-nucleotides (29-mer). In some embodiments, ASO is the nonacosa potassium salt of a fully phosphorothioate-linked oligonucleotide of 30-nucleotides (30-mer). In some embodiments, ASO is the triaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 31-nucleotides (31-mer). In some embodiments, ASO is the potassium hentriaconta salt of a fully phosphorothioate-bonded oligonucleotide of 32-nucleotides (32-mers).In some embodiments, ASO is a dotriaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 33-nucleotide (33-mer). In some embodiments, ASO is a tritriaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 34-nucleotide (34-mer). In some embodiments, ASO is a tetratriaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 35-nucleotide (35-mer). In some embodiments, ASO is a pentatriaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 36-nucleotide (36-mer). In some embodiments, ASO is a hexatriaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 37-nucleotide (37-mer). In some embodiments, ASO is a heptatriaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 38-nucleotide (38-mer). In some embodiments, ASO is the octa-triaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 39-nucleotides (39-mer). In some embodiments, ASO is the nona-atriaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 40-nucleotides (40-mer). In some embodiments, ASO is the tetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 41-nucleotides (41-mer). In some embodiments, ASO is the hentetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 42-nucleotides (42-mer). In some embodiments, ASO is the dotetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 43-nucleotides (43-mer). In some embodiments, ASO is the tritetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 44-nucleotides (44-mer). In some embodiments, ASO is a tetratetraconta potassium salt of a fully phosphorothioate-bonded oligonucleotide of a 45-nucleotide (45-mer).In some embodiments, ASO is a 46-nucleotide (46-mer). ASO is the pentatetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide. In some embodiments, ASO is the hexatetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 47-nucleotide (47-mer). In some embodiments, ASO is the heptatetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 48-nucleotide (48-mer). In some embodiments, ASO is the octatetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 49-nucleotide (49-mer). In some embodiments, ASO is the nonatetraconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 50-nucleotide (50-mer). In some embodiments, ASO is the pentaconta potassium salt of a fully phosphorothioate-linked oligonucleotide of 51-nucleotide (51-mer).

[0234] SCN1A

[0340] The SCN1A gene can encode the SCN1A (sodium channel, voltage-gated, type I, alpha subunit) protein, which is a voltage-gated sodium channel Na V It can also be called the alpha-subunit of 1.1. Furthermore, as mentioned above, SCN1A mutations in DS are widespread throughout the protein. Over 100 novel mutations have been identified throughout the gene, and more debilitation is occurring in de novo studies. These consist of shortenings (47%), missense mutations (43%), deletions (3%), and splice site mutations (7%). The percentage of subjects with SCN1A mutations varies from 33% to 100%. The majority of mutations are novel changes (88%).

[0235]

[0341] In some embodiments, the methods described herein are functional Na V1.1 Used to modulate protein production, for example, to increase or decrease it. As used herein, the term “functional” means any one or more symptoms of the condition being treated, e.g., Dravet syndrome, epilepsy, generalized, with febrile seizures plus, type 2; febrile seizures, familial, type 3A; autism; epileptic encephalopathy, early infancy, type 13; sick sinus syndrome, type 1; Alzheimer’s disease; or Na required to eliminate SUDEP. V 1.1 Refers to the amount of protein activity or function. In some embodiments, this method refers to partially functional Na V 1.1 Used to increase protein production. As used herein, the term “partial functionality” means less than the amount of activity or function required to eliminate or prevent any one or more symptoms of a disease or condition. V 1.1 This refers to any amount of activity or function of a protein. In some embodiments, a partially functional protein or RNA has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% less activity than a fully functional protein or RNA.

[0236]

[0342] In some embodiments, this method is Na V 1.1 Target cells having NIE containing protein-coding premRNA are Na V 1.1 A method to increase protein expression, targeting Na V 1.1 Dravet syndrome, caused by a deficiency in protein activity, Na V 1.1 Protein deficiency is Na V 1.1 Caused by protein haploinsufficiency. In such embodiments, the subject is functional Na V 1.1 The first allele that codes for a protein, and Na V 1.1 Having a second allele that does not produce protein. In another such embodiment, the subject is functional Na V1.1 A first allele encoding a protein, and non-functional Na V It has a second allele encoding the 1.1 protein. In another such embodiment, the subject is a functional Na V It has a first allele encoding the 1.1 protein, and a partially functional Na V It has a second allele encoding the 1.1 protein. In some embodiments, the subject is a partially functional Na derived from one allele V Expresses the 1.1 protein and is partially functional Na V The 1.1 protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, the subject is a non-functional Na derived from one allele V Expresses the 1.1 protein and is non-functional Na V The 1.1 protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion in one allele. In some embodiments, the subject has a deletion of the entire SCN1A gene in one allele. In any of these embodiments, the antisense oligomer binds to the targeted portion of the NIE containing the pre-mRNA transcribed from the second allele, thereby inducing exon skipping of the pseudoexon from the pre-mRNA and increasing the level of the mature mRNA encoding the functional Na V 1.1 protein, and an increase in the expression of Na V 1.1 protein in the cells of the subject.

[0237]

[0343] In embodiments of the present invention, the subject can have a mutation in SCN1A. The mutation in SCN1A can spread throughout the gene. Na V The 1.1 protein can consist of four domains. The SCN1A domain can have transmembrane segments. The Na V The mutation in the 1.1 protein can occur throughout the protein. The Na V1.1 A protein can consist of at least two isoforms. Mutations in SCN1A may include R931C, R946C, M934I, R1648C, or R1648H. In some cases, the mutation is Na V 1.1 It may be observed at the C-terminus of proteins. Na V 1.1 Mutations in proteins are, as mentioned above, Na V 1.1 Mutations can also be found in the loop between segments 5 and 6 of the first three domains of the protein. In some cases, the mutation occurs in Na V 1.1 These mutations may be observed at the N-terminus of the protein. Exemplary mutations within SCN1A include, but are not limited to, R222X, R712X, I227S, R1892X, W952X, R1245X, R1407X, W1434R, c.4338+1G>A, 51516X, L1670fsX1678, or K1846fsX1856. Mutations that can target the present invention can also encode pores in ion channels.

[0238]

[0344] In some embodiments, the methods and compositions described herein can be used to treat DS. In other embodiments, the methods and compositions described herein can be used to treat severe myoclonic epilepsy of infancy (SMEI). In other embodiments, the methods and compositions described herein can be used to treat borderline Dravet syndrome; epilepsy, generalized, with febrile seizures plus, type 2; febrile seizures, familial, type 3A; migraine, familial hemiplegia, type 3; autism; epileptic encephalopathy, early infancy, type 13; sick sinus syndrome, type 1; Alzheimer's disease or SUDEP.

[0239]

[0345] In related embodiments, this method is a method for increasing the expression of a protein or functional RNA using an ASO. In some embodiments, the ASO is Na V 1.1 In terms of protein quantity and function, Na V1.1 Na in target cells with NIE containing protein-coding premRNA V 1.1 Used to increase protein expression, the target is individuals with deficiencies, e.g., Dravet syndrome (DS) (also known as SMEI); severe myoclonic epilepsy of infants (SMEI) borderline (SMEB); febrile seizures (FS); epilepsy, generalized, with febrile seizures plus (GEFS+); epileptic encephalopathy, early infant, 13; generalized epilepsy of unknown cause; focal epilepsy of unknown cause; myoclonus-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic convulsions; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia in children; unclassified epileptic encephalopathy; sudden unexpected death from epilepsy (SUDEP); sick sinus syndrome 1; early infant SCN1A encephalopathy; early infant epileptic encephalopathy (EIEE); or autism. In some embodiments, the ASO increases the amount or function of the SCN8A protein in the target individual. Na in cells V 1.1 Used to increase protein expression, the target is a deficiency, e.g., epileptic encephalopathy, early infant, 13. In some embodiments, ASO is used to increase the amount or function of SCN5A protein in the target cells Na V 1.1 Used to increase protein expression, the target group is those with deficiencies, e.g., sick sinus syndrome 1.

[0240]

[0346] In some embodiments, the methods and compositions described herein can also be used to treat borderline SMEI. Furthermore, the methods and compositions described herein can be used to treat generalized epilepsy with febrile seizures plus (GEFS+). GEFS+ may be associated with mutations in epilepsy-related ion channel subunits such as SCN1B or GABRG2. The methods and compositions described herein can also be used to treat sodium channel disorders. Sodium channel disorders may be associated with mutations in SCN1A. Sodium channel disorders may also be associated with subunits of SCN1A, such as the beta subunit SCN1B. In some cases, additional disorders associated with SCN1A mutations can also be treated using the present disclosure. Associated SCN1A disorders associated with SCN1A mutations include, but are not limited to, atypical congenital myotonia, hyperkalemic periodic paralysis, and congenital paramyotonia.

[0241]

[0347] In some embodiments, subjects having any SCN1A mutation known in the art and described in the literature (e.g., Hamdan et al., 2009, N.Engl.Med., Vol. 360 (No. 6), p. 599; Mulley et al., 2005, Hum.Muta., Vol. 25, pp. 535-542, which are incorporated herein by reference in their entirety) can be treated with the methods and compositions described herein. In some embodiments, the mutation is located within any SCN1A intron or exon.

[0242]

[0348] In some embodiments, NIEs containing premRNA transcripts encoding a protein that causes a disease or condition are targeted by ASOs described herein. In some embodiments, NIEs containing premRNA transcripts encoding a protein that does not cause a disease are targeted by ASOs. For example, a disease resulting from a mutation or deficiency of a first protein in a particular pathway can be mitigated by targeting an NIE containing premRNA encoding a second protein, thereby increasing the production of the second protein. In some embodiments, the function of the second protein can compensate for the mutation or deficiency of the first protein (which causes the disease or condition).

[0243]

[0349] In some embodiments, the subject is (a) The first mutant allele, (i)Na V 1.1 Is the protein produced at a reduced level compared to production from the wild-type allele? (ii) Na V 1.1 The protein is produced in a form with reduced function compared to the equivalent wild-type protein, or (iii) Na V 1.1 No protein or functional RNA is produced. The first mutant allele; and (b) A second mutant allele, (i)Na V 1.1 Is the protein produced at a reduced level compared to production from the wild-type allele? (ii) Na V 1.1 The protein is produced in a form with reduced function compared to the equivalent wild-type protein, or (iii) Na V 1.1 Protein is not produced Second mutant allele The NIE, which contains premRNA, is transcribed from a first allele and / or a second allele. In these embodiments, ASO binds to a targeted region of the NIE containing premRNA transcribed from the first or second allele, thereby inducing exon skipping of pseudoexons from the premRNA-containing NIE, and Na V 1.1 This results in an increase in the level of protein-coding mRNA and an increase in the expression of the target protein or functional RNA in the target cells. In these embodiments, the target protein or functional RNA having an increased expression level resulting from exon skipping of pseudoexons from the NIE containing premRNA is either a form with reduced function (partially functional) compared to the equivalent wild-type protein, or a form with full function (fully functional) compared to the equivalent wild-type protein.

[0244]

[0350] In some embodiments, Na V 1.1 The level of protein-coding mRNA is compared to the Na produced in control cells. V 1.1 The amount of protein-coding mRNA, for example, increases 1.1 to 10 times compared to untreated mRNA or mRNA treated with an antisense oligomer that does not bind to the targeted region of SCN1A NIE containing premRNA.

[0245]

[0351] In some embodiments, the subject treated using the method of this disclosure is a single-allele mutant Na V 1.1 The protein expresses mutant Na V 1.1 Proteins are caused by frameshift mutations, nonsense mutations, missense mutations, or partial gene deletions, resulting in mutant Na V 1.1 Proteins are Na V1.1 This causes an increase in the activity level. In some embodiments, the subject treated using the method of this disclosure is a single allele with an increased amount of Na resulting from a frameshift mutation, nonsense mutation, missense mutation, or partial gene deletion. V 1.1 Express the protein.

[0246]

[0352] In some embodiments, the subject treated using the method of this disclosure is a partially functional Na derived from one allele. V 1.1 Expressing protein, partially functional Na V 1.1 Proteins are caused by frameshift mutations, nonsense mutations, missense mutations, or partial gene deletions. In some embodiments, the subject treated using the methods of this disclosure is a non-functional Na derived from a single allele. V 1.1 Express protein, non-functional Na V 1.1 Proteins are caused by frameshift mutations, nonsense mutations, missense mutations, or partial gene deletions in a single allele. In some embodiments, the subjects treated using the methods of this disclosure have a complete SCN1A gene deletion in a single allele.

[0247]

[0353] In some embodiments, this method is Na V 1.1 Na by target cells with NIE containing protein-coding premRNA V 1.1 A method to reduce protein expression, targeting Na V 1.1 has a gain-of-function mutation. In such embodiments, the subject is Na V 1.1 Alleles produced when the amount of protein increases, or Na in cells V 1.1 It has an allele encoding the mutant SCN1A that induces increased activity. In some embodiments, Na V 1.1 The increased activity is due to the mutant Na V1.1 These are characterized by prolonged or nearly persistent sodium currents mediated by the channel, delayed rapid inactivation, a positive shift in steady-state inactivation, higher channel availability during repeated stimulation, increased deinactivation-depolarization-induced persistent sodium current, delayed entry into inactivation, accelerated recovery from rapid inactivation, and / or recovery of folding defects by incubation or co-expression of the interacting protein at lower temperatures.

[0248] Target transcript

[0354] Splicing of identified SCN1A NIE premRNA species to produce functional mature Scn1a mRNA can be induced using therapeutic agents such as ASO that stimulate NIE exon skipping. Induction of exon skipping can lead to inhibition of the NMD pathway. The resulting mature Scn1a mRNA is translated normally without activating the NMD pathway, thereby enabling Na in patient cells. V 1.1 It can increase the amount of protein and alleviate symptoms of Dravet syndrome (DS); epilepsy, generalized, with febrile seizures plus, type 2; febrile seizures, familial, type 3A; autism; epileptic encephalopathy, early infancy, 13; sick sinus syndrome, 1; Alzheimer's disease; or conditions associated with SCN1A deficiency such as SUDEP.

[0249]

[0355] In various embodiments, the disclosure provides therapeutic agents that can target SCN1A premRNA transcripts and modulate, for example, enhance or inhibit splicing or protein expression levels. The therapeutic agents may be small molecules, polynucleotides, or polypeptides. In some embodiments, the therapeutic agent is an ASO. Various regions or sequences on SCN1A premRNA can be targeted by therapeutic agents such as ASOs. In some embodiments, the ASO targets SCN1A premRNA transcripts containing an NIE. In some embodiments, the ASO targets sequences within the NIE of the SCN1A premRNA transcript. In some embodiments, the ASO targets sequences upstream (or 5') of the 5' end of the NIE(3'ss) of the SCN1A premRNA transcript. In some embodiments, the ASO targets sequences downstream (or 3') of the NIE(5'ss) of the SCN1A premRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 5' end of the NIE in the SCN1A premRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 3' end of the NIE in the SCN1A premRNA transcript. In some embodiments, the ASO targets a sequence containing the NIE-intron boundary in the SCN1A premRNA transcript. The NIE-intron boundary can refer to the junction between the intron sequence and the NIE region. The intron sequence can be adjacent to the 5' end of the NIE or the 3' end of the NIE. In some embodiments, the ASO targets a sequence within an exon in the SCN1A premRNA transcript. In some embodiments, the ASO targets a sequence within an intron in the SCN1A premRNA transcript. In some embodiments, the ASO targets a sequence containing both a portion of an intron and a portion of an exon.

[0250]

[0356] In some embodiments, the therapeutic agents described herein modulate the binding of factors involved in the splicing of premRNA containing NMD exons. In some embodiments, the therapeutic agents described herein prevent the binding of factors involved in the splicing of premRNA containing NMD exons. In some embodiments, the therapeutic agents described herein prevent the binding of factors involved in the splicing of premRNA containing NMD exons. In some embodiments, the therapeutic agent contains NMD exons and Na V The targeting region is located in the intron region between two standard exon regions of the premRNA encoding 1.1, and the intron region contains an NMD exon. In some embodiments, the therapeutic agent targets a targeting region that at least partially overlaps with the NMD exon. In some embodiments, the therapeutic agent targets a targeting region that at least partially overlaps with an intron upstream of the NMD exon. In some embodiments, the therapeutic agent targets a targeting region within the NMD exon.

[0251]

[0357] In some embodiments, the therapeutic agent comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive NMD exons. The targeting site contains nucleotides. In some embodiments, the therapeutic agent targets a targeting site containing at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of NMD exons.

[0252]

[0358] In some embodiments, the therapeutic agent targets a targeted region located proximal to the NMD exon.

[0359] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO targets approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 250 to 300 nucleotides, 350 to 400 nucleotides, 450 to 500 nucleotides, and 550 to 60 nucleotides upstream from the 5' end of the NIE region. The ASO targets sequences upstream (or 5') of 0 nucleotides, approximately 650–700 nucleotides, approximately 750–800 nucleotides, approximately 850–900 nucleotides, approximately 950–1000 nucleotides, approximately 1050–1100 nucleotides, approximately 1150–1200 nucleotides, approximately 1250–1300 nucleotides, approximately 1350–1400 nucleotides, or approximately 1450–1500 nucleotides. In some embodiments, the ASO can target sequences more than 300 nucleotides upstream from the 5' end of the NIE. In some embodiments, the ASO targets sequences downstream (or 3') of approximately 4–300 nucleotides from the 3' end of the NIE. In some embodiments, the ASO targets sequences downstream of approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 350 to 400 nucleotides, 450 to 500 nucleotides, 550 to 600 nucleotides, 650 to 700 nucleotides, 750 to 800 nucleotides, 850 to 900 nucleotides, 950 to 1000 nucleotides, 1050 to 1100 nucleotides, 1150 to 1200 nucleotides, 1250 to 1300 nucleotides, 1350 to 1400 nucleotides, or 1450 to 1500 nucleotides from the 3' end of the NIE. In some embodiments, the ASO targets sequences more than 300 nucleotides downstream from the 3' end of the NIE.

[0253]

[0360] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5') from the 5' end of the NIE. In some cases, the ASO is located upstream of the 5' end of the NIE region at least approximately 1 nucleotide, at least approximately 10 nucleotides, at least approximately 20 nucleotides, at least approximately 50 nucleotides, at least approximately 80 nucleotides, at least approximately 85 nucleotides, at least approximately 90 nucleotides, at least approximately 95 nucleotides, at least approximately 96 nucleotides, at least approximately 97 nucleotides, at least approximately 98 nucleotides, at least approximately 99 nucleotides, at least approximately 100 nucleotides, at least approximately 101 nucleotides, at least approximately 102 nucleotides, at least approximately 103 nucleotides, at least approximately 104 nucleotides, at least approximately 105 nucleotides, at least approximately 110 nucleotides, at least approximately 120 nucleotides, at least approximately 150 nucleotides, at least approximately 200 nucleotides, at least approximately 300 nucleotides, at least approximately 400 nucleotides, at least approximately 500 nucleotides, at least approximately 600 nucleotides, at least approximately 700 nucleotides, at least approximately 800 nucleotides, at least approximately 900 nucleotides, or at least approximately 1000 nucleotides. The sequence is targeted at the (or 5') end. In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides downstream (or 3') from the 3' end of the NIE. In some embodiments, the ASO targets a sequence at least approximately 1 nucleotide, at least approximately 10 nucleotides, at least approximately 20 nucleotides, at least approximately 50 nucleotides, at least approximately 80 nucleotides, at least approximately 85 nucleotides, at least approximately 90 nucleotides, at least approximately 95 nucleotides, at least approximately 96 nucleotides, at least approximately 97 nucleotides, at least approximately 98 nucleotides, at least approximately 99 nucleotides, at least approximately 100 nucleotides, at least approximately 101 nucleotides, at least approximately 102 nucleotides, at least approximately 103 nucleotides, at least approximately 104 nucleotides, at least approximately 105 nucleotides, at least approximately 110 nucleotides, at least approximately 120 nucleotides, at least approximately 150 nucleotides, at least approximately 200 nucleotides, at least approximately 300 nucleotides, at least approximately 400 nucleotides, at least approximately 500 nucleotides, at least approximately 600 nucleotides, at least approximately 700 nucleotides, at least approximately 800 nucleotides, at least approximately 900 nucleotides, or at least approximately 1000 nucleotides downstream from the 3' end of the NIE.

[0254]

[0361] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5') from the 5' end of the NIE. In some embodiments, the ASO targets up to approximately 10 nucleotides, up to approximately 20 nucleotides, up to approximately 50 nucleotides, up to approximately 80 nucleotides, up to approximately 85 nucleotides, up to approximately 90 nucleotides, up to approximately 95 nucleotides, up to approximately 96 nucleotides, up to approximately 97 nucleotides, up to approximately 98 nucleotides, up to approximately 99 nucleotides, up to approximately 100 nucleotides, up to approximately 101 nucleotides, up to approximately 102 nucleotides, up to approximately 103 nucleotides, up to approximately 104 nucleotides, up to approximately 105 nucleotides, and up to approximately 1 The sequence targets 10 nucleotides, up to approximately 120 nucleotides, up to approximately 150 nucleotides, up to approximately 200 nucleotides, up to approximately 300 nucleotides, up to approximately 400 nucleotides, up to approximately 500 nucleotides, up to approximately 600 nucleotides, up to approximately 700 nucleotides, up to approximately 800 nucleotides, up to approximately 900 nucleotides, up to approximately 1000 nucleotides, up to approximately 1100 nucleotides, up to approximately 1200 nucleotides, up to approximately 1300 nucleotides, up to approximately 1400 nucleotides, or up to approximately 1500 nucleotides upstream (or 5'). In some embodiments, the ASO targets the sequence approximately 4 to approximately 300 nucleotides downstream (or 3') from the 3' end of the NIE.In some embodiments, the ASO is located at a maximum of approximately 10 nucleotides, 20 nucleotides, 50 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 96 nucleotides, 97 nucleotides, 98 nucleotides, 99 nucleotides, 100 nucleotides, 101 nucleotides, 102 nucleotides, 103 nucleotides, 104 nucleotides, 105 nucleotides, and so on from the 3' end of the NIE. It targets sequences downstream of 110 nucleotides, up to approximately 120 nucleotides, up to approximately 150 nucleotides, up to approximately 200 nucleotides, up to approximately 300 nucleotides, up to approximately 400 nucleotides, up to approximately 500 nucleotides, up to approximately 600 nucleotides, up to approximately 700 nucleotides, up to approximately 800 nucleotides, up to approximately 900 nucleotides, or up to approximately 1000 nucleotides, up to approximately 1100 nucleotides, up to approximately 1200 nucleotides, up to approximately 1300 nucleotides, up to approximately 1400 nucleotides, or up to approximately 1500 nucleotides. In some embodiments, the ASO targets sequences downstream of more than 300 nucleotides from the 3' end of the NIE.

[0255]

[0362] In some embodiments, the NIE described herein is GRCh37 / hg19:chr2:166,863,740 and GRCh37 / hg19 as shown in Figure 2. It is located between :chr2:166,863,803. In some embodiments, the 5' end of the NIE is located at GRCh37 / hg19:chr2:166,863,803. In some embodiments, the 3' end of the NIE is located at GRCh37 / hg19:chr2:166,863,740.

[0256]

[0363] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5') from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 250 to 300 nucleotides, 350 to 400 nucleotides, and 450 to 500 nucleotides from the genomic site GRCh37 / hg19:chr2:166,863,803. The ASO targets sequences approximately 550–600 nucleotides, 650–700 nucleotides, 750–800 nucleotides, 850–900 nucleotides, 950–1000 nucleotides, 1050–1100 nucleotides, 1150–1200 nucleotides, 1250–1300 nucleotides, 1350–1400 nucleotides, or approximately 1450–1500 nucleotides upstream (or 5'). In some embodiments, the ASO can target sequences more than 300 nucleotides upstream from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets sequences approximately 4–300 nucleotides downstream (or 3') from GRCh37 / hg19:chr2:166,863,740. In some embodiments, ASO is derived from GRCh37 / hg19:chr2:166,863,740 in the following quantities: approximately 1 to approximately 20 nucleotides, approximately 20 to approximately 50 nucleotides, approximately 50 to approximately 100 nucleotides, approximately 100 to approximately 150 nucleotides, approximately 150 to approximately 200 nucleotides, approximately 200 to approximately 250 nucleotides, approximately 250 to approximately 300 nucleotides, approximately 350 to approximately 400 nucleotides, approximately 450 to approximately 500 nucleotides, and approximately The target is a sequence downstream of approximately 550-600 nucleotides, approximately 650-700 nucleotides, approximately 750-800 nucleotides, approximately 850-900 nucleotides, approximately 950-1000 nucleotides, approximately 1050-1100 nucleotides, approximately 1150-1200 nucleotides, approximately 1250-1300 nucleotides, approximately 1350-1400 nucleotides, or approximately 1450-1500 nucleotides.In some embodiments, the ASO targets a downstream sequence of more than 300 nucleotides of GRCh37 / hg19:chr2:166,863,740.

[0257]

[0364] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5') from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence upstream (or 5') of at least approximately 1 nucleotide, at least approximately 10 nucleotides, at least approximately 20 nucleotides, at least approximately 50 nucleotides, at least approximately 80 nucleotides, at least approximately 85 nucleotides, at least approximately 90 nucleotides, at least approximately 95 nucleotides, at least approximately 96 nucleotides, at least approximately 97 nucleotides, at least approximately 98 nucleotides, at least approximately 99 nucleotides, at least approximately 100 nucleotides, at least approximately 101 nucleotides, at least approximately 102 nucleotides, at least approximately 103 nucleotides, at least approximately 104 nucleotides, at least approximately 105 nucleotides, at least approximately 110 nucleotides, at least approximately 120 nucleotides, at least approximately 150 nucleotides, at least approximately 200 nucleotides, at least approximately 300 nucleotides, at least approximately 400 nucleotides, at least approximately 500 nucleotides, at least approximately 600 nucleotides, at least approximately 700 nucleotides, at least approximately 800 nucleotides, at least approximately 900 nucleotides, or at least approximately 1000 nucleotides from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, ASO is approximately 4 to 300 nuclei from GRCh37 / hg19:chr2:166,863,740 Target the sequence downstream (or 3') of ochide. In some embodiments, the ASO targets a sequence downstream of at least approximately 1 nucleotide, at least approximately 10 nucleotides, at least approximately 20 nucleotides, at least approximately 50 nucleotides, at least approximately 80 nucleotides, at least approximately 85 nucleotides, at least approximately 90 nucleotides, at least approximately 95 nucleotides, at least approximately 96 nucleotides, at least approximately 97 nucleotides, at least approximately 98 nucleotides, at least approximately 99 nucleotides, at least approximately 100 nucleotides, at least approximately 101 nucleotides, at least approximately 102 nucleotides, at least approximately 103 nucleotides, at least approximately 104 nucleotides, at least approximately 105 nucleotides, at least approximately 110 nucleotides, at least approximately 120 nucleotides, at least approximately 150 nucleotides, at least approximately 200 nucleotides, at least approximately 300 nucleotides, at least approximately 400 nucleotides, at least approximately 500 nucleotides, at least approximately 600 nucleotides, at least approximately 700 nucleotides, at least approximately 800 nucleotides, at least approximately 900 nucleotides, or at least approximately 1000 nucleotides from GRCh37 / hg19:chr2:166,863,740. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.

[0258]

[0365] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5') from the genomic site GRCh37 / hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence up to approximately 10 nucleotides, up to approximately 20 nucleotides, up to approximately 50 nucleotides, up to approximately 80 nucleotides, up to approximately 85 nucleotides, up to approximately 90 nucleotides, up to approximately 95 nucleotides, up to approximately 96 nucleotides, up to approximately 97 nucleotides, up to approximately 98 nucleotides, up to approximately 99 nucleotides, up to approximately 100 nucleotides, up to approximately 101 nucleotides, up to approximately 102 nucleotides, up to approximately 103 nucleotides, up to approximately 104 nucleotides, up to approximately 105 nucleotides. The creotide targets a sequence up to approximately 110 nucleotides, up to approximately 120 nucleotides, up to approximately 150 nucleotides, up to approximately 200 nucleotides, up to approximately 300 nucleotides, up to approximately 400 nucleotides, up to approximately 500 nucleotides, up to approximately 600 nucleotides, up to approximately 700 nucleotides, up to approximately 800 nucleotides, up to approximately 900 nucleotides, up to approximately 1000 nucleotides, up to approximately 1100 nucleotides, up to approximately 1200 nucleotides, up to approximately 1300 nucleotides, up to approximately 1400 nucleotides, or up to approximately 1500 nucleotides upstream (or 5'). In some embodiments, the ASO targets a sequence up to approximately 4 to approximately 300 nucleotides downstream (or 3') of GRCh37 / hg19:chr2:166,863,740.In some embodiments, ASO is derived from GRCh37 / hg19:chr2:166,863,740 in a maximum of approximately 10 nucleotides, a maximum of approximately 20 nucleotides, a maximum of approximately 50 nucleotides, a maximum of approximately 80 nucleotides, a maximum of approximately 85 nucleotides, a maximum of approximately 90 nucleotides, a maximum of approximately 95 nucleotides, a maximum of approximately 96 nucleotides, a maximum of approximately 97 nucleotides, a maximum of approximately 98 nucleotides, a maximum of approximately 99 nucleotides, a maximum of approximately 100 nucleotides, a maximum of approximately 101 nucleotides, a maximum of approximately 102 nucleotides, a maximum of approximately 103 nucleotides, a maximum of approximately 104 nucleotides, and a maximum of approximately 105 nucleotides. The creotide targets sequences up to approximately 110 nucleotides, 120 nucleotides, 150 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, or up to approximately 1000 nucleotides, 1100 nucleotides, 1200 nucleotides, 1300 nucleotides, 1400 nucleotides, or up to approximately 1500 nucleotides downstream. In some embodiments, the ASO targets sequences more than 300 nucleotides downstream from GRCh37 / hg19:chr2:166,863,740.

[0259]

[0366] The SCN1A gene (SEQ ID NO: 1) was analyzed for NIEs, and the inclusion of a portion of intron 20 (SEQ ID NO: 4) (this portion will be referred to as exon 20x throughout this disclosure) was observed. In some embodiments, the ASO disclosed herein targets NIEs containing premRNA (SEQ ID NO: 2) transcribed from the SCN1A genome sequence. In some embodiments, the ASO targets NIEs containing premRNA transcripts derived from the SCN1A genome sequence that include a portion of intron 20. In some embodiments, the ASO targets NIEs containing premRNA transcripts derived from the SCN1A genome sequence that include exon 20x (SEQ ID NO: 6). In some embodiments, the ASO targets NIEs containing premRNA transcripts of SEQ ID NO: 2 or 12. In some embodiments, the ASO targets NIEs containing premRNA transcripts of SEQ ID NO: 2 or 12 that include the NIE. In some embodiments, the ASO targets NIEs containing premRNA transcripts of SEQ ID NO: 2 or 12 that include the NIE. In some embodiments, the ASO disclosed herein targets an SCN1A premRNA sequence (SEQ ID NO: 2 or 12). In some embodiments, the ASO targets an SCN1A premRNA sequence containing NIE (SEQ ID NO: 10 or 20). In some embodiments, the ASO targets an SCN1A premRNA sequence by any one of SEQ ID NOs: 7-10 or 17-20. In some embodiments, the ASO has a sequence by any one of SEQ ID NOs: 21-67. In some embodiments, the ASO has a sequence by any one of SEQ ID NOs: 68-114. In some embodiments, the ASO has a sequence by any one of SEQ ID NOs: 115-209. In some embodiments, the ASO has a sequence by any one of SEQ ID NOs: 210-256. In some embodiments, the ASO has a sequence by any one of SEQ ID NOs: 257-303. In some embodiments, the ASO has a sequence by any one of SEQ ID NOs: 304-341. In some embodiments, the ASO has a sequence represented by one of sequence numbers 342 to 379. In some embodiments, the ASO has a sequence represented by one of sequence numbers 380 to 1099.In some embodiments, the ASO has a sequence consisting of one of sequence numbers 304 to 1099. In some embodiments, the ASO has a sequence consisting of one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b.

[0260]

[0367] In some embodiments, the SCN1A NIE containing a premRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 1 or 11. In some embodiments, the SCN1A NIE premRNA transcript contains a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs. 2-10 and 12-20.

[0261]

[0368] In some embodiments, the ASO targets exon 20 of the SCN1A NIE containing premRNA including NIE exon 20x. In some embodiments, the ASO targets the exon 21 sequence downstream (or 3') of NIE exon 20x. In some embodiments, the ASO targets the sequence approximately 4 to 300 nucleotides upstream (or 5') from the 5' end of exon 20x. In some embodiments, the ASO targets the sequence approximately 4 to 300 nucleotides downstream (or 3') from the 3' end of exon 20x. In some embodiments, the ASO has a sequence according to any one of sequence numbers 21 to 67. In some embodiments, the ASO has a sequence according to any one of sequence numbers 210 to 256. In some embodiments, the ASO has a sequence according to any one of sequence numbers 380 to 1099. In some embodiments, the ASO has a sequence according to any one of sequence numbers 304 to 1099. In some embodiments, the ASO is one of the arrays listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b. It has an arrangement with a single offset.

[0262]

[0369] In some embodiments, the ASO targets sequences upstream of the 5' end of the NIE. For example, an ASO targeting a sequence upstream of the 5' end of the NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include sequences having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 21-38. In another example, an ASO targeting a sequence upstream of the 5' end of the NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include sequences having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 68-85. In some embodiments, the ASO targets sequences containing exon-intron boundaries (or junctions). For example, an ASO targeting a sequence containing an exon-intron boundary may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 39-41, 51, 52, 228-230, 240, or 241. In another example, an ASO targeting a sequence containing an exon-intron boundary may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 86-88 and 98-99. In some embodiments, the ASO targets a sequence downstream from the 3' end of the NIE. For example, an ASO that targets a sequence downstream from the 3' end of NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 53–67.In another example, an ASO targeting a sequence downstream of the 3' end of an NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 100–114. In some embodiments, the ASO targets a sequence within the NIE. For example, an ASO targeting a sequence within the NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 42–50 or 231–239. In another example, an ASO targeting a sequence within the NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A) may include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of sequence numbers 89–97.

[0263]

[0370] In some embodiments, the ASO targets exon 20x in the SCN1A NIE containing premRNA that includes exon 20x. In some embodiments, the ASO targets the exon 20x sequence downstream (or 3') of the 5' end of exon 20x in the SCN1A premRNA. In some embodiments, the ASO targets the exon 20x sequence upstream (or 5') of the 3' end of exon 20x in the SCN1A premRNA.

[0264]

[0371] In some embodiments, the SCN1A NIE containing a premRNA transcript contains a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 2, 7-10, 12, and 17-20. In some embodiments, the SCN1A NIE containing a premRNA transcript is encoded by a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs: 1, 3-6, 11, and 13-16. In one embodiment, it contains NMD exons, and Na V The targeted portion of the premRNA encoding 1.1 contains a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to a region containing at least eight consecutive nucleic acids, namely sequence numbers 2, 7-10, 12, and 17-20.

[0265]

[0372] In some embodiments, the ASO targets an NIE containing a premRNA transcript. In some embodiments, the ASO targets an NIE containing a premRNA transcript that includes an NIE. In some embodiments, the ASO targets an NIE containing a premRNA transcript that includes exon 20x. In some embodiments, the ASO disclosed herein targets an SCN1A premRNA sequence. In some embodiments, the ASO targets an SCN1A premRNA sequence that includes an NIE. In some embodiments, the ASO targets an SCN1A premRNA sequence. In some embodiments, the ASO has a sequence that is one of sequence numbers 21-67, 210-256, or 304-1099. In another example, the ASO has a sequence that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to one of sequence numbers 21-67, 210-256, or 304-1099. In another example, an ASO contains a sequence that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any one of the sequences listed in Tables 4a, 4b, 5a, 5b, 6a, 6b, 7, 8a, and 8b.

[0266]

[0373] In some embodiments, the ASO targets exon 20 of the SCN1A NIE containing premRNA including NIE exon 20x. In some embodiments, the ASO targets the exon 21 sequence downstream (or 3') of NIE exon 20x. In some embodiments, the ASO targets the sequence approximately 4 to 300 nucleotides upstream (or 5') from the 5' end of exon 20x. In some embodiments, the ASO targets the sequence approximately 4 to 300 nucleotides downstream (or 3') from the 3' end of exon 20x.

[0267]

[0374] In some embodiments, the ASO targets sequences upstream from the 5' end of the NIE. In some embodiments, the ASO targets sequences containing exon-intron boundaries (or junctions). In some embodiments, the ASO targets sequences downstream from the 3' end of the NIE (e.g., exon 20x in human SCN1A, or exon 21x in mouse SCN1A). In some embodiments, the ASO targets sequences within the NIE.

[0268]

[0375] In some embodiments, the ASO targets exon 20x in the SCN1A NIE containing premRNA that includes exon 20x. In some embodiments, the ASO targets the exon 20x sequence downstream (or 3') of the 5' end of exon 20x in the SCN1A premRNA. In some embodiments, the ASO targets the exon 20x sequence upstream (or 5') of the 3' end of exon 20x in the SCN1A premRNA.

[0269]

[0376] In some embodiments, the targeted region of the SCN1A NIE containing premRNA is located in introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 (intron numbering corresponding to the mRNA sequence of NM_006920). In some embodiments, hybridization of the ASO to the targeted region of the NIE premRNA results in at least one exon skipping of the NIE within introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, followed by Na V 1.1 Increase protein production. In some embodiments, AS is directed to the targeted region of NIE premRNA. O hybridization inhibits or blocks exon skipping of at least one NIE within intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and subsequently Na V 1.1 Reduce protein production. In some embodiments, the targeted portion of the SCN1A NIE containing premRNA is located within intron 20. Those skilled in the art can determine the corresponding intron number in any isoform based on the intron sequences provided herein, or using the numbers provided for the mRNA sequences in NM_006920, NM_001202435, NM_001165964, or NM_001165963. Those skilled in the art can also determine the sequences of adjacent exons in any SCN1A isoform for targeting using the method of the present invention based on the intron sequences provided herein, or using the intron numbers provided for the mRNA sequences in NM_006920, NM_001202435, NM_001165964, or NM_001165963.

[0270] Therapeutic drugs

[0377] In various embodiments of this disclosure, compositions and methods comprising therapeutic agents are provided for modulating the protein expression level of SCN1A. In some embodiments, compositions and methods for modulating alternative splicing of SCN1A premRNA are provided. In some embodiments, compositions and methods for inducing exon skipping during splicing of SCN1A premRNA are provided, for example, for inducing pseudoexon skipping during splicing of SCN1A premRNA. In other embodiments, therapeutic agents may be used to induce exon inclusion to reduce the protein expression level.

[0271]

[0378] In some embodiments, the therapeutic agent disclosed herein is a small molecule, a polypeptide, or a polynucleic acid polymer. In some examples, the therapeutic agent is a small molecule. In some examples, the therapeutic agent is a polypeptide. In some examples, the therapeutic agent is a polynucleic acid polymer. In some cases, the therapeutic agent is a repressor. In further cases, the therapeutic agent is an enhancer.

[0272]

[0379] The therapeutic agents disclosed herein may be NIE repressors. The therapeutic agents may include polynucleic acid polymers.

[0380] According to one aspect of this disclosure, functional Na V 1.1 Functional Na, including administering NIE repressors to target protein levels V 1.1 Methods are provided for treating or preventing conditions associated with protein deficiency, wherein NIE repressors bind to the region of the premRNA transcript to reduce the inclusion of NIE in the mature transcript. For example, as specified herein, functional Na V 1.1 Functional Na, including administering NIE repressors to target protein levels V1.1 A method is provided for treating or preventing a condition associated with protein deficiency, wherein an NIE repressor agent binds to an intron region containing NIE in a premRNA transcript (e.g., intron 20 in the human SCN1A gene) or to an NIE activation regulatory sequence within the same intron.

[0273]

[0381] The sequence of the polynucleic acid polymer may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to the target sequence of the mRNA transcript, e.g., a partially processed mRNA transcript. The sequence of the polynucleic acid polymer may be 100% complementary to the target sequence of the premRNA transcript.

[0274]

[0382] The sequence of the polynucleic acid polymer may have four or fewer mismatches with respect to the target sequence of the premRNA transcript. The sequence of the polynucleic acid polymer may have three or fewer mismatches with the target sequence of the NA transcript. The sequence of the polynucleic acid polymer may have two or fewer mismatches with the target sequence of the premRNA transcript. The sequence of the polynucleic acid polymer may have one or fewer mismatches with the target sequence of the premRNA transcript. The sequence of the polynucleic acid polymer may have no mismatches with the target sequence of the premRNA transcript.

[0275]

[0383] Polynucleotide polymers can specifically hybridize to target sequences of pre-mRNA transcripts. For example, polynucleotide polymers may have sequence complementarity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% to target sequences of pre-mRNA transcripts. Hybridization may occur under highly stringent hybridization conditions.

[0276]

[0384] A polynucleic acid polymer may have a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. A polynucleic acid polymer may have a sequence with 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. In some examples, a polynucleic acid polymer may have a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 68–114. In some cases, polynucleic acid polymers may have sequences that have 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs. 68 to 114.

[0277]

[0385] In some cases, polynucleic acid polymers may have sequences with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 21-67, 210-256, or 304-1099. In some cases, polynucleic acid polymers may have sequences with 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 21-67, 210-256, or 304-1099. In some cases, polynucleic acid polymers may have sequences with 100% se...

Claims

1. Na Ｖ 1.1 A method for treating or reducing the likelihood of developing a disease or condition characterized by reduced protein expression or function in a human subject requiring treatment or reduction of the likelihood of developing such a disease or condition, comprising the step of administering to a human subject a pharmaceutical composition comprising an antisense oligomer (ASO) in a first dose of about 0.5 milligrams to about 500 milligrams, wherein the ASO comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 21-67, 210-256, or 304-1099, thereby treating or reducing the likelihood of developing such a disease or condition in a human subject.

2. Na Ｖ 1.1 A method for treating or reducing the likelihood of developing a disease or condition characterized by reduced protein expression or function in a human subject requiring treatment or reduction of the likelihood of developing such a disease or condition, comprising the step of administering to the human subject a pharmaceutical composition comprising a first dose of an antisense oligomer (ASO), wherein the ASO comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 21-67, 210-256, or 304-1099, thereby treating or reducing the likelihood of developing such a disease or condition in a human subject, wherein the human subject is up to 18 years of age at the time of the first dose.

3. Na Ｖ 1.1 A method for treating or reducing the likelihood of developing a disease or condition characterized by reduced protein expression or function in a human subject requiring treatment or reduction of the likelihood of developing such a disease or condition, comprising the step of administering to the human subject a pharmaceutical composition comprising multiple doses of an antisense oligomer (ASO), wherein the ASO comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 21-67, 210-256, or 304-1099, thereby treating or reducing the likelihood of developing such a disease or condition in a human subject.

4. The method according to claim 1 or 2, wherein the first dose is the first of a series of doses.

5. The method according to claim 1 or 3, wherein the human subject is at most 18 years of age at the time of the first dose.

6. In human subjects, approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 6 7.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 130, 132.5, 135, 137.5, 140, 1 The method according to any one of claims 1 to 3, comprising the step of administering a pharmaceutical composition comprising a first dose of ASO of 42.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg.

7. The method according to any one of claims 1 to 6, wherein the disease or condition is Dravet syndrome.

8. The target is, (i) Seizures that often become prolonged and are triggered by high fever, with recurrent focal motor, hemipteral, or generalized tonic-clonic seizures, onset before 12 months of age, (ii) No prior medical history of a lesion detected by magnetic resonance imaging that could be the cause. (iii) No other known etiology of any disease or condition other than Dravet syndrome, (iv) Normal development at the onset of seizure, (v) Pathogenicity variants or variants of uncertain importance in the SCN1A gene, (vi) At least two prior treatments for epilepsy, either lacking adequate seizure management. (vii) Four or more seizures during the 28 days prior to administration, which are selected from unilateral clonic seizures, focal seizures with motor signs, focal to bilateral tonic-clonic seizures, generalized tonic-clonic seizures, tonic seizures, tonic or atonic (falling seizures), and clonic seizures. (viiii) Current interventions for epilepsy, including ketogenic diet therapy, vagus stimulants, or cannabinoids or marijuana-derived products, or medication using at least one antiepileptic drug at a dose that has been stable for at least four weeks, or Any combination of (ix)(i) to (viiii) The method according to any one of claims 1 to 7, characterized by having the following:

9. The targets are further as follows: (a) One of the following mutations in the SCN1A gene: Thr226Met, Leu263Val, Val422Leu, Thr1174Ser, Trp1204Arg, Pro1345Ser, Gln1489Lys, Phe1499Leu, Arg1575Cys, Val1611Phe, Leu1624Pro, Arg1648Cys, Leu1649Gln, Leu1670Trp, Gly1674Arg, and Asp1866Tyr. (b) Known pathogenic mutations in another gene that causes epilepsy, which are homozygous in the case of known recessive genetic diseases. (c) The patient is currently being treated with sodium channel blockers and anticoagulants as maintenance therapy, the sodium channel blockers being phenytoin, carbamazepine, oxycarbazepine, lamotrigine, lacosamide, or rufinamide, and the anticoagulant being not aspirin. (d) Clinically significant medical conditions other than epilepsy, (e) Clinically relevant symptoms or clinically significant illnesses other than epilepsy in the four weeks prior to administration, (f) A history of brain or spinal cord disorders other than epilepsy or Dravet syndrome, or a history of bacterial meningitis or brain malformations. (g) Spinal deformities, or other conditions that alter the free flow of cerebrospinal fluid (CSF) or involve the implantation of a CSF drainage shunt. (h) Clinically significant abnormal clinical laboratory values ​​before administration, (i) Aspartate aminotransferase or alanine aminotransferase levels exceeding 2.5 times the upper limit of normal, serum creatinine levels higher than the upper limit of normal, or platelet count levels lower than the lower limit of normal. (j) Clinically relevant abnormalities in a 12-lead electrocardiogram (ECG) measured before administration. (k) Mental disorder or behavioral disorder, (l) Prescription of anticoagulants other than aspirin, either currently or in the past four weeks, Any combination of (m), (a) to (l) The method according to any one of claims 1 to 8, characterized in that it does not have one or more of the above.

10. The method according to any one of claims 1 to 9, wherein the human subject is 1-18, 2-18, 3-18, 4-18, 5-18, 6-18, 7-18, 8-18, 9-18, 10-18, 11-18, 12-18, 13-18, 14-18, 15-18, 16-18, or 17-18 years of age.

11. The method according to any one of claims 1 to 10, wherein the human subject is a human being 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 years of age.

12. The method according to any one of claims 1 to 11, wherein the human subject is less than 1 year of age or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age.

13. The method according to any one of claims 1 to 12, wherein the pharmaceutical composition is administered into the subarachnoid space of a human subject.

14. The method according to any one of claims 1 to 13, wherein the pharmaceutical composition is administered into the cerebrospinal fluid of a human subject.

15. The method according to any one of claims 1 to 14, wherein the pharmaceutical composition is administered into the brain of a human subject.

16. The method according to any one of claims 1 to 15, wherein the pharmaceutical composition is administered into the cerebrospinal fluid in the brain of a human subject.

17. The method according to any one of claims 1 to 16, wherein the pharmaceutical composition is administered as a bolus injection.

18. The method according to any one of claims 1 to 17, wherein the pharmaceutical composition is administered by injection using a delivery pump.

19. The method according to any one of claims 1 to 18, wherein the pharmaceutical composition is administered by intracerebral injection.

20. The method according to any one of claims 1 to 18, wherein the pharmaceutical composition is administered by intraarachnoid injection.

21. The method according to any one of claims 1 to 20, for reducing or improving at least one symptom of Dravet syndrome in a human subject.

22. The method according to claim 21, wherein the symptom of Dravet syndrome is a seizure.

23. The method according to any one of claims 1 to 22, wherein the administration reduces or improves the frequency, intensity, or duration of seizures.

24. The method according to any one of claims 1 to 23, wherein the ASO includes a sequence having at least 83%, 88%, 94%, or 100% sequence identity with any one of sequence numbers 21 to 67, 210 to 256, or 304 to 1099.

25. ASO is any of the sequence numbers 21-67, 210-256, or 304-1099 The method according to any one of claims 1 to 24, comprising sequences having at least 83%, 88%, 94%, or 100% sequence identity with respect to one of them.

26. The method according to any one of claims 1 to 25, wherein the ASO comprises at least one modified sugar moiety.

27. The method according to any one of claims 1 to 26, wherein the ASO comprises a T-methoxyethyl sugar moiety.

28. The method according to any one of claims 1 to 27, wherein the T-methoxyethyl sugar moiety is a T-2'-methoxyethyl sugar moiety.

29. The method according to any one of claims 1 to 28, wherein the ASO comprises a 2'-O-methoxyethyl moiety.

30. The method according to any one of claims 1 to 29, wherein the ASO comprises a thymidine containing a 2'-O-methoxyethyl moiety.

31. The method according to any one of claims 1 to 30, wherein each nucleic acid base of ASO contains a 2'-O-methoxyethyl moiety.

32. The method according to any one of claims 1 to 31, wherein the ASO consists of 8 to 50 nucleic acid bases.

33. The method according to any one of claims 1 to 32, wherein the ASO consists of 16 to 20 nucleic acid bases.

34. The method according to any one of claims 1 to 33, wherein the ASO consists of 12 to 20 nucleic acid bases.

35. The method according to any one of claims 1 to 34, wherein the ASO consists of 8 to 20 nucleic acid bases.

36. The method according to any one of claims 1 to 35, wherein the ASO comprises 5'-methylcytosine (5'-MeC).

37. The method according to any one of claims 1 to 36, wherein each cytosine in ASO is 5'-methylcytosine (5'-MeC).

38. The method according to any one of claims 1 to 37, wherein the ASO comprises a phosphorothioate bond.

39. The method according to any one of claims 1 to 38, wherein the internucleoside bonds of each ASO are phosphorothioate bonds.

40. The method according to any one of claims 1 to 39, wherein the ASO comprises locked nucleic acid (LNA).

41. The method according to any one of claims 1 to 40, further comprising the step of evaluating the tolerance or efficacy of a pharmaceutical composition.

42. The method according to any one of claims 1 to 41, further comprising the step of administering a pharmaceutical composition containing ASO to a human subject in a subsequent dose of about 0.5 milligrams to about 500 milligrams.

43. Subsequent doses are 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 130, 132 The method according to claim 42, wherein the amount is 5, 135, 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg.

44. The method according to claim 42 or 43, wherein a subsequent dose is lower than the previous dose after it has been shown that the administration of a previous dose is intolerable.

45. The method according to claim 42 or 43, wherein a subsequent dose is the same as the previous dose after the administration of a previous dose has been shown to be tolerable.

46. The method according to claim 42 or 43, wherein a subsequent dose is higher than the previous dose after the administration of a previous dose has been shown to be tolerable.

47. The method according to claim 42 or 43, wherein a subsequent dose is the same as the previous dose after an earlier dose has been shown to be effective.

48. The method according to claim 42 or 43, wherein a subsequent dose is lower than the previous dose after an earlier dose has been shown to be effective.

49. The method according to claim 42 or 43, wherein a subsequent dose is higher than the previous dose after it has been shown that the previous dose was ineffective.

50. The method according to any one of claims 42 to 49, wherein the subsequent dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the administration of the previous dose.

51. The method according to any one of claims 1 to 50, wherein the frequency of administration is maintained or reduced after a previous dose has been shown to be effective.

52. The method according to any one of claims 1 to 51, wherein the frequency of administration is increased after it has been shown that the previous dose was ineffective.

53. The method according to any one of claims 1 to 52, further comprising the step of administering at least one additional therapeutic agent or therapy.

54. The method according to claim 53, wherein at least one additional therapeutic agent or therapy is administered concurrently with the dose.

55. The method according to claim 53, wherein at least one additional therapeutic agent or therapy is administered before the dose.

56. The method according to claim 53, wherein at least one additional therapeutic agent or therapy is administered after the dose is administered.

57. The method according to any one of claims 1 to 56, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient, carrier, or diluent.

58. The method according to any one of claims 1 to 57, wherein the pharmaceutical composition is a liquid composition.

59. The method according to any one of claims 1 to 58, wherein the pharmaceutical composition comprises 0.1 mL to 50 mL of a diluent, and the ASO is solubilized or diluted in the diluent.

60. The method according to claim 59, wherein the pharmaceutical composition comprises about 0.1, 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mL of a diluent.

61. The method according to claim 59, wherein the pharmaceutical composition comprises 1 mL to 20 mL of a diluent, 2 mL to 10 mL of a diluent, or 1 mL to 5 mL of a diluent.

62. The method according to any one of claims 59 to 61, wherein the diluent comprises a cerebrospinal fluid (CSF) sample or an artificial cerebrospinal fluid (aCSF) solution derived from the subject.

63. The method according to any one of claims 1 to 62, comprising the step of obtaining a cerebrospinal fluid sample from a subject.

64. The method according to any one of claims 1 to 63, comprising the step of solubilizing or diluting ASO in a CSF sample from the subject.

65. The method according to any one of claims 1 to 64, comprising the step of administering a pharmaceutical composition as a bolus injection.

66. The method according to any one of claims 1 to 65, comprising the step of administering a pharmaceutical composition as a bolus injection over a period of 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

67. The method according to any one of claims 1 to 66, comprising the step of administering a pharmaceutical composition as a bolus injection using a spinal anesthetic needle.

68. The method according to any one of claims 1 to 67, wherein ASO is solubilized or diluted in an artificial cerebrospinal fluid (aCSF) solution.

69. The method according to any one of claims 1 to 68, wherein the solution comprises a cerebrospinal fluid (CSF) sample derived from the subject.

70. Any one of claims 1 to 69, wherein the ASO is solubilized or diluted in an isotonic solution. Methods used.

71. The method according to any one of claims 1 to 70, wherein the ASO is solubilized or diluted in a phosphate buffer solution having a pH of at least 5.

8.

72. The method according to any one of claims 1 to 70, wherein the ASO is solubilized or diluted in a phosphate buffer solution (pH 6.6 to 7.6).

73. The method according to any one of claims 1 to 72, wherein the ASO is solubilized or diluted in a buffer containing 25 to 250 mM NaCl.

74. The method according to any one of claims 1 to 73, wherein the ASO is solubilized or diluted in a buffer containing 0.1 to 20 mM KCl.

75. ASO is 0.1 to 50 mM Na ２ HPO ４ The method according to any one of claims 1 to 74, wherein the substance is solubilized or diluted in a buffer containing the substance.

76. ASO contains 0.1–50 mM NaH ２ PO ４ The method according to any one of claims 1 to 75, wherein the substance is solubilized or diluted in a buffer containing the substance.

77. ASO is 0.1–50 mM CaCl ２ The method according to any one of claims 1 to 76, wherein the substance is solubilized or diluted in a buffer containing the substance.

78. The method according to any one of claims 1 to 77, wherein the ASO is solubilized or diluted in a buffer containing 0.1 to 50 mM MgCl ２ .

79. ASO contains 25–250 mM NaCl, 0.1–20 mM KCl, and 0.1–50 mM Na ２ HPO ４ , 0.1 to 50 mM NaH ２ PO ４ , 0.1 to 50 mM CaCl ２ and 0.1–50 mM MgCl ２ The method according to any one of claims 1 to 78, wherein the substance is solubilized or diluted in a buffer containing the substance.

80. ASO contains 150 mM NaCl, 3.0 mM KCl, and 0.7 mM Na ２ HPO ４ , 0.3 mM NaH ２ PO ４ , 0.79 mM MgCl ２ and 1.4 mM CaCl ２ The method according to any one of claims 1 to 79, wherein the substance is solubilized or diluted in a buffer containing the substance.

81. The method according to any one of claims 68 to 80, wherein the ASO is solubilized or diluted in a buffer further containing carbohydrates.

82. The method according to claim 81, wherein the carbohydrate comprises D-glucose.

83. The method according to any one of claims 68 to 80, wherein the ASO is solubilized or diluted in a buffer further containing 1 to 100 mM D-glucose.

84. ASO is 1-100 mM NaHCO ３ , 1-100 mM KHCO ３ The method according to any one of claims 68 to 83, wherein the substances are solubilized or diluted in a buffer further comprising a combination thereof.

85. The method according to any one of claims 1 to 84, wherein the ASO is solubilized or diluted in a buffer further containing an antioxidant.

86. The method according to claim 85, wherein the antioxidant is t-butylhydroxyquinoline (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin E, or any combination thereof.

87. The method according to any one of claims 1 to 86, wherein the pharmaceutical preparation does not contain a preservative.

88. The method according to any one of claims 1 to 87, wherein ASO is present in the pharmaceutical composition at a concentration of 0.1 mg / mL to 250 mg / mL.

89. The method according to any one of claims 1 to 88, wherein ASO is present in the pharmaceutical composition at a concentration of approximately 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL.

90. The method according to any one of claims 1 to 88, wherein the ASO is present in the pharmaceutical composition at a concentration of about 22.5 mg / mL, 25 mg / mL, 27.5 mg / mL, 30 mg / mL, 32.5 mg / mL, 35 mg / mL, 37.5 mg / mL, 40 mg / mL, 42.5 mg / mL, 45 mg / mL, 47.5 mg / mL, 50 mg / mL, 52.5 mg / mL, 55 mg / mL, 57.5 mg / mL, 60 mg / mL, 62.5 mg / mL, 65 mg / mL, 67.5 mg / mL, 70 mg / mL, 72.5 mg / mL, 75 mg / mL, 77.5 mg / mL, 80 mg / mL, 82.5 mg / mL, 85 mg / mL, 87.5 mg / mL, 90 mg / mL, 92.5 mg / mL, 95 mg / mL, 97.5 mg / mL, 100 mg / mL, 102.5 mg / mL, 105 mg / mL, 107.5 mg / mL, 110 mg / mL, 112.5 mg / mL, 115 mg / mL, 117.5 mg / mL, 120 mg / mL, 122.5 mg / mL, 125 mg / mL, 127.5 mg / mL, 130 mg / mL, 132.5 mg / mL, 135 mg / mL, 137.5 mg / mL, 140 mg / mL, 142.5 mg / mL, 145 mg / mL, 147.5 mg / mL, 150 mg / mL, 152.5 mg / mL, 155 mg / mL, 157.5 mg / mL, 160 mg / mL, 162.5 mg / mL, 165 mg / mL, 167.5 mg / mL, 170 mg / mL, 172.5 mg / mL, 175 mg / mL, 177.5 mg / mL, 180 mg / mL, 182.5 mg / mL, 185 mg / mL, 187.5 mg / mL, 190 mg / mL, 192.5 mg / mL, 195 mg / mL, 197.5 mg / mL, 200 mg / mL, 202.5 mg / mL, 205 mg / mL, 207.5 mg / mL, 210 mg / mL, 212.5 mg / mL, 215 mg / mL, 217.5 mg / mL, 220 mg / mL, 222.5 mg / mL, 225 mg / mL, 227.5 mg / mL, 230 mg / mL, 232.5 mg / mL, 235 mg / mL, 237.5 mg / mL, 240 mg / mL, 242.5 mg / mL, 245 mg / mL, 247.5 mg / mL or 250 mg / mL.

91. The method according to any one of claims 1 to 88, wherein ASO is present in the pharmaceutical composition at a concentration of 11 mg / mL, 22 mg / mL, 33 mg / mL, 44 mg / mL, 55 mg / mL, 66 mg / mL, 77 mg / mL, 88 mg / mL, 99 mg / mL, or 100 mg / mL.

92. Na Ｖ 1.1 Reduction in protein expression or function, containing NMD exons, Na Ｖ 1.1 The changes in splicing of nonsense mutation-dependent RNA decay-inducible exons from protein-coding premRNA, as described in any one of claims 1 to 91. Method of loading.

93. ASO contains NMD exons, Na Ｖ 1.1 The method according to any one of claims 1 to 92, which facilitates the elimination of NMD exons from protein-coding premRNA.

94. ASO contains NMD exons, Na Ｖ 1.1 The method according to any one of claims 1 to 93, wherein the target is bound to a premRNA that codes for a protein.

95. ASO contains NMD exons, Na Ｖ 1.1 The method according to claim 94, which facilitates the elimination of NMD exons from protein-coding premRNA.

96. When ASO is introduced into cells, ASO becomes Na Ｖ 1.1 The method according to any one of claims 94 to 95, for increasing the level of processed mRNA encoding a protein.

97. When ASO is introduced into cells, ASO becomes Na Ｖ 1.1 The method according to any one of claims 94 to 96, for increasing the level of protein.

98. The method according to any one of claims 94 to 97, wherein the targeted portion is located within an intron sequence adjacent to an NMD exon.

99. The method according to any one of claims 94 to 97, wherein the targeted portion comprises at least one nucleotide of an NMD exon.

100. The method according to any one of claims 94 to 97, wherein the targeted portion is located within an NMD exon.

101. A method according to any one of claims 94 to 100 for treating a disease or condition.

102. (a) Antisense oligomers (ASOs) containing a sequence having at least 80% sequence identity with any one of sequence numbers 21-67, 210-256, or 304-1099, (b) Pharmaceutically acceptable diluents Includes, A pharmaceutical preparation in which ASO is dissolved or suspended in a solution at a concentration of 0.1 to 200 mg / mL.

103. The pharmaceutical formulation according to claim 102, wherein the pharmaceutically acceptable diluent comprises an artificial cerebrospinal fluid (aCSF) solution.

104. The pharmaceutical formulation according to claim 102 or 103, wherein the solution comprises a cerebrospinal fluid (CSF) sample derived from the subject.

105. The pharmaceutical formulation according to any one of claims 102 to 104, wherein ASO is present in the pharmaceutical composition at a concentration of approximately 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL.

106. ASO is about 22.5 mg / mL, 25 mg / mL, 27.5 mg / mL, 30 mg / mL, 32.5 mg / mL, 35 mg / m L, 37.5 mg / mL, 40 mg / mL, 42.5 mg / mL, 45 mg / mL, 47.5 mg / mL, 50 mg / mL, 52. 5mg / mL, 55mg / mL, 57.5mg / mL, 60mg / mL, 62.5mg / mL, 65mg / mL, 67.5mg / m L, 70mg / mL, 72.5mg / mL, 75mg / mL, 77.5mg / mL, 80mg / mL, 82.5mg / mL, 85m g / mL, 87.5 mg / mL, 90 mg / mL, 92.5 mg / mL, 95 mg / mL, 97.5 mg / mL, 100 mg / m L, 102.5mg / mL, 105mg / mL, 107.5mg / mL, 110mg / mL, 112.5mg / mL, 115mg / mL, 117.5 mg / mL, 120 mg / mL, 122.5 mg / mL, 125 mg / mL, 127.5 mg / mL, 130 mg / mL, 132.5mg / mL, 135mg / mL, 137.5mg / mL, 140mg / mL, 142.5mg / mL, 145m g / mL, 147.5 mg / mL, 150 mg / mL, 152.5 mg / mL, 155 mg / mL, 157.5 mg / mL, 16 0mg / mL, 162.5mg / mL, 165mg / mL, 167.5mg / mL, 170mg / mL, 172.5mg / mL, 1 75mg / mL, 177.5mg / mL, 180mg / mL, 182.5mg / mL, 185mg / mL, 187.5mg / mL, 190mg / mL, 192.5mg / mL, 195mg / mL, 197.5mg / mL, 200mg / mL, 202.5mg / mL A pharmaceutical formulation according to any one of claims 102 to 104, wherein the pharmaceutical composition contains the following: 205 mg / mL, 207.5 mg / mL, 210 mg / mL, 212.5 mg / mL, 215 mg / mL, 217.5 mg / mL, 220 mg / mL, 222.5 mg / mL, 225 mg / mL, 227.5 mg / mL, 230 mg / mL, 232.5 mg / mL, 235 mg / mL, 237.5 mg / mL, 240 mg / mL, 242.5 mg / mL, 245 mg / mL, 247.5 mg / mL, or 250 mg / mL.

107. The pharmaceutical formulation according to any one of claims 102 to 104, wherein ASO is present in the pharmaceutical composition at a concentration of 11 mg / mL, 22 mg / mL, 33 mg / mL, 44 mg / mL, 55 mg / mL, 66 mg / mL, 77 mg / mL, 88 mg / mL, 99 mg / mL, or 100 mg / mL.

108. A pharmaceutical preparation according to any one of claims 102 to 107, wherein ASO is solubilized or diluted in an isotonic solution.

109. The pharmaceutical formulation according to any one of claims 102 to 108, wherein the ASO is solubilized or diluted in a phosphate buffer solution having a pH of at least 5.

8.

110. The pharmaceutical formulation according to any one of claims 102 to 108, wherein ASO is solubilized or diluted in a phosphate buffer solution (pH 6.6 to 7.6).

111. The pharmaceutical formulation according to any one of claims 102 to 110, wherein ASO is solubilized or diluted in a buffer containing 25 to 250 mM NaCl.

112. The pharmaceutical formulation according to any one of claims 102 to 111, wherein ASO is solubilized or diluted in a buffer containing 0.1 to 20 mM KCl.

113. ASO is 0.1 to 50 mM Na ２ HPO ４ A pharmaceutical formulation according to any one of claims 102 to 112, which is solubilized or diluted in a buffer containing the above.

114. ASO contains 0.1–50 mM NaH ２ PO ４ Solubilized or diluted in a buffer containing A pharmaceutical preparation according to any one of claims 102 to 113.

115. ASO is 0.1–50 mM CaCl ２ A pharmaceutical formulation according to any one of claims 102 to 114, which is solubilized or diluted in a buffer containing the above.

116. ASO is 0.1–50 mM MgCl ２ A pharmaceutical formulation according to any one of claims 102 to 115, which is solubilized or diluted in a buffer containing the above.

117. ASO contains 25–250 mM NaCl, 0.1–20 mM KCl, and 0.1–50 mM Na ２ HPO ４ , 0.1 to 50 mM NaH ２ PO ４ , 0.1 to 50 mM CaCl ２ and 0.1–50 mM MgCl ２ A pharmaceutical formulation according to any one of claims 102 to 116, which is solubilized or diluted in a buffer containing the above.

118. ASO contains 150 mM NaCl, 3.0 mM KCl, and 0.7 mM Na ２ HPO ４ , 0.3 mM NaH ２ PO ４ , 0.79 mM MgCl ２ and 1.4 mM CaCl ２ A pharmaceutical formulation according to any one of claims 102 to 117, which is solubilized or diluted in a buffer containing the above.

119. The pharmaceutical formulation according to any one of claims 102 to 118, wherein the ASO is solubilized or diluted in a buffer further containing carbohydrates.

120. The pharmaceutical formulation according to claim 119, wherein the carbohydrate comprises D-glucose.

121. The pharmaceutical formulation according to any one of claims 102 to 120, wherein the ASO is solubilized or diluted in a buffer further containing 1 to 100 mM D-glucose.

122. ASO is 1-100 mM NaHCO ３ , 1-100 mM KHCO ３ A pharmaceutical formulation according to any one of claims 102 to 121, which is solubilized or diluted in a buffer further comprising a combination thereof.

123. The pharmaceutical formulation according to any one of claims 102 to 122, wherein the ASO is solubilized or diluted in a buffer further containing an antioxidant.

124. The pharmaceutical formulation according to claim 123, wherein the antioxidant is t-butylhydroxyquinoline (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin E, or any combination thereof.

125. A pharmaceutical preparation according to any one of claims 102 to 124, which does not contain preservatives.

126. A pharmaceutical preparation according to any one of claims 102 to 125, suitable for intraventricular or subarachnoid injection.

127. A pharmaceutical preparation according to any one of claims 102 to 126, packaged in a single-use vial.

128. (i) A concentrate containing an antisense oligomer (ASO), wherein the ASO contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs. 21-67, 210-256, or 304-1099, and (ii) Diluents, wherein the concentrate is miscible with the diluent, and (iii) Instructions for use when diluting or solubilizing ASO in a diluent. A kit that includes this.

129. The kit according to claim 128, wherein the diluent is an artificial cerebrospinal fluid (aCSF) solution.

130. The kit according to any one of claims 128 to 129, wherein the diluent comprises an isotonic solution.

131. The kit according to any one of claims 128 to 130, wherein the diluent comprises a phosphate buffer solution having a pH of at least 5.

8.

132. The kit according to any one of claims 128 to 130, wherein the diluent comprises a phosphate buffer (pH 6.6 to 7.6) solution.

133. The kit according to any one of claims 128 to 132, wherein the diluent contains 25 to 250 mM NaCl.

134. The kit according to any one of claims 128 to 133, wherein the diluent contains 0.1 to 20 mM KCl.

135. The diluent is 0.1 to 50 mM Na ２ HPO ４ A kit according to any one of claims 128 to 134, comprising:

136. The diluent is 0.1 to 50 mM NaH ２ PO ４ A kit according to any one of claims 128 to 135, comprising:

137. The diluent is 0.1 to 50 mM CaCl ２ A kit according to any one of claims 128 to 136, comprising:

138. The diluent is 0.1 to 50 mM MgCl ２ A kit according to any one of claims 128 to 137, comprising:

139. The diluents are 25-250 mM NaCl, 0.1-20 mM KCl, and 0.1-50 mM Na. ２ HPO ４ , 0.1 to 50 mM NaH ２ PO ４ , 0.1 to 50 mM CaCl ２ and 0.1–50 mM MgCl ２ A kit according to any one of claims 128 to 138, comprising:

140. The diluents are 150 mM NaCl, 3.0 mM KCl, and 0.7 mM Na. ２ HPO ４ , 0.3 mM NaH ２ PO ４ , 0.79 mM MgCl ２ and 1.4 mM CaCl ２ A kit according to any one of claims 128 to 139, comprising:

141. The kit according to any one of claims 128 to 140, wherein the diluent further comprises a carbohydrate.

142. The kit according to claim 141, wherein the carbohydrate comprises D-glucose.

143. The kit according to any one of claims 128 to 142, wherein the diluent further comprises 1 to 100 mM D-glucose.

144. The diluent is 1 to 100 mM NaHCO3 ３ , 1-100 mM KHCO ３ A kit according to any one of claims 128 to 143, further comprising a combination thereof.

145. The kit according to any one of claims 128 to 144, wherein the diluent further comprises an antioxidant.

146. The kit according to claim 145, wherein the antioxidant is t-butylhydroxyquinoline (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin E, or any combination thereof.

147. The kit according to any one of claims 128 to 146, wherein the diluent does not contain a preservative.

148. The kit according to any one of claims 128 to 147, wherein the instructions for use for diluting or solubilizing ASO in a diluent include instructions for diluting or solubilizing ASO in a diluent to a concentration of 0.1 mg / mL to 250 mg / mL.

149. The kit according to any one of claims 128 to 148, wherein the instructions for use for diluting or solubilizing ASO in a diluent include instructions for diluting or solubilizing ASO in a diluent to concentrations of approximately 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL.

150. The instructions for diluting or solubilizing ASO in a diluent state that ASO should be added to the diluent at concentrations of approximately 22.5 mg / mL, 25 mg / mL, 27.5 mg / mL, 30 mg / mL, 32.5 mg / mL, 35 mg / mL, 37.5 mg / mL, 40 mg / mL, 42.5 mg / mL, 45 mg / mL, 47.5 mg / mL, 50 mg / mL, 52.5 mg / mL, 55 mg / mL, 57.5 mg / mL, 60 mg / mL, 62.5 mg / mL, 65 mg / mL, 67.5 mg / mL, 70 mg / mL, 72.5 mg / mL, 75 mg / mL, and 77.5 mg / mL. L, 80mg / mL, 82.5mg / mL, 85mg / mL, 87.5mg / mL, 90mg / mL, 92.5mg / mL, 95mg / mL, 97.5 mg / mL, 100 mg / mL, 102.5 mg / mL, 105 mg / mL, 107.5 mg / mL, 110 mg / mL, 112.5mg / mL, 115mg / mL, 117.5mg / mL, 120mg / mL, 122.5mg / mL, 125mg / mL, 1 27.5mg / mL, 130mg / mL, 132.5mg / mL, 135mg / mL, 137.5mg / mL, 140mg / mL, 142 .. 5mg / mL, 145mg / mL, 147.5mg / mL, 150mg / mL, 152.5mg / mL, 155mg / mL, 157.5 mg / mL, 160 mg / mL, 162.5 mg / mL, 165 mg / mL, 167.5 mg / mL, 170 mg / mL, 172.5 m g / mL, 175 mg / mL, 177.5 mg / mL, 180 mg / mL, 182.5 mg / mL, 185 mg / mL, 187.5 mg / mL, 190mg / mL, 192.5mg / mL, 195mg / mL, 197.5mg / mL, 200mg / mL, 202.5mg / mL A kit according to any one of claims 128 to 148, comprising instructions for use for diluting or solubilizing to concentrations of mL, 205 mg / mL, 207.5 mg / mL, 210 mg / mL, 212.5 mg / mL, 215 mg / mL, 217.5 mg / mL, 220 mg / mL, 222.5 mg / mL, 225 mg / mL, 227.5 mg / mL, 230 mg / mL, 232.5 mg / mL, 235 mg / mL, 237.5 mg / mL, 240 mg / mL, 242.5 mg / mL, 245 mg / mL, 247.5 mg / mL, or 250 mg / mL.

151. Instructions for use for diluting or solubilizing ASO in a diluent state that ASO should be added to the diluent in concentrations of approximately 11 mg / mL, 22 mg / mL, 33 mg / mL, 44 mg / mL, 55 mg / mL, 66 mg / mL, 77 mg / mL, 88 mg / mL, 99 mg / mL, or 100 mg / mL. A kit according to any one of claims 128 to 148, comprising instructions for use for diluting or solubilizing to a concentration of mL.

152. The kit according to any one of claims 128 to 151, wherein the instructions for diluting or solubilizing ASO in a diluent include instructions for diluting or solubilizing about 0.5 milligrams to about 500 milligrams of ASO in a diluent.

153. Instructions for diluting or solubilizing ASO in a diluent are provided for approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 5 7.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 130, 132.5, 135, 13 7.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200, 202.5, 205, 207.5, A kit according to any one of claims 128 to 152, comprising instructions for use for diluting or solubilizing 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg of ASO in a diluent.

154. Na Ｖ 1.1 Use of an antisense oligomer (ASO) for the manufacture of a medicament for treating or preventing a disease or condition characterized by reduced protein expression or function in a human subject requiring treatment or prevention of said disease or condition, wherein the medicament is administered in a first dose of about 0.5 milligrams to about 500 milligrams, and the ASO comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 21-67, 210-256, or 304-1099.

155. Pharmaceuticals are approximately 0.1, 0.5, 1, 2.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5, 125, 127.5, 130, 132.5, 135 , 137.5, 140, 142.5, 145, 147.5, 150, 152.5, 155, 157.5, 160, 162.5, 165, 167.5, 170, 172.5, 175, 177.5, 180, 182.5, 185, 187.5, 190, 192.5, 195, 197.5, 200 The use according to claim 154, administered in a first dose of 202.5, 205, 207.5, 210, 212.5, 215, 217.5, 220, 222.5, 225, 227.5, 230, 232.5, 235, 237.5, 240, 242.5, 245, 247.5, or 250 mg.

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