Splice-switching oligonucleotides for treating cacna1a-associated disorders

Abstract

Aspects of the disclosure relate to the discovery that antisense oligonucleotides (ASOs) that bind to single regions of a CACNA1A pre-mRNA to affect alternative splicing lead to an efficient modulation of protein produced from matured CACNA1A pre-mRNA. This modulation, which can be as an increase or decrease, can alleviate issues related to gain-of-function or loss-of-function mutations in the CACNA1A gene, respectively, including those that lead to haploinsufficiencies. Alleviating such issues can be useful to treat neurological disorders that result from such mutations.

Description

SPLICE-SWITCHING OLIGONUCLEOTIDES FOR TREATING CACNA1A- ASSOCIATED DISORDERS

[0001] This application claims priority of U.S. Provisional Application No. 63 / 633,000 filed April 11, 2024, which is hereby incorporated by reference in its entirety. BACKGROUND I. Sequence Listing

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 7, 2025, is named ARCD.P0836WO – SEQ LISTING.xml and is 24,797 bytes in size. II. Field of the Invention

[0003] This disclosure relates to the fields of neurology, molecular biology, and medicine. III. Background

[0004] The CACNA1A gene encodes the pore-forming subunit of the voltage-gated P / Q type calcium channel, which plays a fundamental role in synaptic transmission1. Dominant CACNA1A mutations cause a range of neurological symptoms: Loss-of-function CACNA1A mutations cause episodic ataxia type 2 (EA2), developmental and epileptic encephalopathy 42 (DEE42), and some cases of autism spectrum disorder (ASD)2-4. On the other hand, gain-of- function mutations of CACNA1A can lead to familial hemiplegic migraine 1 (FHM1) and seizure2-4. Additionally, the abnormal C-terminal CAG repeat expansion causes spinocerebellar ataxia 6 (SCA6)2,5. Treating CACNA1A-associated diseases requires precise up-regulation or down-regulation of CACNA1A expression for loss- and gain-of-function mutations, respectively.

[0005] Alternative splicing coupled with nonsense-mediated mRNA decay (AS-NMD) presents a unique mechanism to regulate gene expression6. Antisense oligonucleotides (ASOs) regulating splicing have been successfully developed to treat diseases such as spinal muscular atrophy7-11. The proven chemistry of FDA-approved ASOs forms the foundation to develop treatments for other neurological disorders by altering the nucleotide sequences.297599530.1 - 1 -BRIEF SUMMARY

[0006] Aspects of the disclosure relate, in part, to the discovery that certain ASOs that bind proximal to a nonsense-mediated decay exon on a CACNA1A pre-mRNA are capable of modulating the levels of a protein translated from the CACNA1A pre-mRNA. Such modulation is useful for, among others, increasing or decreasing the levels of CACNA1A protein in a cell. Increasing or decreasing the levels of CACNA1A protein in a cell can be beneficial for certain diseases associated with loss-of-function or gain-of-function mutations in the CACNA1A gene, respectively.

[0007] Aspects of the disclosure relate, in part, to the identification an alternatively spliced exon in CACNA1A that inserts premature translational stop codons and triggers mRNA degradation in the mouse and human brains. We have screened dozens of ASOs that target the alternative exon and its flanking intronic regions. We have identified and validated ASOs that suppress or increase the CACNA1A NMD exon inclusion and thus up- or down-regulate CACNA1A expressions.

[0008] Aspects herein demonstrate that the ASO can up- or down-regulate CACNA1A protein in disease settings to treat loss- and gain-of-function CACNA1A mutations. Specifically, the ASOs that suppress the NMD exon and up-regulate CACNA1A expression can be used to treat EA2, DEE42, and ASD2-4that are caused by loss-of-function CACNA1A mutations. Conversely, the ASOs that increase the NMD exon inclusion and down-regulate CACNA1A expression can be used to treat FHM1, seizures, and SCA62-4that are caused by CACNA1A gain-of-function.

[0009] Certain aspects relate to methods of increasing a CACNA1A protein in a cell. Certain aspects relate to methods of decreasing a CACNA1A protein in a cell. In some aspects, the CACNA1A protein is produced from a CACNA1A pre-mRNA. In certain aspects, the CACNA1A protein is increased in the cell by contacting the cell with an ASO. The ASO may be any ASO described herein. The cell may be any cell that expresses the CACNA1A pre- mRNA. In some aspects, the cell is a neuron. The cell may comprise a mutation in the CACNA1A gene, which may be a loss-of-function mutation or a gain-of-function mutation. Certain aspects relate to methods where the cell is contacted with an ASO. In some aspects, the cell is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more (or any range derivable therein) different ASOs each comprising a unique sequence.

[0010] Certain aspects herein relate to methods of modulating levels of a protein produced from a CACNA1A pre-mRNA in a cell. In some aspects, the method comprises contacting the297599530.1 - 2 -cell with one or more antisense oligonucleotides (ASOs). In some aspects, at least one of the ASOs are fully complementary to a portion of only one contiguous region of the CACNA1A pre-mRNA in the cell. In certain aspects, the cell is a neuron. In certain aspects, the cell comprises a gain-of-function mutation in a CACNA1A gene in the cell’s genome.

[0011] Certain aspects concern methods of contacting a cell, such as a neuron, containing a gain-of-function mutation in a CACNA1A gene with one or more ASOs from YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, YRW598, or a combination thereof. Certain aspects concern methods of contacting a cell, such as a neuron, containing a gain-of-function mutation in a CACNA1A gene with one or more ASOs from YRW549, YRW550, YRW551, YRW571, YRW574, YRW575, or a combination thereof.

[0012] In certain aspects, the cell comprises a loss-of-function mutation in a CACNA1A gene in the cell’s genome. Certain aspects concern methods of contacting a cell, such as a neuron, containing a loss-of-function mutation in a CACNA1A gene with one or more ASOs from YRW519, YRW520, YRW521, YRW525, YRW527, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546, or a combination thereof. Certain aspects concern methods of contacting a cell, such as a neuron, containing a loss-of-function mutation in a CACNA1A gene with one or more ASOs from YRW520, YRW521, YRW533, YRW534, YRW535, YRW536, YRW537, or a combination thereof.

[0013] Aspects also concern methods of treating a neurological disease in a patient. In some aspects, the method comprises administering one or more antisense oligonucleotides (ASOs) to the patient. In certain aspects, at least one of the ASOs are fully complementary to a portion of only one contiguous region of a CACNA1A pre-mRNA in the patient. In certain aspects, the patient has a mutation in a gene encoding the CACNA1A pre-mRNA. In certain aspects, the mutation comprises a gain-of-function mutation in a CACNA1A gene in the patient’s genome. In certain aspects, such as where the patient has a gain-of-function mutation in a CACNA1A gene, the one or more ASOs administered to the patient comprise YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, YRW598, or a combination thereof. In certain aspects, such as where the patient has a gain-of-function mutation in a CACNA1A gene, the one or more ASOs administered to the patient comprise YRW549, YRW550, YRW551, YRW571, YRW574, YRW575, or a combination thereof. In certain aspects, where the patient has a gain-of-function mutation in the CACNA1A gene, the patient has, is suspected of having, or has been diagnosed with having297599530.1 - 3 -familial hemiplegic migraine 1 (FHM1). In certain aspects, where the patient has a gain-of- function mutation in the CACNA1A gene, the patient has progressive cerebellar ataxia. In certain aspects, where the patient has a gain-of-function mutation in the CACNA1A gene, the patient does not have progressive cerebellar ataxia. In certain aspects, where the patient has a gain-of-function mutation in the CACNA1A gene, the patient has, is suspected of having, or has been diagnosed with having spinocerebellar ataxia 6 (SCA6).

[0014] In certain aspects, the mutation comprises a loss-of-function mutation in a CACNA1A gene in the patient’s genome. In certain aspects, such as where the patient has a loss-of-function mutation in a CACNA1A gene, the one or more ASOs administered to the patient comprise YRW519, YRW520, YRW521, YRW525, YRW527, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546, or a combination thereof. In certain aspects, such as where the patient has a loss-of- function mutation in a CACNA1A gene, the one or more ASOs administered to the patient comprise YRW520, YRW521, YRW533, YRW534, YRW535, YRW536, YRW537, or a combination thereof. In certain aspects, where the patient has a loss-of-function mutation in the CACNA1A gene, the patient has, is suspected of having, or has been diagnosed with having episodic ataxia type 2 (EA2), developmental and epileptic encephalopathy 42 (DEE42), and / or autism spectrum disorder (ASD).

[0015] In some aspects, the patient is further administered an additional therapeutic.

[0016] In certain aspects, the one contiguous region of the CACNA1A pre-mRNA is a nonsense-mediated decay exon, or flanking region thereof, of the CACNA1A pre-mRNA.

[0017] Also disclosed are compositions comprising at least one of YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, YRW598.

[0018] Also disclosed are antisense oligonucleotides having 90%, 95%, or 100% sequence identity to the sequence of YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, or YRW598, wherein the antisense oligonucleotide is isolated.

[0019] Also disclosed are compositions comprising at least one of YRW519, YRW520, YRW521, YRW525, YRW527, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546.

[0020] Also disclosed are antisense oligonucleotides having 90%, 95%, or 100% sequence identity to the sequence of YRW519, YRW520, YRW521, YRW525, YRW527, YRW533,297599530.1 - 4 -YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546, wherein the antisense oligonucleotide is isolated.

[0021] The one contiguous region may be an intronic region of the CACNA1A gene, such as a region proximal to the NMD exon of the CACNA1A gene. A region proximal to the NMD exon may be a region within the exon or a region flanking the exon. The one contiguous region may be an alternative splice site in the CACNA1A gene. The one contiguous region may be in an alternatively spliced exon, i.e. an exon that comprises a different 5’ or 3’ sequence depending on which splice site is used during pre-mRNA processing. The one contiguous region may be a binding site for one or more proteins involved in splicing. It is specifically contemplated that, in certain aspects, the ASO does not bind in more than one contiguous region to the pre-mRNA. It is also specifically contemplated that, in certain aspects, the ASO is not complementary to a sequence in the pre-mRNA that is not proximal to an alternative splice site.

[0022] In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:2. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:3. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:4. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:5. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:6. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:7. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:8. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:9. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:10. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:11. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:12. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:13. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:14. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:15. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:16. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:17. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:18. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:19. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:20. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ297599530.1 - 5 -ID NO:21. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:22. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:23. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:24. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:25. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:26. In some aspects, the ASO has at least 90%, 95%, or 100% sequence identity to SEQ ID NO:27. In some aspects, the ASO has 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 (or any range derivable therein) nucleotides. In certain aspects, the ASO is isolated. In some aspects, the ASO consists of an ASO with at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or a combination thereof.

[0023] In some aspects, the ASO has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (or any range derivable therein) nucleotides inserted, contiguously or separately, into the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. The insertions may be on the 5’ and / or 3’ end of the sequence, and / or be inserted within the sequence. In some aspects, the ASO has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (or any range derivable therein) mutations to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27,. In some aspects, the ASO has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more (or any range derivable therein) deletions to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.297599530.1 - 6 -

[0024] In certain aspects, the ASO consists of an ASO with at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In certain aspects, the ASOs consist of or comprise two or more ASOs having at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some aspects, one or more ASO, including one or more ASO described herein, is specifically excluded from the method.

[0025] Certain methods herein modulate, such as increase or decrease, nonsense-mediated decay of an mRNA derived from a CACNA1A pre-mRNA. In some aspects, the CACNA1A pre- mRNA can be processed into an mRNA, where the mRNA has a stop codon upstream of a splicing junction forming a nonsense mRNA. In such aspects, the nonsense mRNA undergoes nonsense-mediated decay. In certain aspects, the ASO, having complementary to one contiguous region of the CACNA1A pre-mRNA, modulates the processing of the CACNA1A pre-mRNA into the nonsense mRNA. In certain aspects, the ASO specifically binds to a CACNA1A pre-mRNA that gets processed into the nonsense mRNA, but does not bind to a CACNA1A pre-mRNA that does not get processed into the nonsense mRNA. In some aspects, the ASO binds to productive pre-mRNA (pre-mRNA that does not get processed into nonsense mRNA) and binds to unproductive pre-mRNA (pre-mRNA that does get processed into nonsense mRNA). In certain aspects, the ASO blocks at least one sequence that are required to generate unproductive mRNA. The ASO may block one or more proteins involved in splicing, including a muscleblind like splicing regulator 1 (MBNL1). In some aspects, a cell is contacted with an amount of the ASO sufficient to reduce nonsense-mediated decay of an mRNA derived from the CACNA1A pre-mRNA, which may be the nonsense mRNA. The reduction may be at least, or approximately equal to, a 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%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,297599530.1 - 7 -68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) reduction in nonsense-mediated decay of CACNA1A mRNA in the cell compared to a cell not contacted with the ASO. In some aspects, the cell is contacted with an amount of the ASO sufficient to increase the protein produced from the CACNA1A pre-mRNA to an amount that is more than, or approximately equal to, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0-fold, or any range derivable therein, greater than an amount of protein produced from the CACNA1A pre-mRNA present in a cell not contacted with the ASO.

[0026] In some aspects, a cell is contacted with an amount of the ASO sufficient to increase nonsense-mediated decay of an mRNA derived from the CACNA1A pre-mRNA, which may be the nonsense mRNA. The increase may be at least, or approximately equal to, a 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%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) increase in nonsense-mediated decay of CACNA1A mRNA in the cell compared to a cell not contacted with the ASO. In some aspects, the cell is contacted with an amount of the ASO sufficient to decrease the protein produced from the CACNA1A pre-mRNA to an amount that is more than, or approximately equal to, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0-fold, or any range derivable therein, less than an amount of protein produced from the CACNA1A pre-mRNA present in a cell not contacted with the ASO.

[0027] The CACNA1A protein may be any isoform. In some aspects, the CACNA1A protein is a CACNA1A isoform expressed in neurons.

[0028] Certain aspects relate to methods comprising administering a therapeutically effective amount of at least one ASO to a patient. In certain aspects, the ASO consists of or comprises one or more ASOs disclosed herein. In certain aspects, the ASO consists of an ASO with at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID297599530.1 - 8 -NO:26, or SEQ ID NO:27. In certain aspects, the ASOs consist or comprise two or more ASOs having at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some aspects, one or more ASO, including one or more ASO described herein, is specifically excluded from the method.

[0029] In certain aspects, the method comprising administering the ASO is a method of treating the patient, such as treating a neurological disorder in the patient. The neurological disorder may be episodic ataxia type 2 (EA2), developmental and epileptic encephalopathy 42 (DEE42), autism spectrum disorder (ASD), familial hemiplegic migraine 1 (FHM1), seizures, or spinocerebellar ataxia 6 (SCA6). In certain aspects, the method comprising administering the ASO is a method of reducing symptoms or pathologies associated with dysregulated CACNA1A protein levels, including those associated with dysregulated CACNA1A protein levels in a neuron in the patient. In some aspects, the dysregulated protein levels are due to a loss-of-function mutation in the CACNA1A gene in the cell. In some aspects, the dysregulated protein levels are due to a gain-of-function mutation in the CACNA1A gene in the cell.

[0030] Certain aspects relate to the compositions described herein, such as any of the ASOs described herein. In certain aspects, the ASO, which may be isolated, comprises a sequence complementary to one contiguous region of a CACNA1A pre-mRNA, wherein the ASO is capable of binding to the contiguous region of the CACNA1A pre-mRNA in a manner that blocks an alternative splicing of the CACNA1A pre-mRNA that leads to nonsense-mediated decay of an mRNA derived from the CACNA1A pre-mRNA. In certain aspects, the ASO comprises SEQ ID NO:1. In certain aspects, the ASO comprises SEQ ID NO:2. In certain aspects, the ASO comprises SEQ ID NO:3. In certain aspects, the ASO comprises SEQ ID NO:4. In certain aspects, the ASO comprises SEQ ID NO:5. In certain aspects, the ASO comprises SEQ ID NO:6. In certain aspects, the ASO comprises SEQ ID NO:7. In certain aspects, the ASO comprises SEQ ID NO:8. In certain aspects, the ASO comprises SEQ ID NO:9. In certain aspects, the ASO comprises SEQ ID NO:10. In certain aspects, the ASO comprises SEQ ID NO:11. In certain aspects, the ASO comprises SEQ ID NO:12. In certain aspects, the ASO comprises SEQ ID NO:13. In certain aspects, the ASO comprises SEQ ID NO:14. In certain aspects, the ASO comprises SEQ ID NO:15. In certain aspects, the ASO comprises SEQ ID NO:16. In certain aspects, the ASO comprises SEQ ID NO:17. In certain297599530.1 - 9 -aspects, the ASO comprises SEQ ID NO:18. In certain aspects, the ASO comprises SEQ ID NO:19. In certain aspects, the ASO comprises SEQ ID NO:20. In certain aspects, the ASO comprises SEQ ID NO:21. In certain aspects, the ASO comprises SEQ ID NO:22. In certain aspects, the ASO comprises SEQ ID NO:23. In certain aspects, the ASO comprises SEQ ID NO:24. In certain aspects, the ASO comprises SEQ ID NO:25. In certain aspects, the ASO comprises SEQ ID NO:26. In certain aspects, the ASO comprises SEQ ID NO:27. In certain aspects, the composition consists of an ASO with at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In certain aspects, the composition consists of or comprises two or more ASOs having at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some aspects, one or more ASO, including one or more ASO described herein, is specifically excluded from the composition. In certain aspects, the composition comprises one or more ASOs, including one or more ASOs disclosed herein, where none of the ASOs bind more than once to a pre-mRNA. It is also specifically contemplated that, in certain aspects, the CACNA1A pre-mRNA does not contain more than one binding site to the specific ASO or ASOs in the composition.

[0031] Certain aspects relate to pharmaceutical compositions comprising at least one ASO. The pharmaceutical composition may comprise any ASO described herein. In certain aspects, the pharmaceutical composition consists of an ASO with at least 90%, 95%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27 and a pharmaceutical excipient. In certain aspects, the pharmaceutical composition consists of or comprises two or more ASOs having at least 90%, 95%, or 100% sequence identity to SEQ ID297599530.1 - 10 -NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some aspects, one or more ASO, including one or more ASO described herein, is specifically excluded from the pharmaceutical composition. In some aspects, the ASO in the pharmaceutical composition is isolated. The pharmaceutical composition may be formulated to stabilize the ASO. In certain aspects, a therapeutically effective amount of the ASO is administered to a patient, including a patient having a neurological disorder. In certain aspects, the pharmaceutical composition comprises one or more ASOs, including one or more ASOs disclosed herein, where none of the ASOs bind more than once to a pre-mRNA. It is also specifically contemplated that, in certain aspects, the CACNA1A pre-mRNA does not contain more than one binding site to the specific ASO or ASOs in the pharmaceutical composition.

[0032] The following enumerated aspects are also disclosed:

[0033] Aspect 1 includes a method of modulating levels of a protein produced from a CACNA1A pre-mRNA in a cell, the method comprising contacting the cell with one or more antisense oligonucleotides (ASOs), wherein at least one of the ASOs are fully complementary to a portion of only one contiguous region of the CACNA1A pre-mRNA in the cell. Aspect 2 includes the method of Aspect 1, wherein the cell is a neuron. Aspect 3 includes the method of Aspect 1 or 2, wherein the cell comprises a gain-of-function mutation in a CACNA1A gene in the cell’s genome. Aspect 4 includes the method of Aspect 3, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW555 (SEQ ID NO:20), YRW569 (SEQ ID NO:21), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), YRW596 (SEQ ID NO:25), YRW597 (SEQ ID NO:26), YRW598 (SEQ ID NO:27), or a combination thereof. Aspect 5 includes the method of Aspect 3, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), or a combination thereof. Aspect 6 includes the method of Aspect 1 or 2, wherein the cell comprises a loss-of-function mutation in a CACNA1A gene in the cell’s genome. Aspect 7 includes the method of Aspect 6, wherein the one or more ASOs comprise YRW519 (SEQ ID NO:1), YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW525 (SEQ ID NO:4), YRW527 (SEQ ID NO:5), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID NO:9), YRW537 (SEQ ID297599530.1 - 11 -NO:10), YRW539 (SEQ ID NO:11), YRW540 (SEQ ID NO:12), YRW543 (SEQ ID NO:13), YRW544 (SEQ ID NO:14), YRW545 (SEQ ID NO:15), YRW546 (SEQ ID NO:16), or a combination thereof. Aspect 8 includes the method of Aspect 6, wherein the one or more ASOs comprise YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID NO:9), YRW537 (SEQ ID NO:10), or a combination thereof. Aspect 9 includes the method of any one of Aspects 1 to 8, wherein the one contiguous region of the CACNA1A pre-mRNA is a nonsense-mediated decay exon, or flanking region thereof, of the CACNA1A pre-mRNA.

[0034] Aspect 10 includes a method of treating a neurological disease in a patient, the method comprising administering one or more antisense oligonucleotides (ASOs) to the patient, wherein at least one of the ASOs are fully complementary to a portion of only one contiguous region of a CACNA1A pre-mRNA in the patient.

[0035] Aspect 11 includes the method of Aspect 10, wherein the patient has a mutation in a gene encoding the CACNA1A pre-mRNA. Aspect 12 includes the method of Aspect 10 or 11, wherein the mutation comprises a gain-of-function mutation in a CACNA1A gene in the patient’s genome. Aspect 13 includes the method of Aspect 12, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW555 (SEQ ID NO:20), YRW569 (SEQ ID NO:21), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), YRW596 (SEQ ID NO:25), YRW597 (SEQ ID NO:26), YRW598 (SEQ ID NO:27), or a combination thereof. Aspect 14 includes the method of Aspect 12, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), or a combination thereof. Aspect 15 includes the method of any one of Aspects 12 to 14, wherein the patient has, is suspected of having, or has been diagnosed with having familial hemiplegic migraine 1 (FHM1). Aspect 16 includes the method of Aspect 15, wherein the patient also has progressive cerebellar ataxia. Aspect 17 includes the method of Aspect 15, wherein the patient does not have progressive cerebellar ataxia. Aspect 18 includes the method of any one of Aspects 12 to 14, wherein the patient has, is suspected of having, or has been diagnosed with having spinocerebellar ataxia 6 (SCA6). Aspect 19 includes the method of Aspect 10 or 11, wherein the mutation comprises a loss-of-function mutation in a CACNA1A gene in the patient’s genome. Aspect 20 includes the method of Aspect 19, wherein the one or more ASOs comprise YRW519 (SEQ ID NO:1), YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW525 (SEQ ID NO:4), YRW527 (SEQ ID NO:5), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID297599530.1 - 12 -NO:9), YRW537 (SEQ ID NO:10), YRW539 (SEQ ID NO:11), YRW540 (SEQ ID NO:12), YRW543 (SEQ ID NO:13), YRW544 (SEQ ID NO:14), YRW545 (SEQ ID NO:15), YRW546 (SEQ ID NO:16), or a combination thereof. Aspect 21 includes the method of Aspect 19, wherein the one or more ASOs comprise YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID NO:9), YRW537 (SEQ ID NO:10), or a combination thereof. Aspect 22 includes the method of any one of Aspects 19 to 21, wherein the patient has, is suspected of having, or has been diagnosed with having episodic ataxia type 2 (EA2), developmental and epileptic encephalopathy 42 (DEE42), and / or autism spectrum disorder (ASD). Aspect 23 includes the method of any one of Aspects 10 to 22, wherein the one contiguous region of the CACNA1A pre-mRNA is a nonsense-mediated decay exon, or flanking region thereof, of the CACNA1A pre-mRNA. Aspect 24 includes the method of any one of Aspects 10 to 23, wherein the patient is further administered an additional therapeutic agent.

[0036] Aspect 25 includes a composition comprising at least one of YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, YRW598.

[0037] Aspect 26 includes an antisense oligonucleotide having 90%, 95%, or 100% sequence identity to the sequence of YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, or YRW598, wherein the antisense oligonucleotide is isolated.

[0038] Aspect 27 includes a composition comprising at least one of YRW519, YRW520, YRW521, YRW525, YRW527, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546.

[0039] Aspect 28 includes an antisense oligonucleotide having 90%, 95%, or 100% sequence identity to the sequence of YRW519, YRW520, YRW521, YRW525, YRW527, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546, wherein the antisense oligonucleotide is isolated.

[0040] The term “one contiguous region,” as used herein, refers to a contiguous, unique region in a nucleic acid, including a pre-mRNA. In some aspects, the one contiguous region is only found once in a pre-mRNA, and includes sequences that are complementary to one or more ASOs described herein. For example, YRW520 comprises a sequence complementary to one contiguous region that occurs only once in a CACNA1A pre-mRNA. In other aspects, there are contiguous regions in the pre-mRNA that are repeated, including those that are297599530.1 - 13 -complementary to previously known ASOs. It is specifically contemplated that, in certain aspects, at least one ASO does not bind to more than one contiguous region.

[0041] The term “CACNA1A protein” used herein refers to a protein produced from a CACNA1A pre-mRNA, a CACNA1A mRNA, and / or a CACNA1A gene. The CACNA1A protein may also be referred to as voltage-dependent P / Q-type calcium channel subunit alpha- 1A or Cav2.1 pore-forming subunit.

[0042] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0043] As used herein, “isolated” means altered or removed from the natural state through human intervention. For example, an ASO, such as an ASO comprising SEQ ID NO:1 or SEQ ID NO:2, naturally present in a living animal is not “isolated,” but a synthetic ASO, or an ASO partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated ASO can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the ASO has been delivered.

[0044] As used herein, the terms “therapeutic composition,” “pharmaceutical composition,” “therapeutic agent” and “pharmaceutical agent” may be used interchangeably and refer to a composition that is used therapeutically to affect a response in a patient.

[0045] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprises plural forms and vice versa.

[0046] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0047] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.297599530.1 - 14 -

[0048] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of” excludes any element, step, or ingredient not specified. The phrase “consisting essentially of” limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.”

[0049] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0050] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of” any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0051] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0052] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description, Claims, and Brief Description of the Drawings.

[0053] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.297599530.1 - 15 -BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0055] FIG. 1 shows a sashimi plot showing alternative splicing of the Cacna1a exon (mm10, chr8:84600494:84600661) in mouse embryonic brain samples (embryonic day 11.5, E11.5; embryonic day 18.5, E18.5). The exon inclusion introduces a premature termination codon in the Cacna1a transcript.

[0056] FIGs. 2A-2C show (A) PCR results showed that cycloheximide (CHX) treatment increased the inclusion of the Cacna1a exon in mouse primary neurons, compared to the control groups treated with dimethyl sulfoxide (DMSO). PSI: percentage spliced in. (B). Statistical analysis of results in panel A by a t-test: ****, p < 0.0001. (C). Statistical analysis of quantitative-PCR (Q-PCR) results of the control group (DMSO) and CHX treatment group. T-test: **, 0.001 ≤ p < 0.01.

[0057] FIG. 3 shows a plot showing the inclusion of the cryptic CACNA1A NMD exon (GRCh38, chr19:13236449:13236618) in human induced pluripotent stem cell (iPSC)-derived neurons and its increase after CHX treatment. The inclusion of this exon introduces a premature termination codon indicated by a red arrow.

[0058] FIGs. 4A-4E show PCR validation of increased CACNA1A NMD exon inclusion in human iPSC, iPSC-derived neurons and HTB186 cells after CHX treatment. (A) PCR results showed that CHX treatment increased CACNA1A NMD exon inclusion in human iPSCs (left) and iPSC-derived neurons (right). (B) Statistical analyses of results in panel A by t-tests. *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01. (C) PCR results showed that CHX treatment increased CACNA1A NMD exon inclusion in a human cerebellar medulloblastoma cell line HTB186. (D) Statistical analysis of results in panel C by a one-way ANOVA test. ***, 0.0001 ≤ p < 0.001. (E) Statistical analysis of quantitative-PCR (Q-PCR) results of the control group (DMSO) and CHX treatment group of HTB186 cells. T-test: *, 0.01 ≤ p < 0.05.

[0059] FIG. 5 shows an overview of CACNA1A ASOs design. This SnapGene figure shows the exon 31 to exon 37 of human CACNA1A. The NMD exon is labeled in red color. ASOs are located within the NMD exon and its upstream and downstream regions.

[0060] FIG.6 shows the sequences and positions of CACNA1A ASOs. The NMD exon is labeled in red color. “-NMD” means the ASO is effective in suppressing the inclusion of the297599530.1 - 16 -NMD exon in CACNA1A transcripts, while “+NMD” means the ASO is effective in promoting the inclusion of the NMD exon.

[0061] FIG.7 shows PCR results of ASOs screening in HTB186 cells. NT, no treatment; NA, no ASO, with transfection reagents; SC, scramble ASO, with transfection reagents. Y# represents YRW#.

[0062] FIGs.8A-8B shows A. PCR results of the second round of ASO tests in HTB186 cells. NC: no ASO, with transfection reagents. NT: no treatment. B. Statistical analyses of the results in panel A by a one-way ANOVA test. NS: not statistically significant. ***, 0.0001 ≤ p < 0.001; ****, p < 0.0001.

[0063] FIGs.9A-9B show (A) PCR results of the second round of ASO tests in HTB186 cells. NC: no ASO, with transfection reagents. (B) Statistical analysis of the results in panel A by a one-way ANOVA test. NS: not statistically significant. **, 0.001 ≤ p < 0.01; ****, p < 0.0001.

[0064] FIGs. 10A-10C show (A) PCR results of ASO tests at 100 nM in HTB186 cells. NC: negative control, no ASO, with transfection reagents. (B) Statistical analyses of the results in panels A by an one-way ANOVA test. *, 0.01 ≤ p < 0.05; ****, p < 0.0001. (C) Statistical analyses of Q-PCR results of ASO tests at 100 nM in HTB186 cells. One-way ANOVA tests; *, 0.01 ≤ p < 0.05; ***, 0.0001 ≤ p < 0.001; ****, p < 0.0001. NS results were not labeled in the figures. E32 represents exon 32. Y# represents YRW#.

[0065] FIGs. 11A-11D show (A) PCR results of ASO tests at 100 nM in HTB186 cells. NC: negative control, no ASO, with transfection reagents. / , too weak signals to calculate. (B) PCR results of ASO tests at 50 nM in HTB186 cells. (C) Statistical analyses of the results in panels A and B by one-way ANOVA tests. NS: not statistically significant. **, 0.001 ≤ p < 0.01; ***, 0.0001 ≤ p < 0.001; ****, p < 0.0001. (D) Statistical analyses of Q-PCR results of ASO tests at 100 nM and 50 nM in HTB186 cells. One-way ANOVA tests; *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, 0.0001 ≤ p < 0.001; ****, p < 0.0001. NS results were not labeled in the figures. E32 represents exon 32.

[0066] FIGs.12A-12C show (A) PCR results of ASO YRW520 at 25 nM, 50 nM, and 100 nM in HTB186 cells. NC: negative control, no ASO, with transfection reagents. Statistical analyses were shown on the right panel. One-way ANOVA test: ***, 0.0001 ≤ p < 0.001; ****, p < 0.0001. (B) PCR results of ASO YRW521 at 25 nM, 50 nM, and 100 nM in HTB186 cells. Statistical analyses were shown on the right panel. One-way ANOVA test: ****, p < 0.0001. (C) Statistical analyses of Q-PCR results of ASO tests at 25 nM, 50 nM, and 100 nM in297599530.1 - 17 -HTB186 cells. One-way ANOVA tests; *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, 0.0001 ≤ p < 0.001. NS results were not labeled in the figures.

[0067] FIGs.13A-13C show (A) PCR results of ASO YRW549 at 10 nM, 25 nM, and 50 nM in HTB186 cells. NC: negative control, no ASO, with transfection reagents. Statistical analyses were shown on the right panel. One-way ANOVA test: ****, p < 0.0001. (B) PCR results of ASO YRW550 at 10 nM, 25 nM, and 50 nM in HTB186 cells. Statistical analyses were shown on the right panel. One-way ANOVA test: ****, p < 0.0001. (C) Statistical analyses of Q-PCR results of ASO tests at 10 nM, 25 nM, and 50 nM in HTB186 cells. One- way ANOVA tests; *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, 0.0001 ≤ p < 0.001; ****, p < 0.0001. NS results were not labeled in the figures.

[0068] FIGs. 14A-14D. show (A) PCR results of the CACNA1A NMD exon inclusion ratios in HEK293T and HEK293TCAM cells, detected by minigene-specific or general primers, respectively. (B and C) PCR results of CACNA1A NMD exon inclusion ratios in HEK293TCAM cells transfected with different concentrations of ASO YRW520. (D) Q-PCR analyses of CACNA1A NMD and non-NMD transcripts in HEK293TCAM cells transfected with ASO YRW520 at different concentrations. Results at each concentration were compared with the NC group by t-tests. *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, 0.0001 ≤ p < 0.001.

[0069] FIG.15 shows a Q-PCR analyses of CACNA1A NMD and non-NMD transcripts in iNGN-induced neurons transfected with ASO YRW549 at different concentrations. DETAILED DESCRIPTION

[0070] Aspects herein relate to the identification and utilization of an alternatively spliced exon in CACNA1A that inserts premature translational stop codons and triggers mRNA degradation in the mouse and human brains. Aspects herein encompass dozens of ASOs that target the alternative exon and its flanking intronic regions. Among others, certain ASOs have been identified and validated as ASOs that suppress or increase the CACNA1A NMD exon inclusion and thus up- or down-regulate CACNA1A expressions.

[0071] Certain aspects herein use ASOs that are capable of up- or down-regulating CACNA1A protein, including in disease settings to treat loss- and gain-of-function CACNA1A mutations. In certain aspects, the ASOs that suppress the NMD exon and up-regulate CACNA1A expression can be used to treat EA2, DEE42, and ASD2-4that are caused by loss- of-function CACNA1A mutations. Conversely, the ASOs that increase the NMD exon inclusion297599530.1 - 18 -and down-regulate CACNA1A expression can be used to treat FHM1, seizures, and SCA62-4that are caused by CACNA1A gain-of-function.

[0072] Alternative splicing coupled with NMD (AS-NMD) can selectively remove unproductive transcripts and has been reported to regulate the expression of neuronal genes (Carvill et al., 2018; Yan et al., 2015; Zhang et al., 2016; Zheng et al., 2012). The therapeutic potential of targeting AS-NMD with SSOs has evoked unprecedented enthusiasm, and SSO screens in cultured cells have been reported in recent studies (Han et al., 2020; Lim et al., 2020). Certain aspects show that it may be safe to target naturally occurring AS-NMD for therapy. Certain aspects relate to whether an AS-NMD exon is required for normal physiological functions in animal development and whether it is safe to suppress or completely block such an AS-NMD exon in vivo. It is important to understand the in vivo expression patterns, regulatory mechanisms, and organismal functions of AS-NMD exons. I. Antisense Oligonucleotides

[0073] In some aspects, the disclosure relates to antisense oligonucleotides (ASOs) that inhibit the binding of certain splicing machinery, such as PTBP1 or PTBP2, which can affect the amount of a protein in a cell. In some embodiments, the ASO is a splice-switching oligonucleotide (SSO). In some aspects, the protein comprises CACNA1A. In some aspects, the disclosure relates to expression systems capable of expressing the ASO. An ASO may increase the translation of a gene transcript in a cell. An ASO may be from 16 to 1000 nucleotides long, and in certain aspects from 15 to 100 nucleotides long. The ASO may have at least or may have at most 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, 36, 37, 38, 40, 50, 60, 70, 80, or 90 (or any range derivable therein) nucleotides. The ASO may comprise any nucleic acid form such as DNA, RNA, LNA, or BNA. The ASO may comprise synthetic or non-natural nucleotides. The ASO may be synthetic and / or isolated. In some aspects, the ASO is single-stranded. In some aspects, the ASO is double-stranded. In some aspects, the ASO comprises single-stranded DNA. In some aspects, the ASO comprises single-stranded RNA. In some aspects, the ASO comprises double- stranded DNA. In some aspects, the ASO comprises double-stranded RNA. In some aspects, the ASO comprises an antisense strand.

[0074] Aspects herein include one or more of the following ASOs and corresponding SEQ ID NO.297599530.1 - 19 -297599530.1 - 20 -

[0075] Particularly, an ASO may be capable of decreasing the nonsense-mediated decay of an mRNA by at least 10%, 20%, 30%, or 40%, more particularly by at least 50%, 60%, or 70%, and most particularly by at least 75%, 80%, 90%, 95%, 99%, or 100% more or any range or value in between the foregoing. In some aspects, the ASO is capable of increasing protein levels of a protein of interest, such as Cav2.1, by modulating the splicing of a pre-mRNA transcript encoding the protein. The ASO may be partially or fully complementary to a sequence in the pre-mRNA that is involved in splicing, such as an alternative splicing site or a splicing factor- binding site.

[0076] In some aspects, the ASO is, or comprises, an oligonucleotide analog and may include modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and / or improve binding specificity. For example, when the sugar portion of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar. Moreover, when other substitutions, such a substitution for the inter-sugar phosphodiester linkage are made, the resulting material is no longer a true species. All such compounds are considered to be analogs. Throughout this specification, reference to the sugar portion of a nucleic acid species shall be understood to refer to either a true sugar or to a species taking the structural place of the sugar of wild type nucleic acids. Moreover, reference to inter-sugar linkages shall be taken to include moieties serving to join the sugar or sugar analog portions in the fashion of wild type nucleic acids.

[0077] The present disclosure concerns modified oligonucleotides, i.e., oligonucleotide analogs or oligonucleosides, and methods for effecting the modifications. These modified oligonucleotides and oligonucleotide analogs may exhibit increased chemical and / or enzymatic stability relative to their naturally occurring counterparts. Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to the backbone-modified compounds. When present as the protonated acid form, the lack of a negatively charged backbone may facilitate cellular penetration.297599530.1 - 21 -

[0078] The modified internucleoside linkages may replace naturally-occurring phosphodiester-5’-methylene linkages with four atom linking groups to confer nuclease resistance and enhanced cellular uptake to the resulting compound.

[0079] Modifications may be achieved using solid supports which may be manually manipulated or used in conjunction with a DNA synthesizer using methodology commonly known to those skilled in DNA synthesizer art. Generally, the procedure involves functionalizing the sugar moieties of two nucleosides which will be adjacent to one another in the selected sequence. In a 5’ to 3’ sense, an “upstream” synthon such as structure H is modified at its terminal 3’ site, while a “downstream” synthon such as structure H1 is modified at its terminal 5’ site.

[0080] Oligonucleosides linked by hydrazines, hydroxylarnines, and other linking groups, are contemplated herein for use in the ASOs, and can be protected by a dimethoxytrityl group at the 5’-hydroxyl and activated for coupling at the 3’-hydroxyl with cyanoethyldiisopropyl- phosphite moieties. These compounds can be inserted into any desired sequence by standard, solid phase, automated DNA synthesis techniques. One of the most popular processes is the phosphoramidite technique. Oligonucleotides containing a uniform backbone linkage can be synthesized by use of CPG-solid support and standard nucleic acid synthesizing machines such as Applied Biosystems Inc.® 380B and 394 and Milligen / Biosearch® 7500 and 8800s. The initial nucleotide (number 1 at the 3’-terminus) is attached to a solid support such as controlled pore glass. In sequence specific order, each new nucleotide is attached either by manual manipulation or by the automated synthesizer system.

[0081] Free amino groups can be alkylated with, for example, acetone and sodium cyanoboro hydride in acetic acid. The alkylation step can be used to introduce other, useful, functional molecules on the macromolecule. Such useful functional molecules include but are not limited to reporter molecules, RNA cleaving groups, groups for improving the pharmacokinetic properties of an oligonucleotide, and groups for improving the pharmacodynamic properties of an oligonucleotide. Such molecules can be attached to or conjugated to the macromolecule via attachment to the nitrogen atom in the backbone linkage. Alternatively, such molecules can be attached to pendent groups extending from a hydroxyl group of the sugar moiety of one or more of the nucleotides. Examples of such other useful functional groups are provided by WO1993007883, which is herein incorporated by reference, and in other of the above-referenced patent applications.

[0082] Solid supports may include any of those known in the art for polynucleotide synthesis, including controlled pore glass (CPG), oxalyl controlled pore glass, TentaGel®297599530.1 - 22 -Support—an aminopolyethyleneglycol derivatized support or Poros—a copolymer of polystyrene / divinylbenzene. Attachment and cleavage of nucleotides and oligonucleotides can be effected via standard procedures. As used herein, the term solid support further includes any linkers (e.g., long chain alkyl amines and succinyl residues) used to bind a growing oligonucleotide to a stationary phase such as CPG. In some aspects, the oligonucleotide may be further defined as having one or more locked nucleotides, ethylene bridged nucleotides, peptide nucleic acids, or a 5’(E)-vinyl-phosphonate (VP) modification. In some aspects, the oligonucleotides has one or more phosphorothioated DNA or RNA bases. II. Obtaining Nucleotides A. Synthesis

[0083] The nucleic acid molecules, including an ASO described herein, may be generated by nucleic acid synthesis. The ASOs may be synthesized using any method known in the art, such as phosphoramidite synthesis and / or solid-phase synthesis. The ASO analogs may be synthesized. B. Expression

[0084] The nucleic acid molecules, including any ASO described herein, may be generated by expression vectors. The expression vectors used herein may contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, and a selectable marker element. Such sequences and methods of using the same are well known in the art. 1. Expression Systems

[0085] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with an aspect to produce nucleic acid sequences. Commercially and widely available systems include but are not limited to bacterial, mammalian, yeast, and insect cell systems. Those skilled in the art are able to express a vector to produce a nucleic acid sequence using an appropriate expression system.297599530.1 - 23 -2. Methods of Gene Transfer

[0086] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction. 3. Host Cells

[0087] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors.297599530.1 - 24 -

[0088] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts. III. Administration of Therapeutic Compositions

[0089] The therapy provided herein may comprise the administration of one or a combination of therapeutic agents, such as one or a combination of unique antisense oligonucleotides (ASOs) and / or a combination of ASOs and other therapeutic compositions, including those useful for treating disorders disclosed herein, such as any neurological disorder, to a patient. In some aspects, the therapy is a cocktail of ASOs. In some aspects, the other therapeutic compositions are useful for reducing symptoms of the neurological disorder and / or reducing side effects of the other therapeutic agents administered. The therapies may be administered in any suitable manner known in the art. In some aspects, a first therapeutic composition (such as an ASO) and a second composition (such as another ASO or another therapeutic composition) may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, the first and second therapeutic compositions are administered in a separate composition. In some aspects, the first and second therapeutic compositions are in the same composition.

[0090] In some aspects, the first therapeutic composition and the second therapeutic composition are administered substantially simultaneously. In some aspects, the first therapeutic composition and the second therapeutic composition are administered sequentially. In some aspects, the first therapeutic composition, the second therapeutic composition, and a third therapeutic composition are administered sequentially. In some aspects, the first therapeutic composition is administered before administering the second therapeutic composition. In some aspects, the first therapeutic composition is administered after administering the second therapeutic composition.

[0091] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or297599530.1 - 25 -in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0092] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0093] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0094] In some aspects, a single dose of the ASO or other therapeutic composition is administered. In some aspects, multiple doses of the ASO or other therapeutic composition are administered. In some aspects, the ASO, at least one ASO, multiple ASOs, therapeutic compositions comprising one or more ASO, or other therapeutic composition is administered at a dose of between 1 mg / kg and 5000 mg / kg. In some aspects, the ASO, at least one ASO, multiple ASOs, therapeutic compositions comprising one or more ASO, or other therapeutic composition is administered at a dose of at least, at most, or about 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,297599530.1 - 26 -241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg / kg, or any range derivable therein.

[0095] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 µg / kg, mg / kg, µg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0096] In certain aspects, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 µM to 150 µM. In another aspect, the effective dose provides a blood level of about 4 µM to 100 µM.; or about 1 µM to 100 µM; or about 1297599530.1 - 27 -µM to 50 µM; or about 1 µM to 40 µM; or about 1 µM to 30 µM; or about 1 µM to 20 µM; or about 1 µM to 10 µM; or about 10 µM to 150 µM; or about 10 µM to 100 µM; or about 10 µM to 50 µM; or about 25 µM to 150 µM; or about 25 µM to 100 µM; or about 25 µM to 50 µM; or about 50 µM to 150 µM; or about 50 µM to 100 µM (or any range derivable therein). In other aspects, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM or any range derivable therein. In certain aspects, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0097] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0098] It will be understood by those skilled in the art and made aware that dosage units of µg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of µg / ml or mM (blood levels). It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0099] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 day, week, month, or year intervals, including all ranges there between.

[0100] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other297599530.1 - 28 -untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0101] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0102] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0103] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0104] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally,297599530.1 - 29 -dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0105] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0106] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. A. Pharmaceutical Compositions

[0107] In certain aspects, the compositions or agents, including those for use in the methods disclosed herein, such as one or more antisense oligonucleotides (ASOs), are suitably contained in a pharmaceutically acceptable carrier. The carrier can be non-toxic, biocompatible, and selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as the brain, nervous tissue, or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0108] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition,297599530.1 - 30 -fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0109] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0110] Certain aspects disclosed herein concern compositions comprising a nanoparticle, which may encapsulate a therapeutic agent, which can be any of the therapeutic agents disclosed herein. The nanoparticle compositions may encapsulate therapeutic agents, which may be engineered protein compositions. The engineered proteins formulated in the nanoparticles can have improved pharmacokinetic and / or pharmacodynamic properties. The engineered proteins formulated in the nanoparticles are better tolerated by a patient, including a cancer patient. The engineered proteins formulated in the nanoparticles, in some aspects, are more effectively delivered to cells to effect their function, such as effecting transcriptional changes, than naked engineered proteins. In several aspects, the composition confers water solubility to hydrophobic agents, to combinations of hydrophobic agents, and / or to combinations of hydrophobic and hydrophilic agents. In several aspects, the nanoparticle composition comprises a liposomal and / or nano-emulsion composition of a therapeutic agent.

[0111] In several aspects, as disclosed elsewhere herein, a nanoparticle composition (e.g., a mixed micelle composition, a liposomal composition, solid lipid particles, oil-in-water emulsions, water-in-oil-in-water emulsions, water-in-oil emulsions, oil-in-water-in-oil emulsions, etc.) is provided to aid in the delivery of therapeutic agents. As disclosed elsewhere herein, in several aspects, the nanoparticles comprise one or more therapeutic agents. In several aspects, a composition comprising the nanoparticles disclosed herein comprises a therapeutically effective amount of one or more therapeutic agents. In several aspects, the nanoparticle composition (e.g., when in water or dried) comprises multilamellar nanoparticle vesicles, unilamellar nanoparticle vesicles, multivesicular nanoparticles, emulsion particles, irregular particles with lamellar structures and bridges, partial emulsion particles, combined lamellar and emulsion particles, and / or combinations thereof. In certain aspects, the nanoparticle compositions do not comprise multilamellar nanoparticle vesicles, unilamellar nanoparticle vesicles, multivesicular nanoparticles, emulsion particles, irregular particles with297599530.1 - 31 -lamellar structures and bridges, partial emulsion particles, combined lamellar and emulsion particles, and / or combinations thereof. In several aspects, the composition is characterized by having multiple types of particles (e.g., lamellar, emulsion, irregular, etc.). In other aspects, a majority of the particles present are emulsion particles. In several aspects, a majority of the particles present are lamellar (multilamellar and / or unilamellar). In other aspects, a majority of the particles present are irregular particles. In still other aspects, a minority of the particles present are emulsion particles. In several aspects, a minority of the particles present are lamellar (multilamellar and / or unilamellar). In other aspects, a minority of the particles present are irregular particles.

[0112] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0113] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0114] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0115] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.297599530.1 - 32 -

[0116] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0117] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0118] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0119] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance. B. Proteins

[0120] The nucleotides as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at297599530.1 - 33 -uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. C. Other Agents

[0121] It is contemplated that other agents may be used in combination with certain aspects of the present aspects to improve the therapeutic efficacy of treatment. These additional agents include agents that act in combination and / or synergistically with the ASOs described herein. The additional agents may comprise agents that reduce symptoms of the disorders disclosed herein, or may comprise agents that reduce side effects associated with the therapeutic compositions disclosed herein. Examples

[0122] The following examples are included to demonstrate certain aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1: Materials and Methods for Certain Aspects Herein Primary mouse neurons

[0123] Primary mouse neurons were isolated from mouse brains at the embryonic day 15.5 through the Papain Dissociation System (Worthington Biochemical, LK003150). Primary neurons were resuspended in neurobasal A medium supplemented with GlutaMax (1X), N2 (1X), B27 (1X), and penicillin-streptomycin (100 U / mL) and then plated in 12-well plates coated with poly D-lysine.1 μM AraC were added into culture media during the first three days (DIV1 to DIV3). Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons

[0124] iPSCs (iNGN12) were cultured in Essential 8 (Thermo Fisher, A1517001) with penicillin-streptomycin (100 U / mL, Thermo Fisher, 15140122) in 10-cm dishes coated with GelTrex (Thermo Fisher, A1413301) in an incubator at 37 C with 5% CO2. A rock inhibitor Y-27632 dihydrochloride (10 μM, Tocris, 1254) was added into culture media for 24 hours297599530.1 - 34 -after passaging. For neuron induction, iNGN iPSCs were grown in Essential 8 supplemented with doxycycline (1 μg / mL, Sigma-Aldrich, D9891) and penicillin-streptomycin (100 U / mL) for 4 days. HTB186 cells

[0125] HTB186 cell line, a cerebellar medulloblastoma cell line, was purchased from ATCC: The Global Bioresource Center. HTB186 cells were cultured in DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 U / mL) in an incubator at 37 °C with 5% CO2. Cycloheximide treatment

[0126] To analyze the NMD-transcript of CACNA1A, primary mouse neuron (DIV3), iNGN, iNGN-derived neurons, and HTB186 cells were treated with cycloheximide (Sigma- Aldrich, C4859) at the concentration of 200 μg / mL for 5 hours in 12-well plates with 1 mL culture media in each well, followed by RNA extraction and PCR analyses. Total RNAs extracted from iNGN-derived neurons were then subject to RNA-sequencing (RNA-seq) library construction by Illumina® Stranded mRNA Prep, Ligation Kit (Illumina, 20040534). Libraries were sequenced on an Illumina NovaSeq (SP flowcell, 100-bp cassette) in the core facility of The University of Chicago. Antisense oligonucleotides (ASOs)

[0127] ASOs were synthesized by Integrated DNA Technologies (IDT). For ASO transfection, HTB186 cells, HEK293TCAM cells, and iNGN-induced neurons were seeded in 12-well plates (2×105cells / well). ASOs were transfected into cells using jetOPTIMUS transfection reagents (Genesee Scientific, 55-251) according to the manufacturer’s instructions. Total RNA was extracted using TRIzol reagent (Thermo Fisher, 15596018) and Direct-zol RNA Purification Kit (Zymo Research, R2060) 24 hours after transfection. cDNA was synthesized by ProtoScript® II Reverse Transcriptase system (New England Biolabs, M0368X). Quantitative PCR (Q-PCR) was performed using SYBR Green PCR Master Mix (Thermo Fisher, 4344463) in QuanStudio Real-Time PCR Systems (Thermo Fisher, ZG11CQS3STD) according to manufacturers’ instructions. RNA-sequencing analysis

[0128] RNA-seq data in fastq format were cleaned by fastp (version 0.22.0), then aligned using STAR (v2.7.9a) with rMATS recommended parameters (--outSAMstrandField297599530.1 - 35 -intronMotif --outFilterMismatchNmax 3 --alignIntronMax 299999 --alignSJDBoverhangMin 3 --alignEndsType EndToEnd --chimSegmentMin 2) against the human reference genome GRCh38.p13 (hg38), after which SAMTOOLS (v1.12) was used for sorting and indexing reads. FeatureCounts (v2.0.3) was then used to estimate the expression values (raw counts), with GENCODE v38 primary_assembly GTF file for annotation. BAM files of sequenced samples were viewed in IGV (v2.16.0). Example 2: Identification of an AS-NMD exon in CACNA1A

[0129] Here, the inventors report a previously unknown and alternatively spliced exon coupled with nonsense-mediated mRNA decay (AS-NMD, or NMD exon) in the mouse and human CACNA1A genes, the development of ASOs to regulate the CACNA1A NMD exon inclusion levels, and the applications of ASOs to up- and down-regulate CACNA1A expression.

[0130] The inventors first identified the Cacna1a NMD exon (mm10, chr8:84600494:84600661) when analyzing the RNA-sequencing (RNA-seq) data of mouse brain samples (Figure 1). Inclusion of this NMD exon in Cacna1a transcripts introduces a premature translational stop codon that is predicted to trigger NMD (Figure 1). Indeed, the inclusion of this exon was increased when RNA degradation was suppressed by cycloheximide (CHX) treatment, suggesting that inclusion of the Cacna1a exon triggers NMD (Figure 2).

[0131] At the homologous genomic locus, the inventors identified a conserved NMD exon (GRCh38, chr19:13236449:13236618) in the human CACNA1A gene: the inventors identified the exon inclusion in RNA-seq data of human induced pluripotent stem cell (iPSC)-derived neurons (Figure 3) and validated that the exon inclusion was upregulated after CHX treatment in iPSCs (Figures 3 and 4), iPSC-derived neurons, and a human cerebellar medulloblastoma cell line HTB186 (Figures 4). Example 3: Design of ASOs targeting the CACNA1A NMD exon and its adjacent regions

[0132] Based on the above data, the inventors designed ASOs to regulate CACNA1A expressions by redirecting the splicing inclusion of this NMD exon, with a goal to develop effective treatments for CACNA1A-associated diseases and disorders. As shown in Figures 5 and 6, the inventors designed a series of ASOs targeting the CACNA1A NMD exon and its adjacent conservative regions.297599530.1 - 36 -Example 4: ASOs are effective in regulating CACNA1A NMD exon splicing

[0133] The inventors used HTB186 cells as an in vitro model to examine the effects of ASOs on regulating CACNA1A NMD exon splicing and CACNA1A expressions. HTB186 cells were transfected with ASOs according to the jetOPTIMUS protocol (Genesee Scientific, #55- 251). RNA was extracted from HTB186 cells 24 hours after transfection and then used to perform reverse-transcription PCR and Q-PCR.

[0134] The first round of screening included 41 ASOs, with 9 ASOs upstream of the NMD exon, 16 ASOs within the NMD exon, and 16 ASOs downstream of the NMD exon (Figures 6 and 7). The final concentration of ASO in the culture media was 200 nM for each ASO group. The results showed that ASOs YRW493, YRW519, YRW520, YRW521, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW544, YRW545, and YRW546 decreased at least 50% of the CACNA1A NMD exon inclusion in HTB186 cells compared to the control groups (NT, no treatment; NC, no ASO, with transfection reagents; and SC, scramble ASO, with transfection reagents) (Figure 7, upper panel).

[0135] On the other hand, ASOs YRW549, YRW550, YRW551, YRW554, YRW555, YRW569, YRW571, YRW574, and YRW575 increased at least 60% of the CACNA1A NMD exon inclusion in HTB186 cells compared to the control groups (NT, NC, and SC) (Figure 7, lower panel). ASOs YRW492 and YRW508 increased about 40% and 30% of the CACNA1A NMD exon inclusion in HTB186 cells, respectively (Figure 7, upper panel).

[0136] The inventors then performed the second round of ASO tests for the above- mentioned effective ASOs. The final concentration of ASO in the culture media was 200 nM for each ASO group. Except for YRW492, YRW493, and YRW554, the ASOs in the second test showed significant effects on either suppressing or promoting the CACNA1A NMD exon inclusion in HTB186 cells (Figures 8 and 9). Specifically, ASOs YRW519, YRW520, YRW521, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW544, YRW545, and YRW546 significantly suppressed CACNA1A NMD exon inclusion in HTB186 cells (Figure 8). ASOs YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, and YRW575 significantly promoted CACNA1A NMD exon inclusion in HTB186 cells (Figure 9).

[0137] Additionally, the inventors designed and tested ASOs (YRW596, YRW597, and YRW598) targeting another conserved region upstream of the CACNA1A NMD exon. RT-PCR and Q-PCR results showed that YRW596 and YRW597 reduced the CACNA1A non-NMD297599530.1 - 37 -transcripts and increased NMD-in transcripts, while YRW598 significantly reduce the total mRNA of CACNA1A (Figure 10).

[0138] The effects of the tested ASOs on the CACNA1A NMD exon inclusion were summarized in the Table 1. Table 1. Summary of CACNA1A ASO sequences ASO nameEffect on the NMD exon inclusion YRW492 accccctgtctagtcact Weak effect YRW493 tcaaaaaccccctgtcta Weak effect YRW502 ggaggtggggccatccct Weak effect YRW506 ccaaggggcgggtgctgg Weak effect YRW507 ggcccaccaaggggcggg Weak effect YRW508 agcccgggcccaccaagg Weak effect YRW509 gaaatcagcccgggccca Weak effect YRW512 catgggtgggtgagctca Weak effect YRW514 gcactcagctcccatggg Weak effect YRW519 gcacgggaccaggcctgc Decreased NMD exon inclusion YRW520 ggagtagcacgggaccag Decreased NMD exon inclusion YRW521 ctgggtggagtagcacgg Decreased NMD exon inclusion YRW525 gggatggcttctcagctt Decreased NMD exon inclusion YRW527 tcccccctctcagggatg Decreased NMD exon inclusion YRW533 cccgctgagtcggagaag Decreased NMD exon inclusion YRW534 tcgctgcccgctgagtcg Decreased NMD exon inclusion YRW535 gagtcctcgctgcccgct Decreased NMD exon inclusion YRW536 cagggtgagtcctcgctg Decreased NMD exon inclusion YRW537 cggctgcagggtgagtcc Decreased NMD exon inclusion YRW539 gctgggactgttcggctg Decreased NMD exon inclusion YRW540 gagggagctgggactgtt Decreased NMD exon inclusion YRW543 cgggaatggggaggacgg Decreased NMD exon inclusion YRW544 ggcgagcgggaatgggga Decreased NMD exon inclusion YRW545 ccccttggcgagcgggaa Decreased NMD exon inclusion YRW546 tcttacccccttggcgag Decreased NMD exon inclusion YRW549 aagcggaagagcatcttt Increased NMD exon inclusion YRW550 tgggagaagcggaagagc Increased NMD exon inclusion YRW551 gccaattgggagaagcgg Increased NMD exon inclusion YRW552 gctcgagccaattgggag Weak effect YRW553 gcagcggctcgagccaat Weak effect YRW554 agaggagcagcggctcga Weak effect YRW555 cggccaagaggagcagcg Increased NMD exon inclusion YRW556 accccacggccaagagga Weak effect YRW557 gacctcaccccacggcca Weak effect YRW562 ccgagctgcccacccgct Weak effect YRW569 cgggactcaccgggcacc Increased NMD exon inclusion YRW571 atgcatctgtcacgggac Increased NMD exon inclusion YRW573 tccgggccagaaatgcat Weak effect YRW574 ttacgctccgggccagaa Increased NMD exon inclusion297599530.1 - 38 -YRW575 ggcatgttacgctccggg Increased NMD exon inclusion YRW576 tccgagggcatgttacgc Weak effect YRW596 caggcatgcgaaggcaggIncreased NMD exon inclusionYRW597 ggggcggatcataacatg Increased NMD exon inclusion YRW598 tacaagccatccctttcat Increased NMD exon inclusion Example 5: ASOs show dose-dependent effects on regulating the NMD exon splicing and CACNA1A expression

[0139] 200 nM of ASO was used in the most previous ASO tests. The inventors further used ASOs YRW520, YRW537, YRW539, YRW549, YRW550, and YRW555 as examples to test whether lower concentrations (100 nM and 50 nM) of ASOs are effective in regulating CACNA1A splicing and expressions in HTB186 cells.

[0140] As shown in Figure 11, ASOs YRW520, YRW537, and YRW539 effectively suppressed CACNA1A NMD exon inclusion at 100 nM and 50 nM in HTB186 cells (Figure 11A to 11C). Additionally, YRW520 and YRW537 significantly increased CACNA1A non- NMD transcripts at 100 nM in HTB186 cells, and YRW520 kept the efficiency even at 50 nM (Figure 11D).

[0141] On the other hand, ASOs YRW549, YRW550, and YRW555 were effective in promoting CACNA1A NMD exon inclusion, increasing CACNA1A NMD-transcripts, and decreasing CACNA1A non-NMD transcripts in HTB186 cells at both concentrations (Figure 11A-11D).

[0142] In the previous tests, YRW520 and YRW549 were most effective in regulating CACNA1A splicing and expressions, so the inventors further explored the dose-effect relationships of these two ASOs, as well as two ASOs YRW521 and YRW550 that are only 6- bp offset from them, respectively.

[0143] For YRW520 and YRW521, the inventors tested 25 nM, 50 nM, and 100 nM in HTB186 cells. Results showed that both ASOs significantly suppressed CACNA1A NMD exon inclusion even at 25 nM, and the suppression was more robust as the concentration increased (Figure 12A and 12B). Q-PCR results showed that YRW520 and YRW521 also exhibited dose- dependent effects on decreasing CACNA1A NMD-transcripts and increasing CACNA1A non- NMD transcripts (Figure 12C).

[0144] For YRW549 and YRW550, the inventors tested 10 nM, 25 nM, and 50 nM in HTB186 cells. Similarly, YRW549 and YRW550 showed dose-dependent effects on promoting CACNA1A NMD exon inclusion, increasing CACNA1A NMD-transcripts, and decreasing CACNA1A non-NMD transcripts (Figure 13A to 13C).297599530.1 - 39 -

[0145] ASO testing in different cell lines further demonstrated the robustness and dose- dependent efficacy of ASOs, using YRW520 and YRW549 as examples. Specifically, we used HEK293T cells to establish a stable cell line HEK293TCAM (HEK293T with a CACNA1A minigene containing CACNA1A exon 31, NMD exon, and exon 33), where the CACNA1A NMD exon minigene is integrated. CACNA1A NMD exon was not detected in HEK293T cells while highly expressed in HEK293TCAM cells (Figure 14A); thus, the HEK293TCAM cells provide a platform for testing ASOs that can potentially downregulate the CACNA1A NMD exon inclusion. PCR and Q-PCR results showed that YRW520 exhibited dose-dependent effects on decreasing CACNA1A NMD-transcripts and increasing CACNA1A non-NMD transcripts within 200 nM; however, higher concentrations (300 nM, 400 nM, 500 nM, 600 nM, and 700 nM) of YRW520 achieved attenuated increase of non-NMD transcripts compared with the 200 nM, suggesting an efficient window of ASO treatment (Figure 14B to 14D).

[0146] Testing of YRW549 in iNGN-induced neurons showed that YRW549 had a dose- dependent effect on downregulating CACNA1A non-NMD transcripts (Figure 15). Example 6: Potential applications of ASOs that regulate the CACNA1A NMD exon splicing and expression

[0147] As shown in the Table 2, CACNA1A mutations lead to multiple autosomal dominant (AD) disorders2-5.Loss-of-function mutations of CACNA1A can cause episodic ataxia type 2 (EA2), developmental and epileptic encephalopathy (DEE42), and some cases of autism spectrum disorder (ASD). ASOs that can inhibit the CACNA1A NMD exon inclusion and increase non-NMD transcripts will benefit patients with EA2, DEE42, and ASD caused by CACNA1A loss-of-function mutations.

[0148] On the other hand, gain-of-function mutations of CACNA1A can cause familial hemiplegic migraine (FHM1), DEE42, and ASD. Additionally, abnormal CAG repeat expansion causes spinocerebellar ataxia 6 (SCA6). ASOs that can promote the CACNA1A NMD exon inclusion and decrease non-NMD transcripts will benefit patients with FHM1, DEE42, and ASD caused by CACNA1A gain-of-function mutations and patients with SCA6.

[0149] On the other hand, gain-of-function mutations of CACNA1A cause familial hemiplegic migraines (FHM). Additionally, abnormal CAG repeat expansion causes spinocerebellar ataxia 6 (SCA6). ASOs that can promote the CACNA1A NMD exon inclusion and decrease non-NMD transcripts will be beneficial for patients with FHM caused by CACNA1A gain-of-function mutations and patients with SCA6.297599530.1 - 40 -Table 2: Disorders associated with CACNA1A mutations2,3

[0150] Based on the above data, the inventors assigned potential disease indications of CACNA1A ASO sequences in the Table 3 below. Table 3: CACNA1A ASO sequences and potential indications ASO name Sequence Potential indications YRW519 gcacgggaccaggcctgc DEE42, EA2, and ASD with CACNA1A mutations YRW520 ggagtagcacgggaccag DEE42, EA2, and ASD with CACNA1A mutations YRW521 ctgggtggagtagcacgg DEE42, EA2, and ASD with CACNA1A mutations YRW525 gggatggcttctcagctt DEE42, EA2, and ASD with CACNA1A mutations YRW527 tcccccctctcagggatg DEE42, EA2, and ASD with CACNA1A mutations YRW533 cccgctgagtcggagaag DEE42, EA2, and ASD with CACNA1A mutations YRW534 tcgctgcccgctgagtcg DEE42, EA2, and ASD with CACNA1A mutations YRW535 gagtcctcgctgcccgct DEE42, EA2, and ASD with CACNA1A mutations YRW536 cagggtgagtcctcgctg DEE42, EA2, and ASD with CACNA1A mutations YRW537 cggctgcagggtgagtcc DEE42, EA2, and ASD with CACNA1A mutations297599530.1 - 41 -YRW539 gctgggactgttcggctg DEE42, EA2, and ASD with CACNA1A mutations YRW540 gagggagctgggactgtt DEE42, EA2, and ASD with CACNA1A mutations YRW543 cgggaatggggaggacgg DEE42, EA2, and ASD with CACNA1A mutations YRW544 ggcgagcgggaatgggga DEE42, EA2, and ASD with CACNA1A mutations YRW545 ccccttggcgagcgggaa DEE42, EA2, and ASD with CACNA1A mutations YRW546 tcttacccccttggcgag DEE42, EA2, and ASD with CACNA1A mutations YRW549 aagcggaagagcatcttt DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW550 tgggagaagcggaagagc DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW551 gccaattgggagaagcgg DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW555 cggccaagaggagcagcg DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW569 cgggactcaccgggcacc DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW571 atgcatctgtcacgggac DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW574 ttacgctccgggccagaa DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW575 ggcatgttacgctccggg DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW596 caggcatgcgaaggcagg DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW597 ggggcggatcataacatg DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 YRW598 tacaagccatccctttcat DEE42, ASD with CACNA1A mutations, FHM1, and SCA6 * * *

[0151] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and297599530.1 - 42 -modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.297599530.1 - 43 -REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1 Simms, B. A. & Zamponi, G. W. Neuronal voltage-gated calcium channels: structure, function, and dysfunction. Neuron 82, 24-45 (2014). https: / / doi.org:10.1016 / j.neuron.2014.03.016 2 Rajakulendran, S., Kaski, D. & Hanna, M. G. Neuronal P / Q-type calcium channel dysfunction in inherited disorders of the CNS. Nat Rev Neurol 8, 86-96 (2012). https: / / doi.org:10.1038 / nrneurol.2011.228 3 Damaj, L. et al. CACNA1A haploinsufficiency causes cognitive impairment, autism and epileptic encephalopathy with mild cerebellar symptoms. Eur J Hum Genet 23, 1505-1512 (2015). https: / / doi.org:10.1038 / ejhg.2015.21 4 Lipman, A. R., Fan, X., Shen, Y. & Chung, W. K. Clinical and genetic characterization of CACNA1A-related disease. Clin Genet 102, 288-295 (2022). https: / / doi.org:10.1111 / cge.14180 5 Zhuchenko, O. et al. Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 15, 62-69 (1997). https: / / doi.org:10.1038 / ng0197-62 6 Lykke-Andersen, S. & Jensen, T. H. Nonsense-mediated mRNA decay: an intricate machinery that shapes transcriptomes. Nat Rev Mol Cell Biol 16, 665-677 (2015). https: / / doi.org:10.1038 / nrm4063 7 Havens, M. A. & Hastings, M. L. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res 44, 6549-6563 (2016). https: / / doi.org:10.1093 / nar / gkw533 8 Finkel, R. S. et al. Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy. N Engl J Med 377, 1723-1732 (2017). https: / / doi.org:10.1056 / NEJMoa1702752 9 Han, Z. et al. Antisense oligonucleotides increase Scn1a expression and reduce seizures and SUDEP incidence in a mouse model of Dravet syndrome. Sci Transl Med 12 (2020). https: / / doi.org:10.1126 / scitranslmed.aaz6100297599530.1 - 44 -10 Yuan, Y. et al. ASO restores excitability, GABA signalling and sodium current density in a model of Dravet syndrome. Brain (2023). https: / / doi.org:10.1093 / brain / awad349 11 Kim, J. et al. A framework for individualized splice-switching oligonucleotide therapy. Nature 619, 828-836 (2023). https: / / doi.org:10.1038 / s41586-023-06277-0 12 Busskamp, V. et al. Rapid neurogenesis through transcriptional activation in human stem cells. Mol Syst Biol 10, 760 (2014). https: / / doi.org:10.15252 / msb.20145508297599530.1 - 45 -

Claims

WHAT IS CLAIMED IS:

1. A method of modulating levels of a protein produced from a CACNA1A pre-mRNA in a cell, the method comprising contacting the cell with one or more antisense oligonucleotides (ASOs), wherein at least one of the ASOs are fully complementary to a portion of only one contiguous region of the CACNA1A pre-mRNA in the cell.

2. The method of claim 1, wherein the cell is a neuron.

3. The method of claim 1 or 2, wherein the cell comprises a gain-of-function mutation in a CACNA1A gene in the cell’s genome.

4. The method of claim 3, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW555 (SEQ ID NO:20), YRW569 (SEQ ID NO:21), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), YRW596 (SEQ ID NO:25), YRW597 (SEQ ID NO:26), YRW598 (SEQ ID NO:27), or a combination thereof.

5. The method of claim 3, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), or a combination thereof.

6. The method of claim 1 or 2, wherein the cell comprises a loss-of-function mutation in a CACNA1A gene in the cell’s genome.

7. The method of claim 6, wherein the one or more ASOs comprise YRW519 (SEQ ID NO:1), YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW525 (SEQ ID NO:4), YRW527 (SEQ ID NO:5), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID NO:9), YRW537 (SEQ ID NO:10), YRW539 (SEQ ID NO:11), YRW540 (SEQ ID NO:12), YRW543 (SEQ ID NO:13), YRW544 (SEQ ID NO:14), YRW545 (SEQ ID NO:15), YRW546 (SEQ ID NO:16), or a combination thereof.

8. The method of claim 6, wherein the one or more ASOs comprise YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID NO:9), YRW537 (SEQ ID NO:10), or a combination thereof.297599530.1 - 46 -9. The method of any one of claims 1 to 8, wherein the one contiguous region of the CACNA1A pre-mRNA is a nonsense-mediated decay exon, or flanking region thereof, of the CACNA1A pre-mRNA.

10. A method of treating a neurological disease in a patient, the method comprising administering one or more antisense oligonucleotides (ASOs) to the patient, wherein at least one of the ASOs are fully complementary to a portion of only one contiguous region of a CACNA1A pre-mRNA in the patient.

11. The method of claim 10, wherein the patient has a mutation in a gene encoding the CACNA1A pre-mRNA.

12. The method of claim 10 or 11, wherein the mutation comprises a gain-of-function mutation in a CACNA1A gene in the patient’s genome.

13. The method of claim 12, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW555 (SEQ ID NO:20), YRW569 (SEQ ID NO:21), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), YRW596 (SEQ ID NO:25), YRW597 (SEQ ID NO:26), YRW598 (SEQ ID NO:27), or a combination thereof.

14. The method of claim 12, wherein the one or more ASOs comprise YRW549 (SEQ ID NO:17), YRW550 (SEQ ID NO:18), YRW551 (SEQ ID NO:19), YRW571 (SEQ ID NO:22), YRW574 (SEQ ID NO:23), YRW575 (SEQ ID NO:24), or a combination thereof.

15. The method of any one of claims 12 to 14, wherein the patient has, is suspected of having, or has been diagnosed with having familial hemiplegic migraine 1 (FHM1).

16. The method of claim 15, wherein the patient also has progressive cerebellar ataxia.

17. The method of claim 15, wherein the patient does not have progressive cerebellar ataxia.

18. The method of any one of claims 12 to 14, wherein the patient has, is suspected of having, or has been diagnosed with having spinocerebellar ataxia 6 (SCA6).

19. The method of claim 10 or 11, wherein the mutation comprises a loss-of-function mutation in a CACNA1A gene in the patient’s genome.297599530.1 - 47 -20. The method of claim 19, wherein the one or more ASOs comprise YRW519 (SEQ ID NO:1), YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW525 (SEQ ID NO:4), YRW527 (SEQ ID NO:5), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID NO:9), YRW537 (SEQ ID NO:10), YRW539 (SEQ ID NO:11), YRW540 (SEQ ID NO:12), YRW543 (SEQ ID NO:13), YRW544 (SEQ ID NO:14), YRW545 (SEQ ID NO:15), YRW546 (SEQ ID NO:16), or a combination thereof.

21. The method of claim 19, wherein the one or more ASOs comprise YRW520 (SEQ ID NO:2), YRW521 (SEQ ID NO:3), YRW533 (SEQ ID NO:6), YRW534 (SEQ ID NO:7), YRW535 (SEQ ID NO:8), YRW536 (SEQ ID NO:9), YRW537 (SEQ ID NO:10), or a combination thereof.

22. The method of any one of claims 19 to 21, wherein the patient has, is suspected of having, or has been diagnosed with having episodic ataxia type 2 (EA2), developmental and epileptic encephalopathy 42 (DEE42), and / or autism spectrum disorder (ASD).

23. The method of any one of claims 10 to 22, wherein the one contiguous region of the CACNA1A pre-mRNA is a nonsense-mediated decay exon, or flanking region thereof, of the CACNA1A pre-mRNA.

24. The method of any one of claims 10 to 23, wherein the patient is further administered an additional therapeutic agent.

25. A composition comprising at least one of YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, YRW598.

26. An antisense oligonucleotide having 90%, 95%, or 100% sequence identity to the sequence of YRW549, YRW550, YRW551, YRW555, YRW569, YRW571, YRW574, YRW575, YRW596, YRW597, or YRW598, wherein the antisense oligonucleotide is isolated.

27. A composition comprising at least one of YRW519, YRW520, YRW521, YRW525, YRW527, YRW533, YRW534, YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546.

28. An antisense oligonucleotide having 90%, 95%, or 100% sequence identity to the sequence of YRW519, YRW520, YRW521, YRW525, YRW527, YRW533, YRW534,297599530.1 - 48 -YRW535, YRW536, YRW537, YRW539, YRW540, YRW543, YRW544, YRW545, YRW546, wherein the antisense oligonucleotide is isolated.297599530.1 - 49 -

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