Compositions targeting sodium channel 1.6

Abstract

The present invention provides therapeutic compositions that include antisense oligonucleotides (ASOs) that are complementary to identified targets on Nav channel mRNA. ASOs hybridize to their target RNA and form duplexes that recruit RNase H to degrade the RNA, thereby downregulating Nav channel synthesis, which inhibits the ability of neurons to contribute to certain conditions, such as epilepsy and pain sensation. ASOs can be provided as gapmers that bind to one of the specific identified targets and contain a central DNA segment flanked by modified RNA wings.

Description

[Technical field]

[0001] Technical Field The present disclosure relates to compositions that inhibit the activity of voltage-gated sodium channel 1.6. [Background technology]

[0002] background Sodium channels are membrane proteins that function as channels that allow the passage of sodium ions (Na+) across the plasma membrane of cells. Sodium channels can be ligand-gated or voltage-gated, and voltage-gated channels for Na+ are called NaV channels. In cells such as neurons and cardiomyocytes, NaV channels are responsible for the ascending phase of the action potential. NaV channels have three states that are distinguished by the structural conformation of the protein: resting, active, and inactive.

[0003] Because cardiac activity, brain function, and physical sensations require cardiomyocytes and neurons whose functions depend on sodium channels, sodium channels have been considered targets for the treatment of cardiac arrhythmias, neurological conditions, and pain. In one example, procainamide has been used to treat atrial fibrillation and complex tachycardia. In another example, the small molecule Funapide is in development as an analgesic. The anticonvulsants phenytoin and carbamazepine are used to treat epilepsy and are understood to function as sodium channel blockers. The list of reported potential therapeutic targets for sodium channel blockers includes chronic pain, migraine, epilepsy, cardiovascular disease, psychiatric disorders, and even cancer. See Li, 2019, Voltage-gated sodium channels and blockers: an overview and where will they go?, Curr Med Sci 39(6):867-873, incorporated by reference. Unfortunately, the utility of small molecule sodium channel blockers can be limited by state-dependence, binding kinetics, and target accessibility, which requires the drug to find and interact with the target protein in a specific conformational state. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Li, 2019, Voltage-gated sodium channels and blockers: an overview and where will they go?, Curr Med Sci 39(6):867-873 Summary of the Invention [Means for solving the problem]

[0005] Abstract The present invention provides compositions that inhibit or knock down the expression of Nav1.6 protein. The compositions of the present invention are potentially useful for treating or diagnosing conditions involving sodium channel function, such as epilepsy, cardiovascular disease, pain or psychiatric disorders, including in some embodiments developmental epileptic encephalopathy 13 (DEE13). The compositions of the present invention are also useful as prophylactic treatments. In humans, there are nine families of voltage-gated sodium channels, namely Nav1.1 to Nav1.9. Of these nine proteins, Nav1.6, encoded by the SCN8A gene (12q13, 13), regulates the initiation of action potentials and is involved in nerve conduction. Nav1.6 is abundantly expressed in the brain and is important for neuronal function. The compositions of the present disclosure may be used to knock down the expression of Nav1.6 for the treatment of conditions in which neuronal activity and sodium channel function play a role. In particular, the compositions of the present disclosure may find benefit as therapeutic treatments for conditions such as epilepsy, pain, or conditions involving neuronal hyperexcitability.

[0006] In some embodiments, the compositions of the present disclosure can be useful as state-independent anti-epileptic or anti-convulsant drugs.Exome sequencing shows the association between SCN8A and epilepsy.It is believed that mutations in SCN8A can cause epilepsy and related epileptic seizures.Specifically, gain-of-function SCN8A mutations can cause hyperexcitability and impaired channel inactivation.In fact, epileptic seizures are treated with small molecule anti-epileptic drugs that function by blocking sodium channels. See Zaman, 2019, A single-center SCN8A-related epilepsy cohort: clinical, genetic, and physiologic characterization, Ann Clin Trans Neurol 6(8): 1445-1455 and Boerma, 2016, Remarkable phenytoin sensitivity in 4 children with SCN8A-related epilepsy: a molecular neuropharmacological approach, Neurotherapeutics 13(1): 192-197, both of which are incorporated by reference. The compositions of the present disclosure can be used to knock down Nav1.6 expression in a state-independent manner, for example, for the treatment of epilepsy, including for the treatment of epileptic seizures. Such an approach can be used to treat developmental epileptic encephalopathy DEE13, which results from a mutation in the SCN8A gene. Such an approach may also be applicable to treat Dravet syndrome and other forms of epilepsy, including but not limited to those resulting from mechanisms of excessive excitation and inhibition (E / I) balance.

[0007] In certain embodiments, the compositions of the present disclosure may be useful as analgesics for the treatment of pain, including, for example, chronic pain conditions, such as cancer pain or arthritis. The compositions include short nucleic acids or oligonucleotides that prevent the synthesis of proteins involved in neural activity. For example, some neurons act as "pain-sensing" neurons, i.e., nociceptors. These pain-sensing neurons have proteins that function as voltage-dependent sodium channels. When stimulation of the nerve ending exceeds a threshold potential (V), the nociceptor neurons direct sodium ions (Na+) across the cell membrane, which can dedifferentiate the neuron in a regenerative manner, resulting in the "firing" of a propagating electrical signal that underlies the sensation of pain. The compositions of the present disclosure may be used to knock down the expression of Nav1.6 in a state-independent manner for the treatment of pain.

[0008] The present invention provides compositions that inhibit or knock down the expression of Nav1.6 protein. The compositions of the present invention include oligonucleotides that bind to messenger RNA (mRNA) or precursor mRNA (pre-mRNA) used in making Nav1.6 sodium channel protein. The present invention includes the identification of a number of specific validated targets within these RNAs. The oligonucleotides are substantially or completely antisense to the targets, described as antisense oligonucleotides (ASOs). The oligonucleotides prevent these proteins from being made, reducing the sensitivity or activity of cells expressing these sodium channels. Because Nav1.6 expression is knocked down in these cells, these cells do not contribute to conditions such as chronic pain, migraine headaches, epilepsy, cardiovascular disease, or certain psychiatric disorders. Thus, the compositions of the present disclosure provide a condition-independent therapeutic treatment for a variety of conditions, providing an alternative to the limitations of small molecule channel blockers whose usefulness may be limited by binding kinetics or conformational state dependency, or access to the target ion channel.

[0009] The oligonucleotides described by the present disclosure are designed to hybridize to certain targets in the RNA used in the synthesis of Nav1.6 protein. The binding of the oligonucleotide prevents protein synthesis and downregulates the expression of the NaV channel. Specifically, the oligonucleotide has a sequence that is substantially or completely complementary to one of the identified targets on the NaV channel precursor mRNA (pre-mRNA) or mRNA. That is, the oligonucleotide is antisense to the identified target. When antisense oligonucleotides (ASOs) hybridize to their target RNA, they form a double-stranded ASO:RNA duplex that recruits an enzyme (RNase H) that degrades a portion of the double-stranded duplex. Degrading the ASO:RNA duplex depletes the NaV channel mRNA from the cell (e.g., neuron), which reduces the amount of NaV channel synthesized by the cell. Downregulating NaV channel expression interferes with the ability of neurons to contribute to epilepsy activity or pain sensation. Thus, when a composition comprising an oligonucleotide that is antisense to an identified target in Nav1.6 pre-mRNA or mRNA is administered to a patient, the patient may have a reduced risk of experiencing epileptic seizures or pain.

[0010] In some embodiments, the present invention targets the regions of transcripts identified herein as hotspots for the ASOs disclosed herein to be usefully targeted.Experimental data emerges from the results presented herein that show that certain ASOs targeting targets within the first approximately 3700 bases of SCN8A transcripts are highly effective in knocking down desired amounts.Some ASOs disclosed herein are specific to this 3700 base region, and data shows that these ASOs unexpectedly produce good results.

[0011] Another insight of the present invention is that certain ASOs knock down SCN8A by beneficial amounts that are not 100%. Although not bound by any mechanism of action, it can be theorized that complete knockdown (or knockout) is harmful, but beneficial effects can be achieved by using ASOs that reach a saturation profile or plateau that does not reach 100% knockdown, i.e., 0% relative normalized expression. Indeed, certain ASOs of the present disclosure provide their benefits as a result of their sequence, and this is not a strictly dose-dependent effect. As dose concentration increases, these ASOs reach a plateau at some % knockdown below 100. Certain embodiments disclosed herein knock down the expression of SCN8A by between about 50% and 80%, for example, about 60%, even at high concentrations or doses.

[0012] In certain embodiments, the disclosure provides compositions comprising an oligonucleotide that hybridizes to a pre-mRNA or mRNA encoding a sodium channel protein along a segment of that RNA that is at least 75% complementary to one of SEQ ID NOs: 1-156, thereby preventing translation of the RNA into a sodium channel protein. The oligonucleotide can hybridize to Nav1.6 pre-mRNA or mRNA and knock down its expression. Preferably, the sequence of bases in the oligonucleotide has at least 80% identity to one of SEQ ID NOs: 1-144. For example, the sequence of bases in the oligonucleotide can be at least 90% or 95% identical to one of SEQ ID NOs: 1-144, and the oligonucleotide can hybridize to either Nav1.6 pre-mRNA or mRNA and induce RNase H cleavage thereof. The composition can include a plurality of therapeutic oligonucleotides, each having a base sequence at least 80, 90, 95 or 100% identical to one of SEQ ID NOs: 1-156. Certain preferred ASOs include eleven that are complementary to targets within the exons of the human SCN8A gene, and five that are complementary to targets within the introns of the human SCN8A gene. The eleven preferred ASOs that are complementary to targets within the exons of the human SCN8A gene include the ASOs referred to by the following reference numbers: 14-016 (SEQ ID NO: 16); 14-041 (SEQ ID NO: 41); 14-044 (SEQ ID NO: 44); 14-045 (SEQ ID NO: 45); 14-100 (SEQ ID NO: 100), 14-117 (SEQ ID NO: 117), 14-124 (SEQ ID NO: 124), 14-125 (SEQ ID NO: 125), 14-130 (SEQ ID NO: 130), 14-131 (SEQ ID NO: 131), 14-132 (SEQ ID NO: 132), 14-133 (SEQ ID NO: 133), 14-134 (SEQ ID NO: 134), 14-135 (SEQ ID NO: 135), 14-136 (SEQ ID NO: 136), 14-137 (SEQ ID NO: 137), 14-138 (SEQ ID NO: 138), 14-139 (SEQ ID NO: 139), 14-200 (SEQ ID NO: 200), 14-201 (SEQ ID NO: 201), 14-202 (SEQ ID NO: 202), 14-203 (SEQ ID NO: 203), 14-204 (SEQ ID NO: 204), 14-205 (SEQ ID NO: 2 4-126 (SEQ ID NO:126), 14-128 (SEQ ID NO:128), 14-129 (SEQ ID NO:129), 14-130 (SEQ ID NO:130), 14-133 (SEQ ID NO:133), 14-134 (SEQ ID NO:134), 14-135 (SEQ ID NO:135), 14-138 (SEQ ID NO:138), 14-139 (SEQ ID NO:139), 14-142 (SEQ ID NO:142), 14-143 (SEQ ID NO:143) and 14-144 (SEQ ID NO:144).Certain most preferred embodiments may include SEQ ID NOs: 016, 041, 044, 045, 117, 124, 135 or 144.

[0013] Five preferred ASOs complementary to targets within an intron of the human SCN8A gene include ASOs referred to by the following reference numbers: 14-100 (SEQ ID NO: 100); 14-101 (SEQ ID NO: 101); 14-102 (SEQ ID NO: 102); 14-103 (SEQ ID NO: 103); and 14-104 (SEQ ID NO: 104).

[0014] Some embodiments include ASOs that target a region of the first 3700 bases of the SCN8A transcript, such as, for example, one of the following: ASO14-001 (SEQ ID NO:1); 14-002 (SEQ ID NO:2); 14-003 (SEQ ID NO:3); 14-004 (SEQ ID NO:4); 14-005 (SEQ ID NO:5); 14-006 (SEQ ID NO:6); 14-007 (SEQ ID NO:7); 14-008 (SEQ ID NO:8); 14-009 (SEQ ID NO:9); 14-010 (SEQ ID NO:10); 14-011 (SEQ ID NO:11); 14-012 (SEQ ID NO:12); 14-013 (SEQ ID NO:13); 14-014 (SEQ ID NO:14). 4-014 (SEQ ID NO:14); 14-015 (SEQ ID NO:15); 14-016 (SEQ ID NO:16); 14-041 (SEQ ID NO:41); 14-042 (SEQ ID NO:42); 14-043 (SEQ ID NO:43); 14-044 (SEQ ID NO:44); 14-045 (SEQ ID NO:45); 14-046 (SEQ ID NO:46); 14-047 (SEQ ID NO:47); 14-048 (SEQ ID NO:48); 14-049 (SEQ ID NO:49); 14-050 (SEQ ID NO:50); 14-051 (SEQ ID NO:51); 14-115 (SEQ ID NO:115); 14-116 (SEQ ID NO:116); 14-117 (SEQ ID NO: No. 117); 14-118 (SEQ ID NO: 118); 14-119 (SEQ ID NO: 119); 14-120 (SEQ ID NO: 120); 14-121 (SEQ ID NO: 121); 14-122 (SEQ ID NO: 122); 14-123 (SEQ ID NO: 123); 14-124 (SEQ ID NO: 124); 14-125 (SEQ ID NO: 125); 14-126 (SEQ ID NO: 126); 14-127 (SEQ ID NO: 127); 14-128 (SEQ ID NO: 128); 14-129 (SEQ ID NO: 129); 14-130 (SEQ ID NO: 130); 14-131 (SEQ ID NO: 131); 14-132 (SEQ ID NO: 132); 14- 133 (SEQ ID NO:133); 14-134 (SEQ ID NO:134); 14-135 (SEQ ID NO:135); 14-136 (SEQ ID NO:136); 14-137 (SEQ ID NO:137); 14-138 (SEQ ID NO:138); 14-139 (SEQ ID NO:139); 14-140 (SEQ ID NO:140); 14-141 (SEQ ID NO:141); 14-142 (SEQ ID NO:142); 14-143 (SEQ ID NO:143); 14-144 (SEQ ID NO:144); 14-145 (SEQ ID NO:145); 14-146 (SEQ ID NO:146); 14-147 (SEQ ID NO:16); 14-148 (SEQ ID NO:16);14-149 (SEQ ID NO: 41); 14-150 (SEQ ID NO: 41); 14-151 (SEQ ID NO: 44); 14-152 (SEQ ID NO: 44); 14-153 (SEQ ID NO: 45); 14-154 (SEQ ID NO: 45); 14-155 (SEQ ID NO: 117); 14-156 (SEQ ID NO: 117); 14-157 (SEQ ID NO: 124); 14-158 (SEQ ID NO: 124); 14-159 (SEQ ID NO: 126); 14-160 (SEQ ID NO: 126); 14-161 (SEQ ID NO: 129); 14-162 (SEQ ID NO: 129); 14-1 63 (SEQ ID NO:133); 14-164 (SEQ ID NO:133); 14-165 (SEQ ID NO:135); 14-166 (SEQ ID NO:135); 14-167 (SEQ ID NO:138); 14-168 (SEQ ID NO:138); 14-169 (SEQ ID NO:139); 14-170 (SEQ ID NO:139); 14-171 (SEQ ID NO:142); 14-172 (SEQ ID NO:142); 14-173 (SEQ ID NO:143); 14-174 (SEQ ID NO:143); 14-175 (SEQ ID NO:144); or 14-176 (SEQ ID NO:144). Certain embodiments may use one of the following: ASO14-147 (SEQ ID NO: 16); 14-148 (SEQ ID NO: 16); 14-149 (SEQ ID NO: 41); 14-150 (SEQ ID NO: 41); 14-151 (SEQ ID NO: 44); 14-152 (SEQ ID NO: 44); 14-153 (SEQ ID NO: 45); 14-154 (SEQ ID NO: 45); 14-155 (SEQ ID NO: 117); 14-156 (SEQ ID NO: 117); 14-157 (SEQ ID NO: 124); 14-158 (SEQ ID NO: 124); 14-159 (SEQ ID NO: 126); 14-160 (SEQ ID NO: 126); 14-161 (SEQ ID NO: 126); row number 129); 14-162 (SEQ ID NO: 129); 14-163 (SEQ ID NO: 133); 14-164 (SEQ ID NO: 133); 14-165 (SEQ ID NO: 135); 14-166 (SEQ ID NO: 135); 14-167 (SEQ ID NO: 138); 14-168 (SEQ ID NO: 138); 14-169 (SEQ ID NO: 139); 14-170 (SEQ ID NO: 139); 14-171 (SEQ ID NO: 142); 14-172 (SEQ ID NO: 142); 14-173 (SEQ ID NO: 143); 14-174 (SEQ ID NO: 143); 14-175 (SEQ ID NO: 144); or 14-176 (SEQ ID NO: 144). ;

[0015] Certain preferred embodiments may use ASOs in which SCN8A knockdown plateaus below 100% as dose is increased. These embodiments may use one of ASOs 14-135, 14-165, 14-166, 14-144, 14-175 and 14-175, which have been shown herein to never reach 100% knockdown, instead plateauing at between about 70% and 80% even at high dose concentrations, which is desirable.

[0016] Certain embodiments use gapmer ASOs having a sequence given by at least one of SEQ ID NO:16; SEQ ID NO:41; SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:117; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:129; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:138; SEQ ID NO:139; SEQ ID NO:142; SEQ ID NO:143; or SEQ ID NO:144, where the gapmer has a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, each wing having one or two phosphodiester linkages, the remaining interbase linkages are phosphorothioates, and all cytosine bases have 5-methyl modifications. Certain most preferred embodiments (for knockdown plateaus reaching between about 50% and 90% expression normalized to untreated) use 14-165, 14-166, 14-175, or 14-176. Herein, 14-165 is SEQ ID NO: 135 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings where the 2nd, 3rd, and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications. 14-166 is SEQ ID NO: 135 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings where the 2nd, 3rd, 4th, and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications. 14-175 is SEQ ID NO: 144 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, in which the 2nd, 3rd and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications.14-176 is SEQ ID NO: 144 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings in which the 2nd, 3rd, 4th and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications.

[0017] Therapeutic oligonucleotides of the present disclosure may have a gapmer structure that includes a central DNA segment flanked by modified RNA wings. Such therapeutic oligonucleotides may include two wings flanking a central region of DNA bases (e.g., about 8-14, e.g., 12 DNA bases). Preferably, at least one end of the oligonucleotide includes modified RNA bases, e.g., any number or combination of 2'-O-methoxyethyl RNA ("2'-MOE") and / or 2'-O-methyl RNA ("2'-O-Me"). The bases may be modified. For example, a percentage or all cytosines may be methylated (e.g., 5-methylcytosine) in the 5' RNA wing, the central DNA region, the 3' RNA wing, or all three.

[0018] The therapeutic oligonucleotide can be provided in a solution or carrier formulated for delivery, such as by injection. The oligonucleotide can be of any suitable length, for example, at least about 15 bases, preferably between about 15 and 25 bases. The oligonucleotide can have phosphorothioate linkages in the backbone. In a preferred embodiment, the oligonucleotide has a base sequence that has been screened and determined not to meet a threshold match for any long non-coding RNA or other off-target sequence or transcript in humans. The oligonucleotide can have a base sequence that has 0 mismatches with the homologous segment in the non-human primate genome and about 5 or less mismatches in the homologous segment in the rodent genome.

[0019] When the composition is delivered to SK-N-AS neuroblastoma cells in vitro, the cells exhibit a dose-dependent knockdown of Nav1.6. The oligonucleotide has at least a 90% match to one of SEQ ID NOs: 1-156 (preferably 14-016 (SEQ ID NO: 16), 14-041 (SEQ ID NO: 41), 14-044 (SEQ ID NO: 44), 14-045 (SEQ ID NO: 450, 14-100 (SEQ ID NO: 100), 14-117 (SEQ ID NO: 117), 14-124 (SEQ ID NO: 124), 14-125 (SEQ ID NO: 125), 14-126 (SEQ ID NO: 126), 14-128 (SEQ ID NO: 128), 14-129 (SEQ ID NO: 129), 14-130 (SEQ ID NO: 130), 14-131 (SEQ ID NO: 131), 14-132 (SEQ ID NO: 132), 14-133 (SEQ ID NO: 133), 14-134 (SEQ ID NO: 134), 14-135 (SEQ ID NO: 135), 14-136 (SEQ ID NO: 136), 14-137 (SEQ ID NO: 137), 14-138 (SEQ ID NO: 138), 14-139 (SEQ ID NO: 138), 14-200 (SEQ ID NO: 138), 14-201 (SEQ ID NO: 138), 14-202 (SEQ ID NO: 138), 14-203 (SEQ ID NO: 13 129), 14-130 (SEQ ID NO:130), 14-133 (SEQ ID NO:133), 14-134 (SEQ ID NO:134), 14-135 (SEQ ID NO:135), 14-138 (SEQ ID NO:138), 14-139 (SEQ ID NO:139), 14-142 (SEQ ID NO:142), 14-143 (SEQ ID NO:143), and 14-144 (SEQ ID NO:144) base sequences with at least a 90% match to one of the following: 14-130 (SEQ ID NO:130), 14-133 (SEQ ID NO:133), 14-134 (SEQ ID NO:134), 14-135 (SEQ ID NO:135), 14-138 (SEQ ID NO:138), 14-139 (SEQ ID NO:139), 14-142 (SEQ ID NO:142), 14-143 (SEQ ID NO:143), and 14-144 (SEQ ID NO:144) base sequences with the bases linked by phosphorothioate linkages. The linkages may be all phosphorothioate or may be a mixture of phosphorothioate and phosphodiester linkages. The oligonucleotide may further have a central region of between about 8 and about 14 DNA bases flanked by 5' and 3' wings, each of which includes some, e.g., a small number of consecutive 2' modified RNA bases. Preferably, the oligonucleotide has a base sequence matching one of SEQ ID NOs: 1-156, the bases being linked by phosphorothioate linkages, and the structure has a central DNA base flanked by a 5' wing and a 3' wing. The number of RNA bases in the wings and the number of DNA bases in the central segment can be 4-12-4, 5-10-5, 5-9-5, 4-11-4, or a similar suitable pattern. The 5' and 3' wings can each contain several 2'-MOE RNA bases.For example, an oligonucleotide can have 12 central DNA bases, with phosphorothioate linkages throughout the central DNA segment, and a mixture of phosphorothioate and phosphodiester linkages in the wings, along with four consecutive 2'-MOE RNA bases in each wing (a "4-12-4" structure).

[0020] In a combination embodiment, the invention provides a composition comprising multiple copies of multiple distinct therapeutic gapmers, each according to the above description, in a suitable formulation or carrier.

[0021] In some aspects, the present disclosure provides a method for treating epilepsy.The method comprises administering to a subject with epilepsy the composition described herein, thereby knocking down the expression of SCN8A gene.The epilepsy being treated can be DEE13, Dravet syndrome, or any epilepsy that involves the pathogenesis of excessive E / I balance.

[0022] Aspects of the present disclosure provide for the use of antisense oligonucleotides (ASOs) for the manufacture of a medicament for treating a condition such as epilepsy or pain in a patient. In use, the ASO has at least about 75% identity, more preferably at least 90% identity, e.g., 95% or 100% identity, to one of SEQ ID NOs: 1-156. Preferred embodiments use ASOs that are between about 15 and 25 bases long, preferably between about 18 and 22 bases long, or between about 19 and 21 bases long (inclusive). In general, reference to "an ASO" includes multiple copies of a substantially identical molecule. Thus, "an ASO" can be any number, e.g., hundreds of thousands or millions of copies of the indicated ASO. In a preferred embodiment, the ASO is 20 bases long and has the sequence of one of SEQ ID NOs: 1-156, and is used in the manufacture of a medicament for the treatment of a condition. The ASO can be provided in any suitable format, such as lyophilized or in solution in a tube, such as a microcentrifuge tube or test tube. A preferred embodiment of the use targets Nav1.6. One or more (e.g., two, three, four or five or more) ASOs can be used in the manufacture of medicines. One or more ASOs can hybridize to a target in Nav1.6 pre-mRNA or mRNA. In certain embodiments of the use, the sequence of the bases in the ASO is at least 90% identical to one of SEQ ID NOs: 1-156.In certain preferred embodiments, the sequence of bases in the ASO is 14-016 (SEQ ID NO: 16), 14-041 (SEQ ID NO: 41), 14-044 (SEQ ID NO: 44), 14-045 (SEQ ID NO: 450), 14-100 (SEQ ID NO: 100), 14-117 (SEQ ID NO: 117), 14-124 (SEQ ID NO: 124), 14-125 (SEQ ID NO: 125), 14-126 (SEQ ID NO: 126), 14-128 (SEQ ID NO: 128), 14-129 (SEQ ID NO: 129), 14-200 (SEQ ID NO: 200), 14-201 (SEQ ID NO: 201), 14-202 (SEQ ID NO: 202), 14-203 (SEQ ID NO: 203), 14-204 (SEQ ID NO: 204), 14-205 (SEQ ID NO: 205), 14-206 (SEQ ID NO: 206), 14-207 (SEQ ID NO: 207), 14-208 (SEQ ID NO: 208), 14-209 (SEQ ID NO: 209), 14-300 (SEQ ID NO: 300), 14-301 (SEQ ID NO: 301), 14-302 (SEQ ID NO: 302), 14-303 (SEQ ID NO: 303), 14-304 (SEQ ID NO: 304), 14-305 (SEQ ID NO: 305), 14-306 (SEQ ID NO: SEQ ID NO:129), 14-130 (SEQ ID NO:130), 14-133 (SEQ ID NO:133), 14-134 (SEQ ID NO:134), 14-135 (SEQ ID NO:135), 14-138 (SEQ ID NO:138), 14-139 (SEQ ID NO:139), 14-142 (SEQ ID NO:142), 14-143 (SEQ ID NO:143), and 14-144 (SEQ ID NO:144). In an embodiment of use, the ASO may have a gapmer structure having a central DNA segment flanked by RNA wings, e.g., a central region of 12 DNA bases with four modified RNA bases on either side of the central region. Each modified RNA base may be a 2'-MOE. Preferably, the backbone of the ASO has multiple phosphorothioate bonds, for example, several, many, most, or all of the sugar linkages can be phosphorothioate (balanced with phosphodiester) in the use embodiment. The medicament can include the ASO in a form suitable for mixing into a formulation suitable for injection, infusion, or introduction by pump. For example, the ASO (thousands or millions or more copies of one ASO) can be lyophilized in a tube or in a solution at a known molar concentration or concentration. The ASO can be dissolved or diluted in a carrier, for example, a pharma- ceutically acceptable composition in which the solvent and / or excipient comprises the ASO, and filled into an IV bag, syringe, or pump. The medicament can be made using any combination of more than one ASO, for example, two, three, four, or five or more ASOs. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows a composition for treating epilepsy. [Diagram 2] FIG. 2 shows an oligonucleotide having a gapmer structure. [Diagram 3] FIG. 3 shows 2'-O-methoxyethyl (MOE) modified ribose sugars. [Figure 4] FIG. 4 shows phosphorothioate linkages in a segment of DNA. [Diagram 5] Figure 5 shows the expression of SCN8A after treatment with ASOs of sequence numbers 1 to 74 (the first 20 bars in each panel represent treatment with one ASO, the 21st bar is NT siRNA; see legend). [Figure 6] Figure 6 shows the expression of SCN8A after treatment with ASOs of SEQ ID NOs: 75-114. [Figure 7] FIG. 7 provides the results of a reproducibility analysis for the ASOs of the present disclosure in different replicate cultures. [Figure 8] Figure 8 shows the dose response under treatment with ASOs of SEQ ID NOs: 1 to 4, 6, 8, 11, 16, 41, 44, 45 and 100 to 104. [Figure 9] Figure 9 shows the percent knockdown resulting from treatment with SCN8A exon-targeted ASOs that tile the entire transcript. [Figure 10] Figure 10 shows the percent knockdown resulting from treatment with SCN8A intron-targeted ASO. [Figure 11] Figure 11 shows the percent knockdown resulting from treatment with SCN8A ASOs that all target the first 3700 bases of the transcript. [Figure 12] Figure 12 shows the percent knockdown for other sodium channels (encoded by SCN2A, SCN3A, and SCN9A) resulting from treatment with an SCN8A exon-targeted ASO that tiled the entire transcript. [Figure 13] Figure 13 shows the percent knockdown for other sodium channels (encoded by SCN2A, SCN3A, and SCN9A) resulting from treatment with an SCN8A intron-targeted ASO. [Figure 14] Figure 14 shows the percent knockdown for other sodium channels (encoded by SCN2A, SCN3A and SCN9A) resulting from treatment with optimized SCN8A exon-targeted ASO. [Figure 15] Figure 15 shows dose response data for certain ASOs in SK-N-AS neuroblastoma cells. [Figure 16] Figure 16 shows expanded dose-response data for certain ASOs in SK-N-AS neuroblastoma cells. [Figure 17] FIG. 17 shows the dose response percent knockdown for all lead PS scaffold candidates targeting SCN8A exons. [Figure 18] FIG. 18 shows the dose response percent knockdown for PO modified daughter leads as human clinical candidates. [Figure 19] FIG. 19 shows knockdown of SCN8A transcripts in human NGN2 stem cell-derived neurons. [Figure 20] FIG. 20 shows knockdown of SCN8A transcripts in human primary neurons using lead candidates. [Figure 21] FIG. 21 shows knockdown of Scn8a transcripts in mouse primary cortical neurons. [Figure 22] FIG. 22 shows Scn8a transcript knockdown in rat primary hippocampal neurons. [Figure 23] FIG. 23 shows evidence of a plateau in SCN8A transcript knockdown in human NGN2 stem cell-derived neurons. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description FIG. 1 includes a composition 101 for treating epilepsy or pain. The composition 101 includes an oligonucleotide 107 that hybridizes to a target segment 115 in an mRNA 117 or pre-mRNA. The mRNA 117 encodes a sodium channel protein. The segment 115 of the mRNA 117 that includes the target is at least about 75% complementary to one of SEQ ID NOs: 1-156. Hybridization of the oligonucleotide 107 to the segment 115 of the mRNA 117 prevents translation of the mRNA into a sodium channel protein (e.g., by recruiting RNase H, which results in digestion of the double-stranded oligonucleotide 107 / RNA 117 complex, or by disrupting translation of the protein). The oligonucleotide 107 can hybridize to the Nav1.6 pre-mRNA or mRNA and knock down its expression. Preferably, the sequence of bases in the oligonucleotide has at least 80% identity to one of SEQ ID NOs: 1-156, more preferably at least 90% identity, for example at least 95% identity.

[0025] In certain embodiments, the sequence of bases in the oligonucleotide is at least 90% identical to one of SEQ ID NOs: 1-156, and the oligonucleotide is capable of hybridizing to and inducing RNase H cleavage of Nav1.6 pre-mRNA or mRNA.

[0026] The oligonucleotide 107 hybridizes to the segment 115 in the RNA 117 because the oligonucleotide 107 is substantially or completely antisense to the target segment 115 of the mRNA 117. In that sense, the composition includes an antisense oligonucleotide. The composition 101 includes an ASO that binds to the target RNA with base pair complementarity and exerts various effects based on the chemical structure and design of the ASO. Various mechanisms commonly used in preclinical models and human clinical trial development of neurological diseases can be used. These mechanisms include RNA target degradation via recruitment of RNase H enzyme.

[0027] A preferred embodiment of the present disclosure includes an ASO that hybridizes to voltage-gated sodium channel (NaV channel) pre-mRNA or mRNA and recruits RNase H enzyme. RNase H enzyme cleaves NaV channel RNA, which downregulates the expression of NaV channel protein. Thus, the oligonucleotide 107 of the present disclosure targets NaV channel for treatment against conditions such as epilepsy, including, as non-limiting examples, DEE13, Dravet syndrome, and forms of epilepsy with a pathogenic mechanism involving an excessive E / I balance ratio. The present disclosure is based on the insight that clinical and preclinical data support the use of small molecule NaV blockers for treatment, for example, as anticonvulsants or analgesics. For example, dibucaine, methylbenzetheonium chloride, darifenacin hydrobromide, and dimethisoquine hydrochloride have all been investigated for their inhibitory activity against Nav1.6 channel. See Atkin, 2018, A comprehensive approach to identifying repurposed drugs to treat SCN8A epilepsy, Epilepsia 59(4):802-813, which is incorporated by reference. Similarly, sodium channels are associated with pain. Compositions comprising anti-NaV ASOs can be administered to subjects to treat conditions such as epilepsy or pain. Anti-NaV ASOs can be found to offer advantages over other approaches such as small molecule blockers, since they can be condition-independent and subtype-selective.

[0028] Thus, the present disclosure provides the use of antisense oligonucleotides (ASOs) for the manufacture of a medicament for treating a condition in a patient. In the use, the ASO has at least about 75% identity, more preferably at least 90% identity, e.g., 95% or higher identity, to one of SEQ ID NOs: 1-156. A preferred embodiment uses an ASO that is between about 15 and 25 bases long, preferably between about 18 and 22 bases long (inclusive). In general, reference to "an ASO" includes multiple copies of a substantially identical molecule. Thus, "an ASO" can be hundreds of thousands or even more than millions of copies of a defined ASO. In a preferred embodiment, the ASO is 20 bases long and has a sequence of one of SEQ ID NOs: 1-156 and is used in the manufacture of a medicament. The ASO can be provided in any suitable format, such as, for example, lyophilized in a tube, such as a microcentrifuge tube or test tube, or in solution in a tube. A preferred embodiment of the use targets Nav1.6. One or more (e.g., two, three, four or five or more combinations) ASOs can be used in the manufacture of medicines. One or more ASOs can hybridize to a target in Nav1.6 RNA. In certain embodiments of use, the sequence of bases in the ASO is at least 90%, preferably at least 95% or 100% identical to one of SEQ ID NOs: 1-156. For example, the sequence of bases in the ASO can be at least 90%, 95% or 100% identical to one of the preferred sequences.

[0029] In an embodiment of use, the ASO may have a gapmer structure with a central DNA segment flanked by RNA wings, for example, a central region of 12 DNA bases with four modified RNA bases on either side of the central region, i.e., a 4-12-4 structure. The structure may be 5-10-5 or 4-9-4 or 4-10-4- or 5-9-5, etc. Each modified RNA base may be 2'-MOE RNA, 2'-O-Me RNA, or other suitable sugar. Preferably, the backbone of the ASO either exclusively has multiple phosphorothioate linkages or also contains phosphodiester linkages, for example, most or all of the sugar linkages may be phosphorothioate in an embodiment of use. The medicament may be formulated, for example, for delivery via injection. Thus, the ASO may initially be in a suitable form for mixing into a formulation suitable for introduction into an IV bag, syringe, or intrathecal pump. For example, ASO (thousands or millions or more copies of one ASO) can be lyophilized in tube, or can be in solution at known molar concentration or concentration.ASO can be dissolved or diluted in a carrier, for example, a pharma- ceutically acceptable composition, in which a solvent or excipient comprises ASO, and can be filled into an IV bag, a syringe, or an intrathecal pump.Medicine can be made using more than one ASO, for example, any combination of two, three, four, or five or more ASOs.

[0030] Any of the ASO(s) described in the use embodiments may be included in the compositions of the present disclosure. A preferred embodiment of the compositions of the present disclosure includes one or more therapeutic oligonucleotides, each having a base sequence at least 90% identical to one of SEQ ID NOs: 1-156, each of the therapeutic oligonucleotides having a gapmer structure comprising a central DNA segment flanked by modified RNA wings, and the plurality of therapeutic oligonucleotides are provided in a solution or carrier formulated for intrathecal injection.

[0031] 2 shows an oligonucleotide 207 having a gapmer structure. The oligonucleotide 207 includes two wings (first wing 215 and second wing 216) flanking a central region 221 of about 12 DNA bases. In a preferred embodiment, the wings 215, 216 are all or mostly RNA bases, while the central region 221 is all or mostly DNA bases. Preferably, the wings are all RNA bases (modified or unmodified) and the central region is all DNA bases. In some embodiments, each wing consists of four RNA bases, all or most of which are modified RNA bases, e.g., each modified RNA base is selected from the group consisting of 2'-O-methoxyethyl RNA and 2'-O-methyl RNA. The modified RNA bases may include substitutions on the 2' hydroxyl group of the ribose sugar.

[0032] FIG. 3 shows 2'-O-methoxyethyl ("2'-MOE") modified sugars that can be included in RNA bases.

[0033] Oligonucleotide 207 preferably contains at least about 15 bases, and may contain between about 15 and about 25 bases. In some embodiments, oligonucleotide 207 has a backbone that includes a plurality of phosphorothioate linkages.

[0034] 4 shows phosphorothioate linkages 505 in the backbone of a segment of DNA, e.g., central region 221 of oligonucleotide 207. Oligonucleotide 207 can contain one or any number of phosphorothioate linkages 505. For example, all of the backbone linkages in oligonucleotide 207 can be phosphorothioate, or most or about half can be phosphorothioate.

[0035] The composition 101 may be formulated for delivery. Thus, the oligonucleotide 107 may initially be in a suitable form for mixing into a formulation suitable for introduction into a syringe, bag, or injection pump. For example, the oligonucleotide 107 (thousands or millions or more copies of one oligonucleotide 107) may be lyophilized in a tube or in a solution at a known molar concentration or concentration. The oligonucleotide 107 may be dissolved or diluted in a carrier, e.g., a pharma- ceutically acceptable composition in which a solvent or excipient comprises the oligonucleotide 107, and loaded into an IV bag, syringe, or intrathecal pump. As described, the composition 101 includes at least one oligonucleotide 107 having a sequence defined by comparison with one of SEQ ID NOs: 1-156. Thus, the compositions of the present disclosure are defined and indicated by the identified target.

[0036] Specifically, oligonucleotide 107 hybridizes to an mRNA encoding a sodium channel protein along a segment of the mRNA that is at least about 75% complementary to one of SEQ ID NOs: 1-156, thereby preventing translation of the mRNA into a sodium channel protein. This is achieved when the oligonucleotide has at least about 75% identity, preferably at least about 90% or 95% identity, to one of SEQ ID NOs: 1-156. In certain embodiments, the oligonucleotide has the sequence of one of SEQ ID NOs: 1-156, but those skilled in the art will understand that an oligonucleotide with 90% or preferably 95% identity to a complementary target will still tend to hybridize to the target in a sequence-specific manner. Forming a double-stranded structure via Watson-Crick base pairing and base stacking is energetically favorable enough that the double-stranded structure can tolerate roughly about one mismatched base pair every 10 base pairs or so. Therefore, under moderately stringent physiological conditions in cells, 95% identity should be effective, especially if the oligonucleotide has a gapmer structure with at least a small number of modified RNA bases or phosphorothioate backbone linkages to protect the oligonucleotide from enzymatic degradation.

[0037] Indeed, a feature and benefit of the compositions of the present disclosure is that the targets (SEQ ID NOs: 1-156) have been screened to exclude sequences that have complements in molecules other than sodium channel transcripts. For example, the sequences have been screened against a database of RNA transcripts, including long non-coding RNAs (lncRNAs), to eliminate initial sequences that match non-target sequences. Thus, an ASO having a sequence of SEQ ID NOs: 1-156, when administered to a patient, should have a minimized chance of hybridizing to non-target sequences. Thus, in a preferred embodiment, oligonucleotide 107 has a base sequence that has been screened and determined not to meet a threshold match for any off-target coding RNA or long non-coding RNA in humans. A composition or use that meets the above criteria should not bind to off-target material, e.g., lncRNAs, in vivo, because the included sequences have been screened against a database of lncRNAs. The sequences of the present disclosure have been screened for target specificity. Preferably, oligonucleotide 107 has a base sequence that has zero mismatches to the homologous segment in the non-human primate genome and about five or less mismatches in the homologous segment in the rodent genome.

[0038] When the composition is delivered to cells in vitro, the cells exhibit a dose-dependent knockdown of Nav1.6.

[0039] FIG. 5 shows the relative normalized expression of SCN8A (on the y-axis) for exonic ASOs of SEQ ID NOs: 1-74 delivered at 100 nM to SK-N-AS cells. In each panel, the first 20 bars correspond to one respective ASO. The 21st and subsequent bars correspond to one of the controls indicated in the legend. The ASO bars are in numerical order by SEQ ID NO: i.e., in the third panel labeled "ASOs 041->060," the first bar is the result from ASO of SEQ ID NO: 41 and the fourth bar is the result from ASO of SEQ ID NO: 44. These bars show that all of the ASOs showed some knockdown compared to vehicle, and that ASOs of SEQ ID NOs: 1-4, 6-11, 16, 41 and 43-45 showed very good knockdown of about 60% or more.

[0040] In all cases, the ASO is identified by the last three digits, so that ASO-001 is 001 and 14-001 is QS-Ts14-ASO-001. These are equivalent designations that refer to the same thing. In some places, the shorter version is used for formatting purposes.

[0041] Figure 6 shows the relative normalized expression of SCN8A (on the y-axis) for intronic ASOs of SEQ ID NOs: 75-114 delivered at 100 nM to SK-N-AS cells. In the figure, for comparison, expression is measured for cells treated with untreated, small interfering RNA, vehicle (no ASO), off-target ASO (e.g., against NaV1.7) and "scrambled" ASO. The bars show that the majority of the ASOs showed some expression knockdown compared to vehicle, and that the ASOs of SEQ ID NOs: 75, 77, 82, 85, 87, 88, 90, 98, 100-104 and 209 showed very good knockdown, e.g., about 60% or more.

[0042] Figure 7 gives the results of a combined reproducibility analysis for ASOs with SEQ ID NOs: 1-74 targeting SCN8A exons and ASOs with SEQ ID NOs: 75-114 targeting SCN8A introns. The scatter plot shows the knockdown across two replicate experiments along with the prioritization of 16 ASOs (11 exonic and 5 intronic) showing >60% transcript knockdown at a single dose. These experiments included a single dose ASO screen (by transfection at 100 nM) in SK-N-AS neuroblastoma cells. The axes are the different replicate experiments. Exonic ASOs with SEQ ID NOs: 1-3, 4, 6, 8, 11, 16, 41, 44 and 45 and intronic ASOs with SEQ ID NOs: 100-104 are the 16 that were prioritized and selected for dose-dependent testing.

[0043] Figure 8 shows the dose-dependent effect of selected ASOs. The graph gives the relative normalized expression of SCN8A at day 2 in vitro (DIV2) 48 hours after ASO treatment with selected ASOs of SEQ ID NOs: 1-3, 4, 6, 8, 11, 16, 41, 44, 45 and 100-104 at five concentrations in increments from 6.25 nM to 100 nM, respectively, in SK-N-AS cells in round 2. Dose responses were completed over a 16-fold concentration range (100, 50, 25, 12.5 and 6.25 nM). The ASOs tested for dose-dependence were made according to an embodiment of the present disclosure (20 bases, 12 bases DNA central region flanked by RNA wings with 2'-MOE RNA, 5-methylcytosine and phosphorothioate linkages throughout the ASO). The five rightmost bars show expression levels using siRNA, using scrambled ASO, vehicle alone (no ASO) and no treatment.All 16 ASOs reduce Nav1.6 expression in a dose-dependent manner compared to control.The graph shows that composition 101 of the present disclosure shows dose-dependent knockdown of Nav1.6.

[0044] Because nucleic acid hybridization has some tolerance for mismatches, it can be found that oligonucleotides 107 having a base sequence that is at least 90% a match to one of SEQ ID NOs: 1-156, in which the bases are linked only by phosphorothioate linkages, and in which the 5' and 3' wings have a central segment of DNA bases flanked by 12 DNA bases (e.g., a 4-12-4 structure, in which the 5' and 3' wings each include four consecutive 2' modified RNA bases flanked by 12 DNA bases, or a 5-10-5 structure, etc.), exhibit dose-dependent knockdown that follows the pattern shown in the chart. In some embodiments, oligonucleotide 107 specifically has a base sequence matching one of SEQ ID NOs: 1-156, the bases being linked by phosphorothioate linkages (and optionally by some phosphodiester linkages in the wings), and oligonucleotide 107 has a central 12 DNA bases flanked by a 5' wing and a 3' wing, each of which contains four consecutive 2'-MOE RNA bases.

[0045] Since these compositions are effective in knocking down the expression of sodium channels, the compositions of the present disclosure can be used to treat conditions in which neuronal activity plays a key role, such as epilepsy or pain. In such conditions, neuronal electrophysiology is involved, and in some such conditions, neuronal activity can be characterized by a condition-specific phenotype that can be detected by the shape of action potentials or spike patterns exhibited in neuronal activity. The compositions of the present disclosure can restore the condition-specific phenotype to a healthy phenotype, and the restoration effect can be demonstrable in vitro, for example, on neurons in vitro, via electrophysiological assays. The effect of the compounds on neurons can be demonstrated using optogenetic assays with in vitro neurons. For example, the in vitro neurons can include optogenetic constructs that provide neural activation under light stimulation (e.g., modified algal channelrhodopsin that causes neurons to fire in response to light) and optical reporters of neural activity (modified archaeo-rhodopsin that emits light proportional to neuronal membrane potential and produces signals of neuronal activity). The in vitro neurons can be assayed in a fluorescent microscopy instrument. See US Patent Application Publication No. 2021 / 0138039, which is incorporated by reference. Any suitable optogenetic construct, optogenetic microscope, or pain mediator composition may be used. For example, suitable optogenetic constructs include those described in U.S. Patent No. 9,594,075, which is incorporated by reference. Suitable optogenetic microscopes include those described in U.S. Patent No. 10,288,863, which is incorporated by reference. Suitable pain mediator compositions include those described in WO2018 / 165577, which is incorporated by reference.

[0046] In one example of testing a composition for analgesic properties, an in vitro DRG assay may include measuring light from an optogenetic nerve sample alone under increasing light stimulation. This gives a baseline reading of nerve excitability. The nerve sample is then stimulated with a stimulant (e.g., a pain mediator composition that includes a mixture of cytokines, proteases, pH, necrosis factors, or other factors that may be found in vivo at the site of a painful tumor). Light is measured from the nerve sample under treatment with the stimulant. Finally, the nerve sample is treated with a composition of the present disclosure. If the stimulant moves the measured excitability from the measured baseline, it may be found that oligonucleotide 107 tends to restore the measured excitability toward the baseline.

[0047] Oligonucleotides of the disclosure in composition 101, e.g., gapmer, ASO, or therapeutic oligonucleotide 107, can have a sequence defined by reference to one of the sequences shown in Table 1. For example, oligonucleotides of the disclosure can have a sequence that is at least about 75%, 80%, 90%, 95% or completely identical to one of SEQ ID NOs: 1-156 shown in Table 1. Top preferred embodiments for SCN8A include 14-016 (SEQ ID NO: 16); 14-041 (SEQ ID NO: 41); 14-044 (SEQ ID NO: 44); and 14-045 (SEQ ID NO: 45); 14-100 (SEQ ID NO: 100), 14-117 (SEQ ID NO: 117), 14-124 (SEQ ID NO: 124), 14-125 (SEQ ID NO: 125), 14-126 (SEQ ID NO: 126), 14-128 (SEQ ID NO: 128), and 14-130 (SEQ ID NO: 130). Column number 128), 14-129 (SEQ ID NO: 129), 14-130 (SEQ ID NO: 130), 14-133 (SEQ ID NO: 133), 14-134 (SEQ ID NO: 134), 14-135 (SEQ ID NO: 135), 14-138 (SEQ ID NO: 138), 14-139 (SEQ ID NO: 139), 14-142 (SEQ ID NO: 142), 14-143 (SEQ ID NO: 143), and 14-144 (SEQ ID NO: 144). The data show that the compositions of the present disclosure exhibit robust and significant knockdown activity (>70%) of Nav1.6 in a dose-dependent manner.

[0048] ASO sequence [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0049] Other features and embodiments are within the scope of the present disclosure.Embodiments of the present disclosure include oligonucleotides, including locked nucleic acid (LNA) antisense oligonucleotides targeting SCN8A, which can inhibit the expression of Nav1.6.The oligonucleotides of the present invention can be used in the prevention or treatment of conditions such as epilepsy or pain.The present invention further provides an advantageous target site sequence on human Nav1.6 pre-mRNA, which can be targeted by oligonucleotide inhibitors of human Nav1.6, such as antisense oligonucleotides or RNAi agents, such as siRNA or shRNA.

[0050] The present invention provides an oligonucleotide of 10-30 nucleotides in length comprising a contiguous nucleotide sequence of 10-30 nucleotides in length having at least 90% complementarity, preferably 100% complementarity, to human Nav1.6 RNA. Oligonucleotide 107 may be 100% identical, or preferably at least 90% identical, to one of SEQ ID NOs: 1-156.

[0051] Embodiments include pharma- ceutically acceptable salts of an antisense oligonucleotide according to the invention or a conjugate according to the invention.

[0052] The present invention provides a pharmaceutical composition comprising an antisense oligonucleotide of the invention or a conjugate of the invention and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0053] The invention provides an antisense oligonucleotide according to the invention or a conjugate according to the invention or a pharmaceutical salt or pharmaceutical composition according to the invention for use in medicine.

[0054] The present invention provides an antisense oligonucleotide of the present invention, or a pharmaceutical salt thereof.The present invention provides the use of an antisense oligonucleotide of the present invention in the preparation of a medicament for the treatment, prevention or amelioration of a condition such as epilepsy or pain.

[0055] Oligonucleotides can be produced in the laboratory by solid-phase chemical synthesis followed by purification and isolation.When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of the nucleic acid base moieties of covalently linked nucleotides or nucleosides, or their modification.The oligonucleotides of the present invention can be artificial, i.e., chemically synthesized, and typically purified or isolated.The oligonucleotides of the present invention can include one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides.

[0056] Modified nucleotides include deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, and the like. The nucleotides may be independently selected from the group consisting of restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, glycol modified nucleotides, and 2-O-(N-methylacetamido) modified nucleotides, and combinations thereof.

[0057] The nitrogenous bases of the ASO can be selected from naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants, such as substituted purines or substituted pyrimidines, such as isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine. In certain preferred embodiments, the compositions of the invention include oligonucleotides in which some, many, most, or all of the cytosine bases are present in a methylated form, such as 5-methylcytosine.

[0058] Nucleobase moieties can be represented by letter codes for each corresponding nucleobase, such as A, T, G, C, or U, and each letter can optionally include modified nucleobases of equivalent function.For example, in the illustrated oligonucleotide, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine.Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides can be used.

[0059] The oligonucleotide 107 of the present disclosure can downregulate (inhibit) the expression of sodium channel (Nav1.6). In some embodiments, the antisense oligonucleotide of the present invention can modulate the expression of the target by inhibiting or downregulating it. Preferably, such modulation causes at least 20% inhibition of expression compared to the normal expression level of the target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70% inhibition compared to the normal expression level of the target.

[0060] The antisense oligonucleotides of the disclosure may reduce the levels of a target nucleic acid (e.g., via RNase H cleavage) or may reduce (or alter) the functionality of a target nucleic acid, e.g., via modulation of pre-mRNA splicing.

[0061] The oligonucleotide 107 of the present disclosure may include one or more nucleosides with modified sugar moieties, i.e., modified sugar moieties, when compared to the ribose sugar moieties found in DNA and RNA. Numerous nucleosides with modified ribose sugar moieties have been created, primarily for the purpose of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance. Such modifications include those in which the ribose ring structure is modified, for example, by replacement with a hexose ring (HNA), or by replacement with a bicyclic ring that typically has a bridge between the C2 and C4 carbons on the ribose ring (LNA), or by replacement with an unlinked ribose ring that typically lacks a bond between the C2 and C3 carbons (e.g., UNA). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, such as in the case of peptide nucleic acid (PNA), or morpholino nucleic acid.

[0062] Sugar modifications also include modifications made through changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group found naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4' or 5' positions.

[0063] Oligonucleotides may contain one or more locked nucleic acid (LNA) bases. LNA may contain 2' modified nucleosides that contain a biradical (also called "2'-4' bridge") linking the C2' and C4' of the ribose sugar ring of the nucleoside, which constrains or locks the conformation of the ribose ring. These nucleosides are also called bridged nucleic acids or bicyclic nucleic acids (BNA) in the literature. Locking of the ribose conformation is associated with enhanced affinity of hybridization (duplex stabilization) when LNA is incorporated into an oligonucleotide to a complementary RNA or DNA molecule. This can be determined by measuring the melting temperature of the oligonucleotide / complement duplex. Non-limiting exemplary LNA nucleosides are disclosed in WO99 / 014226, WO00 / 66604, WO98 / 039352, WO2004 / 046160, WO00 / 047599, WO2007 / 134181, WO2010 / 077578, WO2010 / 036698, WO2007 / 090071, WO2009 / 006478, WO2011 / 156202, WO2008 / 154401, WO2009 / 067647, and WO2008 / 150729, all of which are incorporated by reference.

[0064] Pharmaceutically acceptable salts of the oligonucleotides of the present disclosure include salts that retain the biological effectiveness and properties of free base or free acid, without being biologically or otherwise undesirable.Salts are formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid, and organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, sulfonic acid or salicylic acid.Furthermore, these salts can be prepared by adding inorganic or organic bases to free acid.Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.

[0065] Oligonucleotide 107 may mediate or promote nuclease-mediated degradation of sodium channel pre-mRNA or mRNA transcript. Nuclease-mediated degradation refers to an oligonucleotide capable of mediating the degradation of a complementary nucleotide sequence when duplexed with such sequence. In some embodiments, oligonucleotides may function through nuclease-mediated degradation of target nucleic acid, where the oligonucleotide of the present invention is capable of recruiting a nuclease, particularly an endonuclease, preferably an endoribonuclease (RNase), such as RNase H. An example of an oligonucleotide design that operates through a nuclease-mediated mechanism is an oligonucleotide, such as a gapmer, that typically contains a region of at least five or six consecutive DNA nucleosides and is flanked on one or both sides by affinity-enhancing nucleosides. The RNase H activity of antisense oligonucleotide 107 refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule.

[0066] The antisense oligonucleotide 107 of the present invention, or its contiguous nucleotide sequence, may be a gapmer, also called a gapmer oligonucleotide or gapmer design. Antisense gapmers are commonly used to inhibit target nucleic acids via RNase H-mediated degradation. A gapmer oligonucleotide comprises at least three distinct structural regions: 5'-flanking, gap and 3'-flanking, FG-F', in a 5'->3' orientation. The "gap" region (G) comprises a stretch of contiguous DNA nucleotides that allows the oligonucleotide to recruit RNase H. The gap region is flanked by a 5'-flanking region (F) that comprises one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides, and a 3'-flanking region (F') that comprises one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides. The one or more sugar-modified nucleosides in regions F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., are affinity-enhancing sugar-modified nucleosides). In some embodiments, one or more sugar modified nucleosides in regions F and F' are 2' sugar modified nucleosides, e.g., high affinity 2' sugar modifications, e.g., independently selected from LNA and 2'-MOE.

[0067] A mixed wing gapmer may be an LNA gapmer in which one or both of regions F and F' comprise a 2'-substituted nucleoside, e.g., a 2'-substituted nucleoside selected from the group consisting of 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, 2'-MOE units, arabinonucleic acid (ANA) units, 2'-fluoro-ANA units, or combinations thereof. In some embodiments in which at least one of regions F and F', or both of regions F and F' comprise at least one LNA nucleoside, the remaining nucleosides of regions F and F' are independently selected from the group consisting of 2'-MOE and LNA. In some embodiments in which at least one of regions F and F', or both of regions F and F' comprise at least two LNA nucleosides, the remaining nucleosides of regions F and F' are independently selected from the group consisting of 2'-MOE and LNA. In some mixed wing embodiments, one or both of regions F and F' may further comprise one or more DNA nucleosides. Gapmer design is discussed in WO2008 / 049085 and WO2012 / 109395, both of which are incorporated by reference.

[0068] Conjugation of oligonucleotide 107 to one or more non-nucleotide moieties can improve the pharmacology of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments, the conjugate moiety can modify or enhance the pharmacokinetic properties of the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability and / or cellular uptake of the oligonucleotide. In particular, the conjugate can target the oligonucleotide to a specific organ, tissue or cell type, thereby enhancing the efficacy of the oligonucleotide in that organ, tissue or cell type. The conjugate can also function to reduce the activity of the oligonucleotide in non-target cell types, tissues or organs, for example, off-target activity or activity in non-target cell types, tissues or organs.

[0069] In certain embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0070] The oligonucleotide 107 of the present disclosure may be provided in a pharmaceutical composition comprising any of the above-mentioned oligonucleotides and / or their oligonucleotide conjugates or salts and a pharma- ceutically acceptable diluent, carrier, salt and / or adjuvant. Pharmaceutically acceptable diluents include artificial cerebrospinal fluid (ACSF), and pharma-ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharma-ceutically acceptable diluent is sterile phosphate buffered saline or sterile sodium carbonate buffer. In some preferred embodiments, diluents for clinical applications include Elliotts B solution and / or ACSF artificial cerebrospinal fluid.

[0071] In some embodiments, the oligonucleotide of the present invention is in the form of a solution in a pharma- ceutically acceptable diluent, for example, dissolved in phosphate-buffered saline (PBS) or sodium carbonate buffer. The oligonucleotide may be preformulated in a solution, or in some embodiments, may be in the form of a dry powder (e.g., a lyophilized powder) that may be dissolved in a pharma-ceutically acceptable diluent prior to administration. Suitably, for example, the oligonucleotide may be dissolved at a concentration of 0.1-100 mg / mL, for example, 1-10 mg / mL.

[0072] The compositions of the present disclosure can be administered to patients for the prevention or treatment of conditions such as epilepsy or pain, for example, chronic pain, neuropathic pain, inflammatory pain, spontaneous pain or nociceptive pain.Preferred embodiments are used for the treatment of epilepsy involving DEE13, Dravet syndrome, or the pathogenesis of an excessive E / I balance ratio.The oligonucleotide of the present invention, or the conjugate, salt or pharmaceutical composition of the present invention can be for use as a local analgesic.

[0073] The present disclosure provides a method for treating or preventing a condition in a subject, e.g., a human, suffering from or likely to suffer from a condition, comprising administering a therapeutically or prophylactically effective amount of a composition of the present disclosure comprising, e.g., an oligonucleotide as described herein, to a subject suffering from or suffering from a condition, such as epilepsy or pain, e.g., cancer pain, osteoarthritis pain, chronic pain, neuropathic pain, inflammatory pain, spontaneous pain or nociceptive pain, wherein the oligonucleotide is targeted to a sequence complementary to one of SEQ ID NOs: 1-114 according to any of the descriptions herein having any combination of the features described herein. EXAMPLES

[0074] Figures 9-11 provide summary data of percent knockdown from single dose screening of SCN8A ASOs. SCN8A-targeting ASOs were screened in vitro by treating SK-N-AS neuroblastoma cells plated at 20,000 cells per well of a 96-well plate with 100 nM of ASO. Data from two rounds (replicates) are shown for each figure. ASOs were delivered by transfection using 0.3 uL of RNAiMax per well of a 96-well plate. The data shown shows a summary table of qPCR readout of SCN8A knockdown (expressed as percent SCN8A knockdown) for 146 ASOs screened in our primary screen. All samples were normalized to the vehicle-only (i.e., RNAiMax only) condition.

[0075] Figure 9 shows an SCN8A exon-targeted ASO that targets the entire transcript.

[0076] Figure 10 shows an SCN8A intron-targeting ASO.

[0077] Figure 11 gives the results from an optimized SCN8A exon-targeted ASO focused on the first 3700 nucleotides of the transcript. All cells were transfected with ASO at the time of plating, and 48 hours after ASO transfection, cells were harvested for qPCR. Transcript levels for the housekeeping gene actin were used to normalize the levels for SCN8A. All data are shown across two independent rounds of plating, ASO treatment, and qPCR.

[0078] Note: ASOs with IDs ending in 115-146 were designed after screening ASOs with IDs ending in 001-074, where we identified the first 3700 nucleotides of the SCN8A transcript (NM_014101.4) as a hotspot for modulation by ASOs. In other words, a larger number of ASOs targeting this region succeeded in knocking down the SCN8A transcript by at least about 60%. The sequence for this part of the transcript is available in GenBank under the accession number NM_014101.4.

[0079] Figures 12-14 provide summary data from a sodium channel counterscreen using SCN8A ASO that achieves at least 60% target knockdown in a single dose screen.

[0080] From the single dose screening experiments (shown in Figures 9-11), ASOs that achieved at least 60% SCN8A transcript knockdown in SK-N-AS cells were prioritized. Due to known homology in transcript sequences across sodium channels, ASO candidates were further screened to quantify their ability to knockdown other sodium channels, which is not a desired feature of lead candidates. The focus of the experiments via qPCR was on SCN2A, SCN3A and SCN9A, which are known to be expressed in this cell type. For other sodium channels not expressed in SK-N-AS cells, a computational alignment score (right column, Figures 12 and 14) was calculated that predicts the off-target effect potential of the ASO on an approximate scale of 11-40 (where 40 indicates a perfect match). Herein, qPCR knockdown across all sodium channels and maximum predicted alignment scores are shown for the prioritized ASOs.

[0081] Figure 12 provides data from an SCN8A exon-targeted ASO that targets the entire transcript.

[0082] Figure 13 provides data from an SCN8A intron-targeted ASO.

[0083] Figure 14 presents data from optimized SCN8A exon-targeted ASOs focusing on the first 3700 nucleotides of the transcript. Because predicted alignment to homologous sodium channels was considered in the design of ASOs 115-146, there is a lower level of overall off-target knockdown in this batch. Note that the maximum predicted alignment score is not shown for intron-targeted ASOs, since it is based on alignment to exons.

[0084] Figures 15 and 16 provide an example of dose-response screening of SCN8A lead ASO candidates.

[0085] Candidate lead SCN8A-targeting ASOs were selected based on at least 60% SCN8A transcript knockdown in single dose screens (Figures 9-11) and less than about 20% transcript knockdown in the homologous sodium channel (Figures 10, 12-14). For each candidate lead sequence, a new ASO with the identical sequence was synthesized with one to three PO backbone modifications in the 3' and 5' 2'-MOE RNA-like wings, respectively (a total of three to four PO modifications per ASO). These candidate leads were then tested for dose-response modulation of SCN8A transcript expression. For these experiments, SK-N-AS neuroblastoma cells plated at a density of 20,000 cells per well of a 96-well plate were treated with a range of concentrations: 800, 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 nM. ASO was delivered by transfection using 0.3uL of RNAiMax per well of a 96-well plate. All cells were transfected with ASO at the time of plating, and cells were harvested for qPCR 48 hours after ASO transfection. Actin was used as a normalization gene to SCN8A. Each data point represents two technical replicates and one biological replicate.

[0086] Figure 15 shows five-point dose-response data for two lead SCN8A ASO parent candidates (117, 124) and their PO-modified daughter molecules (155, 156, 157, 158) in SK-N-AS neuroblastoma cells.

[0087] FIG. 16 shows the nine-point dose-response data for the same PO-modified daughter molecules.

[0088] Figures 17 and 18 provide summary data from dose-response screening of SCN8A ASO lead candidates.

[0089] Candidate lead SCN8A-targeting ASOs were selected based on at least 60% transcript knockdown in the primary single dose screen. For each candidate lead, a new ASO with identical sequence was synthesized with 1-3 PO backbone modifications in the 3' and 5' 2'-MOE RNA-like wings, respectively (3-4 PO modifications in total per ASO). All candidate leads were then tested for dose-response modulation of SCN8A transcript expression. For these experiments, SK-N-AS neuroblastoma cells plated at 20k per well of a 96-well plate were plated on a 96-well plate. ASOs were screened at five doses: 100, 50, 25, 12.5, 6.25nM. ASOs were delivered by transfection using 0.3uL of RNAiMax per well of a 96-well plate. All cells were transfected with ASO at the time of plating, and cells were harvested for qPCR 48 hours after ASO transfection. Actin was used as a normalization gene to SCN8A. All samples were further normalized to the vehicle condition within each experiment. Dose-response data for all lead candidates are shown and analyzed.

[0090] FIG. 17 shows data for lead all PS scaffold candidates targeting SCN8A exons.

[0091] FIG. 18 shows data for the PO-modified daughter leads as human clinical candidates.

[0092] FIG. 19 shows knockdown of SCN8A transcripts in human NGN2 stem cell-derived neurons using SCN8A lead candidates.

[0093] SCN8A is important in neuronal excitability, and as a result, this cell type is important for evaluating the functional effects of targeted transcript knockdown. To show that our ASOs are effective in relevant human cell types, we transfected our SCN8A ASOs into human induced pluripotent stem cell-derived neurons (differentiated via NGN2 overexpression and dual SMAD inhibition). Neurons were plated at 70k per well on 96-well plates and treated with 250 and 100nM SCN8A ASOs. ASOs were transfected into neurons at DIV20 using Endoporter PEG transfection reagent (0.6uL per well). Cells were harvested for qPCR 4 days after treatment at DIV24. Many ASOs show >80% knockdown of SCN8A transcripts in human neurons. Beta-tubulin was used as a normalization gene for SCN8A. Each bar represents two technical and one biological replicates.

[0094] FIG. 20 shows knockdown of SCN8A transcripts in human primary neurons using SCN8A lead candidates.

[0095] SCN8A is important in neuronal excitability, and as a result, this cell type is important for assessing the functional effects of targeted transcript knockdown. To show that our ASOs are effective in relevant human cell types, we transfected human primary neurons (derived from 19-week-old female fetuses; obtained from ScienCell) with selected SCN8A ASOs. Neurons were plated at 30k per well on 96-well plates and treated with 1 uM SCN8A ASO. ASOs were delivered gymnotically on DIV1. Cells were harvested for qPCR 13 days after ASO treatment. Many ASOs show 50-60% knockdown of SCN8A transcripts in human primary neurons with naked delivery. Beta-tubulin was used as a normalization gene for SCN8A. Each bar shows two technical and one biological replicates.

[0096] FIG. 21 shows knockdown of Scn8a transcripts in mouse primary cortical neurons using SCN8A lead candidates.

[0097] Two mouse models of SCN8A-induced encephalopathy with R1872W and N1768D mutations are available (references below). These mouse models are useful for demonstrating proof-of-concept and efficacy in vivo in disease model systems. To show that the selected ASOs are effective in relevant mouse cell types, we treated mouse primary cortical neurons (Brainbits) with the selected SCN8A ASOs. Neurons were plated at 50k per well on 96-well plates and treated with 500nM SCN8A ASOs. ASOs were delivered naked on DIV5. Cells were harvested for qPCR 7 days after ASO treatment (DIV12). Optimized lead candidates of SCN8A with skeletal modifications were screened. ASOs with mouse homology show at least 60% knockdown. Beta-tubulin was used as a normalization gene for Scn8a. Each bar represents two technical and one biological replicates.

[0098] References for the SCN8A-R1872W mouse model are provided in Bunton-Stasyshyn, 2019, Prominent role of forebrain excitatory neurons in SCN8A encephalopathy, Brain 142(2): 362-375, which is incorporated by reference.

[0099] References for the SCN8A-N1768D mouse model are provided in Wagnon, 2015, Convulsive seizures and SUDEP in a mouse model of SCN8A epileptic encephalopathy, Hum Mol Genet 24(2): 506-515, which is incorporated by reference.

[0100] FIG. 22 shows knockdown of Scn8a transcripts in rat primary hippocampal neurons using SCN8A lead candidates.

[0101] Lead ASOs are screened in vivo in rats to test for tolerability, toxicology, PK and PD. To show that selected ASOs are effective in relevant rat cell types, rat primary hippocampal neurons (Brainbits) were treated with selected SCN8A ASOs. Neurons were plated at 12k per well on 96-well plates and treated with 450nM SCN8A ASOs. ASOs were delivered naked on DIV5. Cells were harvested for qPCR 7 days after ASO treatment on DIV12. Optimized lead candidates of SCN8A with backbone modifications were screened. ASOs with rat homology show at least 60% knockdown. Beta-tubulin was used as a normalization gene for Scn8a. Each bar shows two technical replicates and one biological replicate.

[0102] FIG. 23 shows evidence of a plateau in transcript knockdown in human NGN2 stem cell-derived neurons. It has been observed that Nav1.6 null mice died early. See Raman, 1997, Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a Mutant Mice, Neuron, incorporated by reference. Literature suggests that hypomorphic mice expressing 10% of normal levels of Nav1.6 experience severe dystonia. See Kearney, 2002, Molecular and pathological effects of a modified gene on deficiency of the sodium channel Scn8a (Navi.6), Hum Mol Genet, incorporated by reference. Evidence suggests that DEE13 mice treated with ASOs that knock down Scn8a by 50% experience reduced epileptic seizures and increased life span. See Lenk, 2020, Scn8a antisense oligonucleotide is protective in mouse models of SCN8A encephalopathy and Dravet syndrome, Ann Neurol, incorporated by reference. Given that background, we hypothesize that the therapeutic window is within the range of 50-90% protein knockdown. ASOs with a concentration response for SCN8A knockdown that achieves 50% knockdown and plateaus at well-tolerated knockdown levels below 90% may yield an optimal therapeutic profile.

[0103] We transfected our SCN8A ASO into human induced pluripotent stem cell-derived neurons (differentiated via NGN2 overexpression and dual SMAD inhibition). Neurons were plated at 70k per well on 96-well plates and treated with 1000, 800, 500, 250 and 100nM SCN8A ASO in a dose response. ASO was transfected into neurons at DIV20 using Endoporter PEG transfection reagent (0.6uL per well). Cells were harvested for qPCR at DIV24 and DIV30, 4 and 10 days after treatment. Beta-tubulin was used as a normalization gene for SCN8A. Each bar shows two technical replicates and one biological replicate.

[0104] ASOs denoted 165, 166, 175 and 176 show maximal knockdown of 70-80% starting from 500 nM ASO treatment, whereas ASOs denoted 153, 154, 157 and 158 show >80% knockdown starting from 100 nM.

[0105] ASO 165, also known as 14-165, is SEQ ID NO: 135 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings where the 2nd, 3rd, and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications. ASO 166, also known as 14-166, is SEQ ID NO: 135 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings where the 2nd, 3rd, 4th, and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications. ASO 175, also known as 14-175, is SEQ ID NO: 144, a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, where the second, third and eighteenth interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications. ASO 176, also known as 14-176, is SEQ ID NO: 144, a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, where the second, third, fourth and eighteenth interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications. The graph shows that these four compositions show a maximum knockdown of about 70-80% for the concentration range tested (?).

[0106] SCN8A lead candidate ASOs for in vivo studies are shown in the following supplier order format (which can be ordered from suppliers such as Integrated DNA Technologies) and include ASO-147-158, 165, 166, 175 and 176. Unless otherwise indicated, all ASOs are gapmers with a 12 base core and 3-5 base 2'-MOE RNA wings. All ASOs have mostly PS backbones, unless otherwise indicated in the supplier order format. Cytosines are methylated.

[0107] Lead ASOs were selected based on single-dose and dose-response efficacy, sodium channel counterscreen data, sequence motif trends and off-target alignment analysis. ASOs with the highest in vitro efficacy against SCN8A, no knockdown in other sodium channels, lowest off-target alignments and limited sequence motif concerns were prioritized as leads.

[0108] In Table 1, ASOs that fall within the first 3700 nucleotides are marked with an asterisk ( * Of these, ASOs with IDs ASO 14-001 to 14-146 as well as 16-003, 16-004, 16-009, 16-011, 16-013, 16-014, 16-016, 16-017, 16-019 and 16-020 were synthesized using the following chemistry: 4 (2'-MOE) x 12 (DNA) x 4 (2'-MOE); all C bases have 5-methyl modifications; all PS backbone.

[0109] The table below shows certain SCN8A ASO candidates that were synthesized. ASOs 14-147 to 176 were synthesized with the following modified linkages, as shown in the FASTA list: 2MOEr = 2'-O-methoxyethyl RNA; i2MOEr = internal 2'-O-methoxyethyl RNA; iMe-dC / 2MOErC / i2MOErC = 5-methyl modifications; and PS linkages ( * ) vs PO( / / ) linkage.

[0110] As shown in the FASTA catalog, certain embodiments use gapmer ASOs having a sequence given by at least one of SEQ ID NO:16; SEQ ID NO:41; SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:117; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:129; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:138; SEQ ID NO:139; SEQ ID NO:142; SEQ ID NO:143; or SEQ ID NO:144, where a gapmer has a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, each wing having one or two phosphodiester linkages, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications.

[0111] Certain most preferred embodiments (for knockdown plateaus that achieve between about 50% and 90% expression normalized to untreated) use 14-165, 14-166, 14-175 or 14-176.

[0112] As used herein, 14-165 is SEQ ID NO: 135, a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, in which the 2nd, 3rd and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications.

[0113] 14-166 is SEQ ID NO: 135 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings where the 2nd, 3rd, 4th and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications. 14-175 is SEQ ID NO: 144 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings where the 2nd, 3rd and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications. 14-176 is SEQ ID NO: 144 in a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, where the 2nd, 3rd, 4th and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate, and all cytosine bases have 5-methyl modifications. A FASTA catalog is a format that can be submitted to a supplier such as Integrated DNA Technologies to order such ASOs.

[0114] [ka] [ka] [ka] [ka] [ka]

Claims

1. A composition comprising an oligonucleotide that hybridizes to an RNA encoding a sodium channel protein along a segment of RNA that is at least about 75% complementary to one of SEQ ID NOs: 1-156, thereby preventing translation of said RNA into said sodium channel protein.

2. The composition of claim 1, wherein the oligonucleotide hybridizes to Nav1.6 pre-mRNA or mRNA and knocks down expression.

3. The composition of claim 1, wherein the sequence of bases in the oligonucleotide has at least about 80% identity to one of SEQ ID NOs: 1-156.

4. 2. The composition of claim 1, wherein the sequence of bases in the oligonucleotide is at least about 95% identical to one of SEQ ID NOs: 1-156, and the oligonucleotide is capable of hybridizing to Nav1.6 pre-mRNA or mRNA and inducing RNase cleavage.

5. 2. The composition of claim 1, wherein the composition comprises a plurality of therapeutic oligonucleotides, each having a base sequence at least about 80% identical to one of SEQ ID NOs: 1-156, each of the therapeutic oligonucleotides having a gapmer structure comprising a central DNA segment flanked by modified RNA wings, and wherein the plurality of therapeutic oligonucleotides is provided in a solution or carrier formulated for intrathecal injection.

6. The composition of claim 1 , wherein the oligonucleotide comprises two wings flanking a central region of at least about 9 DNA bases.

7. The composition of claim 1 , wherein at least one end of the oligonucleotide comprises a modified RNA base.

8. 8. The composition of claim 7, wherein each modified RNA base is selected from the group consisting of 2'-O-methoxyethyl RNA and 2'-O-methyl RNA.

9. The composition of claim 1 , wherein the oligonucleotide comprises at least about 15 bases.

10. The composition of claim 1 , wherein the oligonucleotide comprises between about 15 and about 25 bases.

11. The composition of claim 1 , wherein the oligonucleotide has a backbone containing a plurality of phosphorothioate linkages.

12. The composition of claim 1 , wherein the oligonucleotide has a base sequence that has been screened and determined to not meet a threshold match for any non-target transcripts in humans.

13. The composition of claim 1, wherein the composition comprises multiple copies of a therapeutic oligonucleotide having a base sequence at least about 95% identical to one of SEQ ID NOs: 1-156, and the therapeutic oligonucleotide has a gapmer structure comprising a central 12-base DNA segment flanked by two wings of four 2'-O-methoxyethyl RNA bases and a backbone of phosphorothioate linkages.

14. The composition of claim 1, wherein when the composition is delivered to a cell in vitro, the cell exhibits dose-dependent knockdown of Nav1.

6.

15. 2. The composition of claim 1, wherein the oligonucleotide has a base sequence having at least about a 90% match to one of SEQ ID NOs: 1-156, the bases being linked exclusively by phosphorothioate linkages, and the oligonucleotide further comprises a central 12 DNA bases flanked by a 5' wing and a 3' wing, each of which comprises four consecutive 2' modified RNA bases.

16. 16. The composition of claim 15, wherein the oligonucleotide has a base sequence having at least about 90% match to one of SEQ ID NOs: 16, 41, 44, 45, 100, 117, 124, 125, 126, 128, 129, 130, 133, 134, 135, 138, 139, 142, 143 and 144.

17. 2. The composition of claim 1, wherein the oligonucleotide has a base sequence that matches one of SEQ ID NOs: 1-156, a majority of the interbase linkages comprise phosphorothioate linkages, and the oligonucleotide further comprises central 12 DNA bases flanked by a 5' wing and a 3' wing, each of which comprises four consecutive 2'-MOE RNA bases.

18. 18. The composition of claim 17, wherein the oligonucleotide has a base sequence that matches one of SEQ ID NOs: 16, 41, 44, 45, 100, 117, 124, 125, 126, 128, 129, 130, 133, 134, 135, 138, 139, 142, 143 and 144.

19. The composition of claim 1 , wherein the oligonucleotide hybridizes to a position within the first 3700 bases of the SCN8A transcript.

20. The composition of claim 1, wherein the oligonucleotide has one sequence selected from the group consisting of SEQ ID NO:16; SEQ ID NO:41; SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:117; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:129; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:138; SEQ ID NO:139; SEQ ID NO:142; SEQ ID NO:143; or SEQ ID NO:

144.

21. 21. The composition of claim 20, further comprising: said oligonucleotide being a gapmer having a central segment of 12 bases of DNA flanked by two 2'-MOE RNA wings, each wing having one, two or three phosphodiester linkages, the remaining interbase linkages being phosphorothioate, and all cytosine bases having 5-methyl modifications.

22. The composition of claim 1, wherein the oligonucleotide knocks down expression of the SCN8A transcript to about 50-90% compared to an untreated control.

23. Furthermore, the oligonucleotide comprises: SEQ ID NO:135, a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, in which the 2nd, 3rd and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications; SEQ ID NO:135, a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, in which the 2nd, 3rd, 4th and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications; SEQ ID NO:144, a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, in which the 2nd, 3rd and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications; and SEQ ID NO:144 for a gapmer having a central segment of 12 bases of DNA flanked by 2'-MOE RNA wings, in which the 2nd, 3rd, 4th and 18th interbase linkages are phosphodiester, the remaining interbase linkages are phosphorothioate and all cytosine bases have 5-methyl modifications. The composition of claim 1, which is one selected from the group consisting of:

24. 24. The composition of claim 23, wherein the oligonucleotide knocks down expression of the SCN8A transcript to about 50-70% compared to a control when delivered to a cell at a concentration of about 1000 nM.

25. A composition described in any one of claims 1 to 24 for use in a method, the method comprising the step of administering the composition to a subject having epilepsy, thereby knocking down expression of the SCN8A gene.

26. 26. The composition of claim 25, wherein the epilepsy comprises Dravet syndrome, DEE13, or epilepsy involving a pathogenic mechanism of excessive E / I balance.

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